Preface

Neurochemistry is a vitally important academic discipline that contributes to our understanding of molecular, cellular, and medical neurobiology. As a field, neurochemistry focuses on the role of the chemical entities that build the nervous system, the function of neurons and glial cells in health and disease, aspects of cell metabolism and neurotransmission, and degenerative processes and aging of the nervous system. Accordingly, this book contains chapters on a variety of topics, written by experts in their respective fields. This book is a valuable resource for neurochemists and other scientists alike. In addition, it contributes to the training of current and future neurochemists and, hopefully, will lead us on the path to curing some of the biggest challenges in human health.

In Chapter 1 ('Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction'), Drs. Thomas Heinbockel and Antonei Csoka introduce the field of neurochemistry as a whole. Holistically it is concerned with the types, structures, and functions of the chemical components of the nervous system, and how the physiology of the nervous system is regulated by said chemicals. Neurological diseases such as Alzheimer's and Parkinson's disease are often a consequence of changes in the body's neurochemistry. Medicine uses neurochemicals to alter brain function and treat disease. Neurochemists study how the components of the nervous system function during processes such as neural plasticity, neural development, and learning and memory formation, and how these components undergo changes during disease, neural dysfunction, and aging. The chapter also includes examples of how external and internal factors impact and modify neurochemistry.

In Chapter 2 ('Synaptic Transmission and Amino Acid Neurotransmitters'), Dr. Manorama Patri reviews the role of amino acids acting as neurotransmitters in the brain. Amino acids, primarily glutamic acid, GABA, aspartic acid, and glycine are released from pre-synaptic nerve terminals in response to action potentials and cross the synaptic cleft to bind with specific receptors on the postsynaptic membrane to elicit responses. Interestingly, unlike the monoamine transmitters (5% of the total synapses in brain), glutamate and GABA are thought to account for at least 50% of the synapses. Also, glutamate and aspartate in particular provide the CNS with many functions essential for learning and memory, structural and functional organisation, neural development, and neurodegeneration.

In Chapter 3 ('Trends of Protein Aggregation in Neurodegenerative Diseases') Dr. Abdulbaki Agbas introduces the reader to protein aggregations that occur in the brain and, thereby, cause neurodegenerative diseases. He outlines the nature of protein aggregation and proteolytic systems such as the proteasome and autophagosome in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Huntington disease, and prion disease, including new studies and findings.

Finally, in Chapter 4 ('Targeting the NO/cGMP/CREB Phosphorylation Signaling Pathway in Alzheimer's Disease'), Dr. Jole Fiorito and colleagues provide

a comprehensive review of the nitric oxide (NO) signaling pathway in the hippocampus and its role in the pathogenesis of Alzheimer's disease. This pathway culminates with the phosphorylation of the transcriptional factor cAMP-responsive element binding (CREB) protein via increase of cyclic guanosine monosphosphate (cGMP) and activation of cGMP-dependent protein kinase. The chapter provides an overview of the progress being made in modulating hippocampal synaptic transmissions, which are critical for learning and memory, by targeting different components of said pathway. Furthermore, the chapter explores recent research on the pathway through the use of phosphodiesterase inhibitors.

We are grateful to IntechOpen for conceiving of this book project and for asking us to serve as editors. Thanks goes to Dolores Kuzelj at IntechOpen for guiding us through the publication process and for moving the book ahead in a timely fashion. Thanks are also due to all contributors of this book for taking the time to write a chapter proposal, compose the chapter, and make the requested revisions. Hopefully all contributors will continue their neurochemistry research with many intellectual challenges in exciting new directions. T.H. would like to thank his wife Dr. Vonnie D.C. Shields, Associate Dean and Professor, Towson University, Towson, MD, and their son Torben Heinbockel for the time that he was able to spend working on this book project during the past year. Finally, T.H. is grateful to his parents Erich and Renate Heinbockel for their continuous support and interest in his work over many years.

## **Thomas Heinbockel, Ph.D.**

Professor & Interim Chair, Department of Anatomy, Howard University College of Medicine, Washington, DC, USA

### **Antonei B. Csoka, Ph.D.**

**1**

**Chapter 1**

**1. Introduction**

Introductory Chapter: The

*Thomas Heinbockel and Antonei B. Csoka*

and Dysfunction

impact and modify these components.

**2. Building blocks of the nervous system**

Chemical Basis of Neural Function

What is neurochemistry? As a field of study, neurochemistry is concerned with the types, structures and functions of the chemical components found in the nervous system [1]. These components in turn regulate the physiology of the nervous system [2–4]. Neurochemistry is mainly concerned with the chemicals that are specifically found in the nervous system such as small organic molecules, neurotransmitters and neuropeptides. Neurological diseases are often a reflection of changes in the body's neurochemistry, e.g., in Alzheimer's disease or Parkinson's disease. Medicine uses neurochemicals to alter brain function and treat disease. Neurochemists study how the components of the nervous system are at work during processes such as neural plasticity, neural development, learning and memory formation and how these components undergo changes during disease processes, neural dysfunction, and aging. This chapter will introduce the chemical components of the nervous system and briefly discuss how external and internal factors

The nervous system comprises a vast array of cells that vary in form and function and how they interact with other cells. The two principal types of cells are nerve cells or neurons and glial cells. Both types have many subtypes that are named based either on their shape or function. Neurons can be broadly classified as unipolar, bipolar, multipolar or pseudounipolar based on their arrangement and presence of dendrites and axons, or they are classified as sensory, motor or interneurons based on their function in neural networks. Dendrites are considered as the recipient portion of a nerve cell while axons carry information to other parts of the nervous system. However, this distinction can be blurred in neural circuits where both axons and dendrites can serve in either function. Axons reach a length of 1.5 m in adult humans and are even longer in larger animals such as giraffes. While axons serve as long-distance communication devices for information through the propagation of action potentials, axons also transport physical material toward the axonal terminal and from the terminal to the cell body. Protein synthesis takes place in the cell body where genetic information is located. Therefore, all proteins and also organelles such as mitochondria that are needed in the axon terminal have to be shipped down the axon with the help of motor proteins. Two motor proteins, kinesin and dynein, move vesicles or organelles along microtubules in the axon.

Associate Professor, Department of Anatomy, Howard University College of Medicine, Washington, DC, USA

## **Chapter 1**

## Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction

*Thomas Heinbockel and Antonei B. Csoka*

## **1. Introduction**

What is neurochemistry? As a field of study, neurochemistry is concerned with the types, structures and functions of the chemical components found in the nervous system [1]. These components in turn regulate the physiology of the nervous system [2–4]. Neurochemistry is mainly concerned with the chemicals that are specifically found in the nervous system such as small organic molecules, neurotransmitters and neuropeptides. Neurological diseases are often a reflection of changes in the body's neurochemistry, e.g., in Alzheimer's disease or Parkinson's disease. Medicine uses neurochemicals to alter brain function and treat disease. Neurochemists study how the components of the nervous system are at work during processes such as neural plasticity, neural development, learning and memory formation and how these components undergo changes during disease processes, neural dysfunction, and aging. This chapter will introduce the chemical components of the nervous system and briefly discuss how external and internal factors impact and modify these components.

### **2. Building blocks of the nervous system**

The nervous system comprises a vast array of cells that vary in form and function and how they interact with other cells. The two principal types of cells are nerve cells or neurons and glial cells. Both types have many subtypes that are named based either on their shape or function. Neurons can be broadly classified as unipolar, bipolar, multipolar or pseudounipolar based on their arrangement and presence of dendrites and axons, or they are classified as sensory, motor or interneurons based on their function in neural networks. Dendrites are considered as the recipient portion of a nerve cell while axons carry information to other parts of the nervous system. However, this distinction can be blurred in neural circuits where both axons and dendrites can serve in either function. Axons reach a length of 1.5 m in adult humans and are even longer in larger animals such as giraffes. While axons serve as long-distance communication devices for information through the propagation of action potentials, axons also transport physical material toward the axonal terminal and from the terminal to the cell body. Protein synthesis takes place in the cell body where genetic information is located. Therefore, all proteins and also organelles such as mitochondria that are needed in the axon terminal have to be shipped down the axon with the help of motor proteins. Two motor proteins, kinesin and dynein, move vesicles or organelles along microtubules in the axon.

Kinesin moves vesicles toward the terminal or away from the center of the neuron (anterograde axonal transport) while dynein moves in the opposite direction, namely from the terminal toward the cell body (retrograde axonal transport).

Glial cells also come in different flavors. Glial cells are increasingly recognized for their physiological functions in the nervous system and have been named "the unsung heroes of the brain" [5]. Certain glial cell types are found only in the peripheral nervous system such as satellite cells or myelin-forming Schwann cells, while others are housed in the central nervous system (brain and spinal cord) such as astrocytes (fibrous and protoplasmic), microglia and myelin-forming oligodendrocytes. Despite these commonly accepted classifications, it should be clear that nerve cells are so varied in their morphology that it has been virtually impossible to adequately classify them based on shape, ultrastructure, neurotransmitter profile, physiology or location. Furthermore, neurons with similar form and function have been described in distant animal taxa that do not share recent phylogenetic relations.

Nerve cells are equipped with cellular machinery that is present in most other cell types such as nucleus, Golgi apparatus, mitochondria, smooth and rough endoplasmic reticulum. Histological staining of nerve cells with dyes such as toluidine blue or cresyl violet (Nissl staining, nucleic acid stain) reveals particularly intense labeling of the nucleolus and Nissl substance in the cytoplasm [6]. Nissl substance refers to free ribosomes in the cytoplasm and bound ribosomes on the ER. This heavy staining pattern is a reflection of the active metabolism and continuous production of peptides and proteins in nerve cells and identifies nerve cells among the most active cells in the body.

#### **3. Excitable cell membranes and channelopathies**

Nerve cells and glial cells are compartmentalized by membranes that are built by lipids and proteins. These lipids and proteins are key elements for the unique functional role each neuron plays in the neural circuit and for the intracellular activities that occur in axons and dendrites distant from the cell nucleus. During development, axonal guidance and remodeling of dendritic spines are shaped in response to signal input at local membrane compartments which is communicated to the cell interior through specific receptors and channels. The inside and outside of the cellular membranes are different from each other, such that an asymmetric distribution of lipids and proteins between cytoplasmic and exoplasmic leaflets allows for an unequal division of labor [1]. Lipids are critically relevant for the structure and function of the nervous system. Membrane lipids are the main component of the myelin that ensheathes axons both in the central and peripheral nervous system. Furthermore, at the connections between nerve cells, the synapses, membranes have unique lipid compositions. The synapse is equipped with a synaptic machinery of vesicles and proteins that contribute to the specialized properties of these membrane compartments and to the plastic changes in synaptic transmission from pre- to postsynaptic neurons (synaptic plasticity) [7–9]. Lipid intermediates and lipid modification play roles in signaling pathways related to cell differentiation and in modulating the activity of trophic factors and receptors [1].

Nerve cells are excitable cells with unique properties in transferring information. In order to do so, the membranes of nerve cells are equipped with highly selective pores or ion channels for sodium, potassium, calcium and chloride ions. These channels are critical for membrane excitation and propagation of action potentials. Ion channels are responsive to changes in voltage (voltage gated channels), binding of a chemical (ligand gated channels) or mechanical perturbation.

**3**

*Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction*

Channelopathies, disorders of ion channels, are resulting from disturbed ion channel function due to problems with ion channel subunits or regulation of the ion channels [10]. The rapidly growing field of channelopathies started with the discovery of voltage gated channelopathies that result in inherited muscle disease due to mutations in a subunit of the sodium channel or a mutation in a gene coding for a chloride channel in skeletal muscle [10]. Channelopathies have also been identified for ligand gated ion channels due to mutations of a subunit of the glycine receptor or a subunit of the nicotinic acetylcholine receptor [11, 12]. The underlying reasons for channelopathies can be traced back to either inherited causes (congenital, resulting from one or more mutations in the genes encoding the ion channel) or

acquired causes such as toxins and autoimmune attack on an ion channel.

The best known neurochemicals are neurotransmitters and neuropeptides since

they modulate brain function [1–4]. One set of neurotransmitters is formed by common amino acids such as glutamate, gamma-aminobutyric acid (GABA) and glycine. These amino acids have a number of functions throughout the body. In nerve terminals of neurons, they are packaged and stored in secretory or synaptic vesicles, so they can be released by exocytosis in a calcium dependent manner. The vesicular membrane is recycled, i.e., endocytosed, for future synaptic release cycles. Glutamate is the most prominent excitatory neurotransmitter. It is released at excitatory synapses, and evokes membrane potential depolarization and, possibly, firing of action potentials in the connected postsynaptic cell. In contrast, GABA is the best known inhibitory neurotransmitter. Its action results in reducing neuronal excitability. Glycine is another inhibitory neurotransmitter found in the spinal cord, brainstem and retina. The monoamines form an important group of neurotransmitters involved in regulation of emotion, arousal, some forms of memory as well as sensory processing [1–4, 13, 14]. Because of their functional roles, drugs are used to regulate their effects in patients with psychiatric as well as neurological disorders [15]. As their name implies, monoamines contain an amino group connected to an aromatic ring by a two-carbon chain. The enzymes monoamine oxidases terminate the action of monoamines. Histamine, serotonin, dopamine, epinephrine (adrenaline), norepinephrine (noradrenaline) are monoamines. The latter three are also grouped together as catecholamines because they contain a catechol group. Trace amines such as octopamine, tryptamine, tyramine, phenethylamine and others, have been identified as neurotransmitters. Neuropeptides include compounds such as oxytocin, substance P, somatostatin, opioid peptides, cocaine and amphetamine regulated transcript (CART), glucagon, orexin, dynorphin, endorphin, enkephalin, neuropeptide Y, neuropeptide S, and others. Nitric oxide, hydrogen sulfide and carbon monoxide act as gaseous neurotransmitters [16]. These neurotransmitters are synthesized de novo in nerve cells and because of their chemical nature are able to rapidly diffuse through the plasma membrane to act on neighboring cells. Acetylcholine is released in the autonomic nervous system and also from motor neurons at the neuromuscular junction to evoke skeletal muscle contraction. Chemically, acetylcholine is an ester of acetic acid and choline. Endogenously produced cannabinoids (endocannabinoids) such as anandamide differ from the above mentioned neurotransmitters because they are formed from membrane lipids and are essentially lipids. They can be rapidly synthesized on demand from the cell membrane and released nonsynaptically and not from synaptic vesicles as the classic neurotransmitters, reviewed in [9, 17, 18]. Endocannabinoids bind to cannabinoid receptors on presynaptic neurons to regulate presynaptic neurotransmitter release. Therefore, endocannabinoids together with the

*DOI: http://dx.doi.org/10.5772/intechopen.89072*

**4. Neurotransmitters and neuropeptides**

*Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.89072*

Channelopathies, disorders of ion channels, are resulting from disturbed ion channel function due to problems with ion channel subunits or regulation of the ion channels [10]. The rapidly growing field of channelopathies started with the discovery of voltage gated channelopathies that result in inherited muscle disease due to mutations in a subunit of the sodium channel or a mutation in a gene coding for a chloride channel in skeletal muscle [10]. Channelopathies have also been identified for ligand gated ion channels due to mutations of a subunit of the glycine receptor or a subunit of the nicotinic acetylcholine receptor [11, 12]. The underlying reasons for channelopathies can be traced back to either inherited causes (congenital, resulting from one or more mutations in the genes encoding the ion channel) or acquired causes such as toxins and autoimmune attack on an ion channel.

### **4. Neurotransmitters and neuropeptides**

*Neurochemical Basis of Brain Function and Dysfunction*

Kinesin moves vesicles toward the terminal or away from the center of the neuron (anterograde axonal transport) while dynein moves in the opposite direction, namely from the terminal toward the cell body (retrograde axonal transport). Glial cells also come in different flavors. Glial cells are increasingly recognized

Nerve cells are equipped with cellular machinery that is present in most other cell types such as nucleus, Golgi apparatus, mitochondria, smooth and rough endoplasmic reticulum. Histological staining of nerve cells with dyes such as toluidine blue or cresyl violet (Nissl staining, nucleic acid stain) reveals particularly intense labeling of the nucleolus and Nissl substance in the cytoplasm [6]. Nissl substance refers to free ribosomes in the cytoplasm and bound ribosomes on the ER. This heavy staining pattern is a reflection of the active metabolism and continuous production of peptides and proteins in nerve cells and identifies nerve cells among

Nerve cells and glial cells are compartmentalized by membranes that are built by lipids and proteins. These lipids and proteins are key elements for the unique functional role each neuron plays in the neural circuit and for the intracellular activities that occur in axons and dendrites distant from the cell nucleus. During development, axonal guidance and remodeling of dendritic spines are shaped in response to signal input at local membrane compartments which is communicated to the cell interior through specific receptors and channels. The inside and outside of the cellular membranes are different from each other, such that an asymmetric distribution of lipids and proteins between cytoplasmic and exoplasmic leaflets allows for an unequal division of labor [1]. Lipids are critically relevant for the structure and function of the nervous system. Membrane lipids are the main component of the myelin that ensheathes axons both in the central and peripheral nervous system. Furthermore, at the connections between nerve cells, the synapses, membranes have unique lipid compositions. The synapse is equipped with a synaptic machinery of vesicles and proteins that contribute to the specialized properties of these membrane compartments and to the plastic changes in synaptic transmission from pre- to postsynaptic neurons (synaptic plasticity) [7–9]. Lipid intermediates and lipid modification play roles in signaling pathways related to cell differentiation and

for their physiological functions in the nervous system and have been named "the unsung heroes of the brain" [5]. Certain glial cell types are found only in the peripheral nervous system such as satellite cells or myelin-forming Schwann cells, while others are housed in the central nervous system (brain and spinal cord) such as astrocytes (fibrous and protoplasmic), microglia and myelin-forming oligodendrocytes. Despite these commonly accepted classifications, it should be clear that nerve cells are so varied in their morphology that it has been virtually impossible to adequately classify them based on shape, ultrastructure, neurotransmitter profile, physiology or location. Furthermore, neurons with similar form and function have been described in distant animal taxa that do not share recent phylogenetic

**2**

relations.

the most active cells in the body.

**3. Excitable cell membranes and channelopathies**

in modulating the activity of trophic factors and receptors [1].

Nerve cells are excitable cells with unique properties in transferring information. In order to do so, the membranes of nerve cells are equipped with highly selective pores or ion channels for sodium, potassium, calcium and chloride ions. These channels are critical for membrane excitation and propagation of action potentials. Ion channels are responsive to changes in voltage (voltage gated channels), binding of a chemical (ligand gated channels) or mechanical perturbation.

The best known neurochemicals are neurotransmitters and neuropeptides since they modulate brain function [1–4]. One set of neurotransmitters is formed by common amino acids such as glutamate, gamma-aminobutyric acid (GABA) and glycine. These amino acids have a number of functions throughout the body. In nerve terminals of neurons, they are packaged and stored in secretory or synaptic vesicles, so they can be released by exocytosis in a calcium dependent manner. The vesicular membrane is recycled, i.e., endocytosed, for future synaptic release cycles. Glutamate is the most prominent excitatory neurotransmitter. It is released at excitatory synapses, and evokes membrane potential depolarization and, possibly, firing of action potentials in the connected postsynaptic cell. In contrast, GABA is the best known inhibitory neurotransmitter. Its action results in reducing neuronal excitability. Glycine is another inhibitory neurotransmitter found in the spinal cord, brainstem and retina. The monoamines form an important group of neurotransmitters involved in regulation of emotion, arousal, some forms of memory as well as sensory processing [1–4, 13, 14]. Because of their functional roles, drugs are used to regulate their effects in patients with psychiatric as well as neurological disorders [15]. As their name implies, monoamines contain an amino group connected to an aromatic ring by a two-carbon chain. The enzymes monoamine oxidases terminate the action of monoamines. Histamine, serotonin, dopamine, epinephrine (adrenaline), norepinephrine (noradrenaline) are monoamines. The latter three are also grouped together as catecholamines because they contain a catechol group. Trace amines such as octopamine, tryptamine, tyramine, phenethylamine and others, have been identified as neurotransmitters. Neuropeptides include compounds such as oxytocin, substance P, somatostatin, opioid peptides, cocaine and amphetamine regulated transcript (CART), glucagon, orexin, dynorphin, endorphin, enkephalin, neuropeptide Y, neuropeptide S, and others. Nitric oxide, hydrogen sulfide and carbon monoxide act as gaseous neurotransmitters [16]. These neurotransmitters are synthesized de novo in nerve cells and because of their chemical nature are able to rapidly diffuse through the plasma membrane to act on neighboring cells. Acetylcholine is released in the autonomic nervous system and also from motor neurons at the neuromuscular junction to evoke skeletal muscle contraction. Chemically, acetylcholine is an ester of acetic acid and choline. Endogenously produced cannabinoids (endocannabinoids) such as anandamide differ from the above mentioned neurotransmitters because they are formed from membrane lipids and are essentially lipids. They can be rapidly synthesized on demand from the cell membrane and released nonsynaptically and not from synaptic vesicles as the classic neurotransmitters, reviewed in [9, 17, 18]. Endocannabinoids bind to cannabinoid receptors on presynaptic neurons to regulate presynaptic neurotransmitter release. Therefore, endocannabinoids together with the gaseous neurotransmitters are unusual neurotransmitters [17, 19, 20]. One key distinction of these novel neurotransmitters is the fact that they act as retrograde messengers at synapses and presynaptically regulate either glutamatergic or GABAergic synapses to alter release-probability in synaptic plasticity. Gaseous neurotransmitters and endocannabinoids have been shown to have a functional role in experiencedependent activity and mediate a variety of forms of short- and long-term synaptic plasticity [21–24].

## **5. Factors that influence neurochemistry**

What are some of the factors that affect the chemistry of the nervous system? Factors that modify neurochemistry include sensory stimuli, environmental signals such as recreational drugs, pharmaceuticals and toxins, and bodily changes such as aging and disease. Listed below, and described in more detail, are examples of some such factors known to influence neurochemistry. It is important to realize that this list is not exhaustive, and that in theory almost any external stimulus or internal state could influence neurochemistry.

#### **5.1 Sleep**

Sleep is controlled by circadian rhythms, which have a neurochemical (and oscillatory epigenetic) basis. During waking, several brain structures participate, namely the basal forebrain, posterior and lateral hypothalamus, and nuclei in the tegmentum and pons. Neurotransmitters that act as significant wakefulness factors are acetylcholine and monoamines, glutamate and hypocretin/orexin. Conversely, the preoptic/anterior hypothalamic area regulates active sleep mechanisms, and sleep is promoted by GABA and peptide factors, including growth hormone-releasing hormone, cytokines, and prolactin [25]. Adenosine is a significant homeostatic factor acting in basal forebrain and preoptic areas via A1 and A2A receptors. Lack of sleep increases inducible nitric oxide synthase in the basal forebrain, which causes adenosine release and recovery sleep. Also, many genes have been found differentially expressed in wakefulness versus sleep, and they relate to neural transmission, energy metabolism, stress protection, and synaptic plasticity [25].

#### **5.2 Exercise**

There has recently been a large amount of research into the neurochemical changes that occur during and after exercise, finding that it stimulates the increase of many chemicals, namely lactate, cortisol, neurotrophins, including BDNF, VEGF and IGF-1, neurotransmitters, including dopamine, serotonin, norepinephrine, GABA, acetylcholine, and glutamate, and neuromodulators, including endocannabinoids and endogenous opioids [26]. However, it should be noted that many of these alterations have been demonstrated only peripherally, and gaps still exist in our knowledge as to exactly where these changes occur in the brain. Therefore, further work is needed to link the exercise-induced changes in peripheral levels to central levels, and to understand how these chemicals are involved in the exerciseinduced changes in cognition, mood, and so forth [26].

#### **5.3 Diet**

The scientific field concerned with the effects various contents of the diet such as macronutrients, and micronutrients such as minerals, vitamins, dietary

**5**

*Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction*

supplements, and food additives have on neurochemistry is called "nutritional neuroscience." Recent research on nutrition and its effect on the brain show that it is involved in almost every aspect of neurological function, modulating neurotrophic factors, neural pathways, neurogenesis, and neuroplasticity [27]. This is not surprising when we consider that the brain consumes a very large amount of energy relative to the rest of the body. Specifically, the human brain is approximately 2% of the body mass but uses up to 25% of the energy input. Therefore, mechanisms involved in the transfer of energy from foods to neurons are likely to be essential to the control of brain function. Furthermore, insufficient intake of certain vitamins and other cofactors, or the consequences of metabolic disorders such as diabetes, affect cognition by altering processes in the body that are associated with the management of energy and synthesis of neurotrophic and neuroendocrine factors (i.e., BDNF and IGF-1) as well as neurotransmitter in neurons, which can subsequently affect neurotransmission, synaptic plasticity,

Stress has been defined as a brain-body reaction to stimuli from the environment or from internal states that are interpreted by the body as disrupted homeostasis [28]. The response to such stress involves both the activity of different neurotransmitters in the limbic system, and the response of neurons there to other chemicals and hormones, mainly glucocorticoids, released from the adrenal cortex. Thus, body-brain integration probably plays a major role in the stress response [28]. Specifically, acute stress is correlated with alterations of neurotransmitters such as dopamine, acetylcholine, GABA and glutamate in areas of the brain associated with the regulation of stress responses. These areas include the prefrontal cortex, amygdala, nucleus accumbens, and hippocampus. Glucocorticoids also play an important role, and interact with several neurotransmitters in those same areas of the brain. Also, the actions of neuromodulators released from peripheral organs such as the liver (IGF-1), pancreas (insulin), or gonads (estrogens) play a role [28]. A permanent increase in the baseline levels of glucocorticoids arising from a stressful lifestyle could exacerbate neuronal damage that occurs in the above areas of the brain during aging. Conversely, stress

One way to counteract stress is through practices such as meditation and yoga. These techniques have recently received increased attention due to the accumulation of research showing both direct and indirect benefits [30]. Based on studies conducted so far, it has been found that the practice of meditation influences the levels of neurotransmitters such as GABA, serotonin, dopamine, and norepinephrine, in a way that positively affects psychological disorders such as anxiety. Also, by reducing baseline levels of stress hormones and neurotransmitters, meditation may

Alcohol has effects on many neurotransmitters in the brain. Its major effect is to stimulate the release of GABA, and it acts principally at the GABAA receptors, and thereby has sedative effects [31]. It also inhibits postsynaptic NMDA excitatory glutamate receptors, and this inhibition further contributes to the sedation.

*DOI: http://dx.doi.org/10.5772/intechopen.89072*

reduction may have an anti-aging effect [29].

act as a form of preventative medicine [30].

and cell survival [27].

**5.4 Stress**

**5.5 Meditation**

**5.6 Alcohol**

#### *Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.89072*

supplements, and food additives have on neurochemistry is called "nutritional neuroscience." Recent research on nutrition and its effect on the brain show that it is involved in almost every aspect of neurological function, modulating neurotrophic factors, neural pathways, neurogenesis, and neuroplasticity [27]. This is not surprising when we consider that the brain consumes a very large amount of energy relative to the rest of the body. Specifically, the human brain is approximately 2% of the body mass but uses up to 25% of the energy input. Therefore, mechanisms involved in the transfer of energy from foods to neurons are likely to be essential to the control of brain function. Furthermore, insufficient intake of certain vitamins and other cofactors, or the consequences of metabolic disorders such as diabetes, affect cognition by altering processes in the body that are associated with the management of energy and synthesis of neurotrophic and neuroendocrine factors (i.e., BDNF and IGF-1) as well as neurotransmitter in neurons, which can subsequently affect neurotransmission, synaptic plasticity, and cell survival [27].

### **5.4 Stress**

*Neurochemical Basis of Brain Function and Dysfunction*

**5. Factors that influence neurochemistry**

state could influence neurochemistry.

plasticity [21–24].

**5.1 Sleep**

**5.2 Exercise**

gaseous neurotransmitters are unusual neurotransmitters [17, 19, 20]. One key distinction of these novel neurotransmitters is the fact that they act as retrograde messengers at synapses and presynaptically regulate either glutamatergic or GABAergic synapses to alter release-probability in synaptic plasticity. Gaseous neurotransmitters and endocannabinoids have been shown to have a functional role in experiencedependent activity and mediate a variety of forms of short- and long-term synaptic

What are some of the factors that affect the chemistry of the nervous system? Factors that modify neurochemistry include sensory stimuli, environmental signals such as recreational drugs, pharmaceuticals and toxins, and bodily changes such as aging and disease. Listed below, and described in more detail, are examples of some such factors known to influence neurochemistry. It is important to realize that this list is not exhaustive, and that in theory almost any external stimulus or internal

Sleep is controlled by circadian rhythms, which have a neurochemical (and oscillatory epigenetic) basis. During waking, several brain structures participate, namely the basal forebrain, posterior and lateral hypothalamus, and nuclei in the tegmentum and pons. Neurotransmitters that act as significant wakefulness factors are acetylcholine and monoamines, glutamate and hypocretin/orexin. Conversely, the preoptic/anterior hypothalamic area regulates active sleep mechanisms, and sleep is promoted by GABA and peptide factors, including growth hormone-releasing hormone, cytokines, and prolactin [25]. Adenosine is a significant homeostatic factor acting in basal forebrain and preoptic areas via A1 and A2A receptors. Lack of sleep increases inducible nitric oxide synthase in the basal forebrain, which causes adenosine release and recovery sleep. Also, many genes have been found differentially expressed in wakefulness versus sleep, and they relate to neural transmission,

There has recently been a large amount of research into the neurochemical changes that occur during and after exercise, finding that it stimulates the increase of many chemicals, namely lactate, cortisol, neurotrophins, including BDNF, VEGF and IGF-1, neurotransmitters, including dopamine, serotonin, norepinephrine, GABA, acetylcholine, and glutamate, and neuromodulators, including endocannabinoids and endogenous opioids [26]. However, it should be noted that many of these alterations have been demonstrated only peripherally, and gaps still exist in our knowledge as to exactly where these changes occur in the brain. Therefore, further work is needed to link the exercise-induced changes in peripheral levels to central levels, and to understand how these chemicals are involved in the exercise-

The scientific field concerned with the effects various contents of the diet such as macronutrients, and micronutrients such as minerals, vitamins, dietary

energy metabolism, stress protection, and synaptic plasticity [25].

induced changes in cognition, mood, and so forth [26].

**4**

**5.3 Diet**

Stress has been defined as a brain-body reaction to stimuli from the environment or from internal states that are interpreted by the body as disrupted homeostasis [28]. The response to such stress involves both the activity of different neurotransmitters in the limbic system, and the response of neurons there to other chemicals and hormones, mainly glucocorticoids, released from the adrenal cortex. Thus, body-brain integration probably plays a major role in the stress response [28]. Specifically, acute stress is correlated with alterations of neurotransmitters such as dopamine, acetylcholine, GABA and glutamate in areas of the brain associated with the regulation of stress responses. These areas include the prefrontal cortex, amygdala, nucleus accumbens, and hippocampus. Glucocorticoids also play an important role, and interact with several neurotransmitters in those same areas of the brain. Also, the actions of neuromodulators released from peripheral organs such as the liver (IGF-1), pancreas (insulin), or gonads (estrogens) play a role [28]. A permanent increase in the baseline levels of glucocorticoids arising from a stressful lifestyle could exacerbate neuronal damage that occurs in the above areas of the brain during aging. Conversely, stress reduction may have an anti-aging effect [29].

#### **5.5 Meditation**

One way to counteract stress is through practices such as meditation and yoga. These techniques have recently received increased attention due to the accumulation of research showing both direct and indirect benefits [30]. Based on studies conducted so far, it has been found that the practice of meditation influences the levels of neurotransmitters such as GABA, serotonin, dopamine, and norepinephrine, in a way that positively affects psychological disorders such as anxiety. Also, by reducing baseline levels of stress hormones and neurotransmitters, meditation may act as a form of preventative medicine [30].

#### **5.6 Alcohol**

Alcohol has effects on many neurotransmitters in the brain. Its major effect is to stimulate the release of GABA, and it acts principally at the GABAA receptors, and thereby has sedative effects [31]. It also inhibits postsynaptic NMDA excitatory glutamate receptors, and this inhibition further contributes to the sedation. However, alcohol also has euphoric effects, and these are more related to increases in dopamine. The effects on dopamine are also thought to be involved in alcohol craving and relapse. In addition, alcohol alters opioid receptors and can lead to a release of β-endorphins. Additional important effects include increased serotonin and decreased nicotinic acetylcholine receptors [31].

#### **5.7 Recreational drugs**

Drugs can alter the regular functions of neurochemicals, inhibit the way they are supposed to act, or disrupt their communication [32]. At first, pleasure is usually increased, but cognitive ability and rationality are decreased. Psychomotor stimulant drugs like amphetamines, methamphetamine, and cocaine cause an overproduction of neurotransmitters, principally the monoamines dopamine and norepinephrine, and may also prevent them from being reabsorbed, causing an abnormally large amount to be present in synapses, and thereby activate the mesolimbic dopamine system. Drugs like ecstasy (3,4-methalynedioxymethamphetamine) similarly interfere with the transmission of serotonin, and the way it is transported along neural pathways. Other drugs, such as heroin, opioids, and marijuana, mimic endogenous brain chemicals and bind to receptors as agonists, activating the neurons and thus disrupting the natural transmission and production of neurotransmitters [32]. With repeated drug abuse, the brain can be rewired via neuroplasticity as it attempts to maintain chemical homeostasis [33].

#### **5.8 Neurodegenerative diseases and aging**

Studies of the neurobiology of aging are beginning to uncover the mechanisms underlying not only the physiology of aging of the brain, but also the mechanisms that make people more vulnerable to cognitive dysfunction and neurodegenerative diseases [29]. Neurotransmission is impaired in age-related disorders, such as Alzheimer's and Parkinson's diseases, which has stimulated investigations into the neurochemistry of the aging human brain. Out of all the neurotransmitter systems studied, age-related changes in the serotonergic, cholinergic, and dopaminergic systems are the most reliably found [34]. The dopamine system in particular, is especially vulnerable to aging [35]. The association of these neurotransmitters with mood, memory, and motor function may contribute to age-associated behavioral changes and predispose older people to age-related diseases. Moreover, age-related neurodegenerative diseases may evolve from the interaction between defects in specific neurochemical mechanisms and other pathophysiologic processes [33].

### **Acknowledgements**

This work was supported in part by grants from the National Science Foundation (NSF IOS-1355034) and the Charles and Mary Latham Trust Fund to TH and from the National Institutes of Health (NIH) to ABC.

### **Conflict of interest**

The authors declare that there is no conflict of interests regarding the publication of this chapter.

**7**

**Author details**

Washington, DC, USA

Thomas Heinbockel\* and Antonei B. Csoka

provided the original work is properly cited.

Department of Anatomy, Howard University College of Medicine,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: theinbockel@howard.edu

*Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction*

*DOI: http://dx.doi.org/10.5772/intechopen.89072*

*Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.89072*

## **Author details**

*Neurochemical Basis of Brain Function and Dysfunction*

and decreased nicotinic acetylcholine receptors [31].

**5.7 Recreational drugs**

tain chemical homeostasis [33].

pathophysiologic processes [33].

the National Institutes of Health (NIH) to ABC.

**Acknowledgements**

**Conflict of interest**

tion of this chapter.

**5.8 Neurodegenerative diseases and aging**

However, alcohol also has euphoric effects, and these are more related to increases in dopamine. The effects on dopamine are also thought to be involved in alcohol craving and relapse. In addition, alcohol alters opioid receptors and can lead to a release of β-endorphins. Additional important effects include increased serotonin

Drugs can alter the regular functions of neurochemicals, inhibit the way they are supposed to act, or disrupt their communication [32]. At first, pleasure is usually increased, but cognitive ability and rationality are decreased. Psychomotor stimulant drugs like amphetamines, methamphetamine, and cocaine cause an overproduction of neurotransmitters, principally the monoamines dopamine and norepinephrine, and may also prevent them from being reabsorbed, causing an abnormally large amount to be present in synapses, and thereby activate the mesolimbic dopamine system. Drugs like ecstasy (3,4-methalynedioxymethamphetamine) similarly interfere with the transmission of serotonin, and the way it is transported along neural pathways. Other drugs, such as heroin, opioids, and marijuana, mimic endogenous brain chemicals and bind to receptors as agonists, activating the neurons and thus disrupting the natural transmission and production of neurotransmitters [32]. With repeated drug abuse, the brain can be rewired via neuroplasticity as it attempts to main-

Studies of the neurobiology of aging are beginning to uncover the mechanisms underlying not only the physiology of aging of the brain, but also the mechanisms that make people more vulnerable to cognitive dysfunction and neurodegenerative diseases [29]. Neurotransmission is impaired in age-related disorders, such as Alzheimer's and Parkinson's diseases, which has stimulated investigations into the neurochemistry of the aging human brain. Out of all the neurotransmitter systems studied, age-related changes in the serotonergic, cholinergic, and dopaminergic systems are the most reliably found [34]. The dopamine system in particular, is especially vulnerable to aging [35]. The association of these neurotransmitters with mood, memory, and motor function may contribute to age-associated behavioral changes and predispose older people to age-related diseases. Moreover, age-related neurodegenerative diseases may evolve from the interaction between defects in specific neurochemical mechanisms and other

This work was supported in part by grants from the National Science Foundation (NSF IOS-1355034) and the Charles and Mary Latham Trust Fund to TH and from

The authors declare that there is no conflict of interests regarding the publica-

**6**

Thomas Heinbockel\* and Antonei B. Csoka Department of Anatomy, Howard University College of Medicine, Washington, DC, USA

\*Address all correspondence to: theinbockel@howard.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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The Proceedings of the Nutrition

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[34] Strong R. Neurochemical changes in the aging human brain: Implications

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[19] Barañano DE, Ferris CD, Snyder SH. Atypical neural messengers. Trends in Neurosciences. 2001;**24**:99-106

[20] Boehning D, Snyder SH. Novel neural modulators. Annual Review of Neuroscience. 2003;**26**:105-131

[21] Hardingham N, Dachtler J, Fox K. The role of nitric oxide in presynaptic plasticity and homeostasis. Frontiers in Cellular Neuroscience.

[22] Cachope R. Functional diversity on synaptic plasticity mediated by endocannabinoids. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences.

[23] Castillo PE, Younts TJ, Chávez AE, Hashimotodani Y. Endocannabinoid signaling and synaptic function.

[24] Katona I, Freund TF. Multiple functions of endocannabinoid signaling

[25] Stenberg D. Neuroanatomy and neurochemistry of sleep. Cellular and Molecular Life Sciences.

[26] Basso JC, Suzuki WA. The effects of acute exercise on mood, cognition, neurophysiology, and neurochemical pathways: A review. Brain Plasticity.

[27] Dauncey MJ. New insights into nutrition and cognitive neuroscience.

in the brain. Annual Review of Neuroscience. 2012;**35**:529-558

978-953-51-2429-0

2013;**31**(7):190

2012;**367**:3242-3253

Neuron. 2012;**76**:70-81

2007;**64**:1187-1204

2017;**28**(2):127-152

*Introductory Chapter: The Chemical Basis of Neural Function and Dysfunction DOI: http://dx.doi.org/10.5772/intechopen.89072*

[18] Heinbockel T, Wang ZJ, Brown EA, Austin PT. Endocannabinoid signaling in neural circuits of the olfactory and limbic system 2. In: Meccariello R, Chianese R, editors. Cannabinoids in Health and Disease. Rijeka, Croatia: InTech Publisher; 2016. pp. 11-37. ISBN: 978-953-51-2429-0

[19] Barañano DE, Ferris CD, Snyder SH. Atypical neural messengers. Trends in Neurosciences. 2001;**24**:99-106

[20] Boehning D, Snyder SH. Novel neural modulators. Annual Review of Neuroscience. 2003;**26**:105-131

[21] Hardingham N, Dachtler J, Fox K. The role of nitric oxide in presynaptic plasticity and homeostasis. Frontiers in Cellular Neuroscience. 2013;**31**(7):190

[22] Cachope R. Functional diversity on synaptic plasticity mediated by endocannabinoids. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2012;**367**:3242-3253

[23] Castillo PE, Younts TJ, Chávez AE, Hashimotodani Y. Endocannabinoid signaling and synaptic function. Neuron. 2012;**76**:70-81

[24] Katona I, Freund TF. Multiple functions of endocannabinoid signaling in the brain. Annual Review of Neuroscience. 2012;**35**:529-558

[25] Stenberg D. Neuroanatomy and neurochemistry of sleep. Cellular and Molecular Life Sciences. 2007;**64**:1187-1204

[26] Basso JC, Suzuki WA. The effects of acute exercise on mood, cognition, neurophysiology, and neurochemical pathways: A review. Brain Plasticity. 2017;**28**(2):127-152

[27] Dauncey MJ. New insights into nutrition and cognitive neuroscience. The Proceedings of the Nutrition Society. 2009;**68**:408-415

[28] Mora F, Segovia G, Del Arco A, de Blas M, Garrido P. Stress, neurotransmitters, corticosterone and body-brain integration. Brain Research. 2012;**1476**:71-85

[29] Mora F. Successful brain aging: Plasticity, environmental enrichment, and lifestyle. Dialogues in Clinical Neuroscience. 2013;**15**:45-52

[30] Krishnakumar D, Hamblin MR, Lakshmanan S. Meditation and yoga can modulate brain mechanisms that affect behavior and anxiety—A modern scientific perspective. Ancient Science. 2015;**2**:13-19

[31] Banerjee N. Neurotransmitters in alcoholism: A review of neurobiological and genetic studies. Indian Journal of Human Genetics. 2014;**20**:20-31

[32] Korpi ER, den Hollander B, Farooq U, Vashchinkina E, Rajkumar R, Nutt DJ, et al. Mechanisms of action and persistent neuroplasticity by drugs of abuse. Pharmacological Reviews. 2015;**67**:872-1004

[33] Leshner AI, Koob GF. Drugs of abuse and the brain. Proceedings of the Association of American Physicians. 1999;**111**:99-108

[34] Strong R. Neurochemical changes in the aging human brain: Implications for behavioral impairment and neurodegenerative disease. Geriatrics. 1998;**53**(Suppl 1):S9-S12

[35] Dreher JC, Meyer-Lindenberg A, Kohn P, Berman KF. Age-related changes in midbrain dopaminergic regulation of the human reward system. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**:15106-15111

**8**

1301559

10.5772/67891

*Neurochemical Basis of Brain Function and Dysfunction*

[10] Rose MR. Dysfunctional ion channels may cause many neurological diseases. BMJ. 1998;**316**(7138):1104- 1105. DOI: 10.1136/bmj.316.7138.1104

[11] Shiang R, Ryan SG, Zhu YZ, Hahn AF, O'Connell P, Wasmuth JJ. Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nature Genetics.

[12] Steinlein OK, Mulley JC,

[13] Bloom FE. The functional significance of neurotransmitter diversity. The American Journal of Physiology. 1984;**246**(3 Pt 1):

[14] Jacob SN, Nienborg H.

Monoaminergic neuromodulation of sensory processing. Front Neural Circuits. 2018;**12**:51. DOI: 10.3389/ fncir.2018.00051. eCollection 2018

[15] Kurian MA, Gissen P, Smith M, Heales SJR, Clayton PT. The monoamine

[16] Haley JE. Gases as neurotransmitters. Essays in Biochemistry. 1998;**33**:79-91

neurotransmitter disorders: An expanding range of neurological syndromes. The Lancet Neurology. 2011;**10**(8):721-733. DOI: 10.1016/

[17] Heinbockel T, editor. Chapter 6: Neurochemical communication: The case of endocannabinoids. In: Neurochemistry. Rijeka, Croatia: InTech Open Access Publisher; 2014. pp. 179-198.

S1474-4422(11)70141-7

ISBN: 978-953-51-1237-2

Propping P, Wallace RH, Phillips HA, Scheffer IE, et al. A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nature Genetics.

1993;**5**:351-358

1995;**11**:201-203

C184-C194

[1] Brady ST, Siegel JG, Albers BW, Price DL. Basic Neurochemistry, Principles of Molecular, Cellular, and Medical Neurobiology. 8th ed. Boston: Elsevier; 2012. DOI: 10.1016/C2009-0- 00066-X. ISBN: 978-0-12-374947-5

[2] Purves D, Augustine GJ,

McGraw-Hill; 2013. ISBN: 978-0-07-139011-8

ISBN: 978-0-7817-7817-6

ISBN: 978-0-323-03388-6

978-981-4307-31-4

[5] Young JK. Introduction to Cell Biology. New Jersey: World Scientific Publishing Co. Pte. Ltd.; 2010. ISBN-13:

[6] Telser AG, Young JK, Baldwin KM. Elsevier's Integrated Histology. Philadelphia: Mosby Elsevier; 2007.

[7] Sheng M, Sabatini BL, Südhof TC. The Synapse. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2012. 397pp

[8] Citri A, Malenka RC. Synaptic

plasticity: Multiple forms, functions, and mechanisms. Neuropsychopharmacology. 2008;**33**:18-41. DOI: 10.1038/sj.npp.

[9] Heinbockel T, editor. Introductory chapter: Mechanisms and function of synaptic plasticity. In: Synaptic Plasticity. Rijeka, Croatia: InTech Publisher; 2017. pp. 3-13. DOI:

978-0-87893-695-3

**References**

Fitzpatrick D, Hall WC, LaMantia AS, White LE. Neuroscience. 5th ed. Sunderland: Sinauer; 2012. ISBN:

[3] Kandel ER, Schwartz JH, Jessell TM, Siegelbaum SA, Hudspeth AJ. Principles of Neural Science. 5th ed. New York:

[4] Bear M, Connors BW, Paradiso MA. Neuroscience—Exploring the Brain. 4th ed. Philadelphia: Wolters Kluwer; 2016.

**11**

**Chapter 2**

**Abstract**

**1. Introduction**

*Manorama Patri*

Synaptic Transmission and Amino

Amino acids are the most abundant neurotransmitters in the brain. Neurotransmitters are synthesized and stored in presynaptic terminals, released from terminals upon stimulation with specific receptors on the postsynaptic cells. Chemical and electrical synapses are specialized biological structures found in the nervous system; they connect neurons together and transmit signals across the neurons. The process of synaptic transmission generates or inhibits electrical impulses in a network of neurons for the processing of information. Glutamate is the primary excitatory neurotransmitter in the brain, while GABA is the principal inhibitory neurotransmitter. The balance of glutamatergic and GABAergic tone is crucial to normal neurologic function. Through synaptic transmission, this information is communicated from the presynaptic cell to the postsynaptic cell. Amino acid neurotransmitters primarily glutamic acid, GABA, aspartic acid, and glycine are single amino acid residues released from presynaptic nerve terminals in response to an action potential and cross the synaptic cleft to bind with specific receptor on the postsynaptic membrane. The integral role of amino acid neurotransmitters is important on the normal functioning of the brain. The presynaptic and postsynaptic events in chemical synapses are subject to use dependent and highly regulated as per

the changes in synaptic neurotransmitter release and function.

**Keywords:** synapse, neurotransmitter, receptor, glutamate, GABA, glycine, aspartate

The nervous system is composed of billions of specialized cells called neurons. Neurons are the cells of chemical communication in the brain. In its most basic form, a neuron has two ends (although either can have multiple branches): an axon and a dendrite (**Figure 1**). Efficient communication between neuronal cells is a crucial process for the normal functioning of the central and peripheral nervous system. Neurotransmitters are chemical substances that act as the mediator for the transmission of nerve impulses from one neuron to another neuron through synapses. Neurotransmitters are stored in the axon (or presynaptic neuron) in little packages called synaptic vesicles. The release of neurotransmitter is triggered by the arrival of nerve impulse (or action potential). Synapses are specialized junctions through which cells of the nervous system signal to one another and to non-neuronal cells such as muscles or glands. The process by which the information

is communicated through synapse is called synaptic transmission [1, 2].

The neurotransmitters are stored in the vesicles within the presynaptic nerve terminal at the synaptic membrane of one nerve cell and released into the synaptic

Acid Neurotransmitters

## **Chapter 2**

## Synaptic Transmission and Amino Acid Neurotransmitters

*Manorama Patri*

## **Abstract**

Amino acids are the most abundant neurotransmitters in the brain. Neurotransmitters are synthesized and stored in presynaptic terminals, released from terminals upon stimulation with specific receptors on the postsynaptic cells. Chemical and electrical synapses are specialized biological structures found in the nervous system; they connect neurons together and transmit signals across the neurons. The process of synaptic transmission generates or inhibits electrical impulses in a network of neurons for the processing of information. Glutamate is the primary excitatory neurotransmitter in the brain, while GABA is the principal inhibitory neurotransmitter. The balance of glutamatergic and GABAergic tone is crucial to normal neurologic function. Through synaptic transmission, this information is communicated from the presynaptic cell to the postsynaptic cell. Amino acid neurotransmitters primarily glutamic acid, GABA, aspartic acid, and glycine are single amino acid residues released from presynaptic nerve terminals in response to an action potential and cross the synaptic cleft to bind with specific receptor on the postsynaptic membrane. The integral role of amino acid neurotransmitters is important on the normal functioning of the brain. The presynaptic and postsynaptic events in chemical synapses are subject to use dependent and highly regulated as per the changes in synaptic neurotransmitter release and function.

**Keywords:** synapse, neurotransmitter, receptor, glutamate, GABA, glycine, aspartate

## **1. Introduction**

The nervous system is composed of billions of specialized cells called neurons. Neurons are the cells of chemical communication in the brain. In its most basic form, a neuron has two ends (although either can have multiple branches): an axon and a dendrite (**Figure 1**). Efficient communication between neuronal cells is a crucial process for the normal functioning of the central and peripheral nervous system. Neurotransmitters are chemical substances that act as the mediator for the transmission of nerve impulses from one neuron to another neuron through synapses. Neurotransmitters are stored in the axon (or presynaptic neuron) in little packages called synaptic vesicles. The release of neurotransmitter is triggered by the arrival of nerve impulse (or action potential). Synapses are specialized junctions through which cells of the nervous system signal to one another and to non-neuronal cells such as muscles or glands. The process by which the information is communicated through synapse is called synaptic transmission [1, 2].

The neurotransmitters are stored in the vesicles within the presynaptic nerve terminal at the synaptic membrane of one nerve cell and released into the synaptic cleft in response to nerve impulses [2]. The secreted neurotransmitters can then act on receptors on the membrane of the postsynaptic neuron through a gap called synaptic gap (0.02 micron). The number of synaptic contacts of an average neuron is approximately 10,000. Thus, there are 3–5 × 1015 synapses in the human brain. There are two types of synapses, electrical and chemical synapses, but chemical synapses (**Figure 2**) far outnumber electrical ones. Electric synapses are gap junction. A gap junction is a junction between neurons that allows different molecules and ions to pass freely between cells. The junction connects the cytoplasm of cells. A gap junction is composed of connexons (each connexon is composed of six connexin proteins) which connect across the intercellular space. Neurons connected by gap junctions sometimes act as though they were equivalent to one large neuron with many output pathways, all of which fire synchronously.

Synapses are made on all regions of a receiving nerve cell and can be classified on the basis where they are located. On spiny dendrites of a nerve cell, each spine is the target of an axon terminal and comprises the postsynaptic component of a single synapse. Synapses between axons and dendrites are called axodendritic. Particularly powerful synapses are made between axons of one neuron and cell body of another postsynaptic cell. These are called axosomatic synapses. Synapses between axon terminals and axons of postsynaptic neurons are said to be axo-axonal.

**Figure 1.** *Structure of neuron.*

**13**

*Synaptic Transmission and Amino Acid Neurotransmitters*

acid, GABA, aspartic acid, and glycine).

adrenaline, dopamine, serotonin, and histamine).

ters present within the central nervous system (CNS).

trations go back to resting levels, and the cell returns to −70 mv.

The action potential is produced by an influx of calcium ions through voltagedependent calcium-selective ion channels. Calcium ions then trigger a biochemical cascade which results in neurotransmitter vesicles fusing with the presynaptic membrane and releasing their contents to the synaptic cleft. Receptors on the opposite side of the synaptic gap bind neurotransmitter molecules and respond

Substances that act as neurotransmitters can be categorized into different groups. The three major categories of substances that act as neurotransmitters are:

1.Amino acids: The neurotransmitters of this group are involved in fast synaptic transmission and are inhibitory and excitatory in action (primarily glutamic

2.Amines: Amines are the modified amino acids such as biogenic amines, e.g., catecholamines. The neurotransmitters of this group involve in slow synaptic transmission and are inhibitory and excitatory in action (noradrenaline,

3.Others: The one which do not fit in any of these categories (acetyl choline and nitric oxide). Amino acids are among the most abundant of all neurotransmit-

Several amino acids have been implicated as neurotransmitters in the CNS, including GABA, glutamic acid, glycine, and aspartic acid [3]. Some (like glutamate) are excitatory, whereas others (like GABA) are primarily inhibitory. Aspartate is closely related to glutamate, and the two amino acids are often are found together at axon terminals. Neurons synthesize glutamate and aspartate and

The amino acid neurotransmitters are common neurotransmitters in the central nervous system. Glycine, glutamate, and GABA are classed under amino acid neurotransmitter. The two amino acids functioning as excitatory neurotransmitter are glutamate and aspartate. GABA acts as a brake to the excitatory neurotransmitters, and thus when it is abnormally low, this can lead to anxiety, and glutamate usually ensures homeostasis with the effects of GABA [4]. Several related amino acids, like homocysteic acid and N-acetylaspartylglutamate, may also serve a neurotransmitter function. Neurotransmitters can be classified as either excitatory or inhibitory. Excitatory neurotransmitters function to activate the receptors on the postsynaptic membrane and enhance the effects of action potential, while inhibitory neurotransmitter functions in a reverse mechanism. If the electrical impulses transmitted inward toward the cell body are large enough, they will generate an action potential. The action potentials are caused by an exchange of ions across the neuron membrane; a stimulus first causes sodium channels to open, because there are many more sodium ions on the outside, and the inside of the neuron is negative relative to the outside; sodium ions rush into the neuron. Since sodium has a positive charge, the neuron becomes more positive and becomes depolarized. It takes longer for potassium channels to open; when they do open, potassium rushes out of the cell, reversing the depolarization. Also at about this time, sodium channels start to close; this causes the action potential to go back toward −70 mv (a repolarization). The action potential actually goes past (overshoots) −70 mv (a hyperpolarization) because the potassium channels stay open a bit too long. Gradually, the ion concen-

*DOI: http://dx.doi.org/10.5772/intechopen.82121*

are independent of dietary supply.

**1.1 Function of amino acid neurotransmitter**

**Figure 2.** *Structure of chemical and electrical synapse.*

*Neurochemical Basis of Brain Function and Dysfunction*

with many output pathways, all of which fire synchronously.

terminals and axons of postsynaptic neurons are said to be axo-axonal.

cleft in response to nerve impulses [2]. The secreted neurotransmitters can then act on receptors on the membrane of the postsynaptic neuron through a gap called synaptic gap (0.02 micron). The number of synaptic contacts of an average neuron is approximately 10,000. Thus, there are 3–5 × 1015 synapses in the human brain. There are two types of synapses, electrical and chemical synapses, but chemical synapses (**Figure 2**) far outnumber electrical ones. Electric synapses are gap junction. A gap junction is a junction between neurons that allows different molecules and ions to pass freely between cells. The junction connects the cytoplasm of cells. A gap junction is composed of connexons (each connexon is composed of six connexin proteins) which connect across the intercellular space. Neurons connected by gap junctions sometimes act as though they were equivalent to one large neuron

Synapses are made on all regions of a receiving nerve cell and can be classified on the basis where they are located. On spiny dendrites of a nerve cell, each spine is the target of an axon terminal and comprises the postsynaptic component of a single synapse. Synapses between axons and dendrites are called axodendritic. Particularly powerful synapses are made between axons of one neuron and cell body of another postsynaptic cell. These are called axosomatic synapses. Synapses between axon

**12**

**Figure 2.**

*Structure of chemical and electrical synapse.*

**Figure 1.** *Structure of neuron.*

Substances that act as neurotransmitters can be categorized into different groups. The three major categories of substances that act as neurotransmitters are:


Several amino acids have been implicated as neurotransmitters in the CNS, including GABA, glutamic acid, glycine, and aspartic acid [3]. Some (like glutamate) are excitatory, whereas others (like GABA) are primarily inhibitory. Aspartate is closely related to glutamate, and the two amino acids are often are found together at axon terminals. Neurons synthesize glutamate and aspartate and are independent of dietary supply.

#### **1.1 Function of amino acid neurotransmitter**

The amino acid neurotransmitters are common neurotransmitters in the central nervous system. Glycine, glutamate, and GABA are classed under amino acid neurotransmitter. The two amino acids functioning as excitatory neurotransmitter are glutamate and aspartate. GABA acts as a brake to the excitatory neurotransmitters, and thus when it is abnormally low, this can lead to anxiety, and glutamate usually ensures homeostasis with the effects of GABA [4]. Several related amino acids, like homocysteic acid and N-acetylaspartylglutamate, may also serve a neurotransmitter function. Neurotransmitters can be classified as either excitatory or inhibitory. Excitatory neurotransmitters function to activate the receptors on the postsynaptic membrane and enhance the effects of action potential, while inhibitory neurotransmitter functions in a reverse mechanism. If the electrical impulses transmitted inward toward the cell body are large enough, they will generate an action potential.

The action potentials are caused by an exchange of ions across the neuron membrane; a stimulus first causes sodium channels to open, because there are many more sodium ions on the outside, and the inside of the neuron is negative relative to the outside; sodium ions rush into the neuron. Since sodium has a positive charge, the neuron becomes more positive and becomes depolarized. It takes longer for potassium channels to open; when they do open, potassium rushes out of the cell, reversing the depolarization. Also at about this time, sodium channels start to close; this causes the action potential to go back toward −70 mv (a repolarization). The action potential actually goes past (overshoots) −70 mv (a hyperpolarization) because the potassium channels stay open a bit too long. Gradually, the ion concentrations go back to resting levels, and the cell returns to −70 mv.

The action potential is produced by an influx of calcium ions through voltagedependent calcium-selective ion channels. Calcium ions then trigger a biochemical cascade which results in neurotransmitter vesicles fusing with the presynaptic membrane and releasing their contents to the synaptic cleft. Receptors on the opposite side of the synaptic gap bind neurotransmitter molecules and respond

by opening nearby ion channels in the postsynaptic cell membrane, causing ions to rush in or out and changing the local transmembrane potential of the cell. The resulting change in voltage is called a postsynaptic potential. The result is excitatory, in the case of depolarizing currents, or inhibitory in the case of hyperpolarizing currents resulting in EPSP or IPSP, respectively (**Figure 3**).

Whether a synapse is excitatory or inhibitory depends on what type(s) of ion channel conduct the postsynaptic current, which in turn is a function of the type of receptors and neurotransmitter employed at the synapse. Neurotransmitters may have excitatory effects if they drive a cell's membrane to the threshold of an action potential (**Figure 4**).

The resting potential of a neuron tells about what happens when a neuron is at rest. An action potential occurs when a neuron sends information down an axon, away from the cell body. The action potential is an explosion of electrical activity that is created by a depolarizing current. This means that a stimulus causes the resting potential to move toward 0 mv.


There are no big or small action potentials in one nerve cell. All action potentials are the same in size in a particular neuron type (it can differ between different types of neurons). Therefore, either the neuron does not reach the threshold or a full action potential is fired—this is the "all or none" principle.

**15**

not for Na<sup>+</sup>

effect.

**Figure 4.** *Action potential.*

*1.1.1 Excitatory neurotransmitters*

excitatory postsynaptic current.

*1.1.2 Inhibitory neurotransmitters*

ions.

*Synaptic Transmission and Amino Acid Neurotransmitters*

A neuron encodes the intensity of a stimulus in the frequency of firing and not in the size of a single impulse. Neurotransmitters may have inhibitory effects if they help to drive the membrane away from threshold. An excitatory postsynaptic potential (EPSP) is a summation of signals that brings the membrane closer to the threshold (depolarizing effect). An inhibitory postsynaptic potential (IPSP) drives the membrane away from threshold by a hyperpolarizing

An excitatory postsynaptic potential (EPSP) is a temporary increase in postsynaptic membrane potential within dendrites or cell bodies caused by the flow of sodium ions into the postsynaptic cell. EPSPs are additive. Larger EPSPs result in greater membrane depolarization and thus increase the likelihood that the postsynaptic cell reaches the threshold for firing an action potential. When an active presynaptic cell releases neurotransmitters into the synapse, some of them bind to receptors on the postsynaptic cell. Many of these receptors contain an ion channel capable of passing positively charged ions either into or out of the cell. At excitatory synapses, the ion channel typically allows sodium into the cell, generating an

GABA and glycine are inhibitory, both instead of depolarizing the postsynaptic

ions and Cl<sup>−</sup> ions but

ions, thereby

membrane and producing an EPSP; they hyperpolarize the postsynaptic membrane and produce IPSP. IPSP is the change in membrane voltage of a postsynaptic neuron which results from synaptic activation of inhibitory neurotransmitter receptors. The most common inhibitory neurotransmitters in the nervous system are γ-aminobutyric acid (GABA) and glycine. At a typical inhibitory synapse, the

bringing the membrane potential closer to the equilibrium potential of these ions.

postsynaptic neural membrane permeability increases for K+

This generally causes an influx of chloride ions and efflux of K<sup>+</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.82121*

**Figure 3.** *EPSP and IPSP on a neuron.*

*Synaptic Transmission and Amino Acid Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.82121*

#### **Figure 4.** *Action potential.*

*Neurochemical Basis of Brain Function and Dysfunction*

potential (**Figure 4**).

the same.

ing potential to move toward 0 mv.

action potential will fire.

by opening nearby ion channels in the postsynaptic cell membrane, causing ions to rush in or out and changing the local transmembrane potential of the cell. The resulting change in voltage is called a postsynaptic potential. The result is excitatory, in the case of depolarizing currents, or inhibitory in the case of hyperpolar-

Whether a synapse is excitatory or inhibitory depends on what type(s) of ion channel conduct the postsynaptic current, which in turn is a function of the type of receptors and neurotransmitter employed at the synapse. Neurotransmitters may have excitatory effects if they drive a cell's membrane to the threshold of an action

The resting potential of a neuron tells about what happens when a neuron is at rest. An action potential occurs when a neuron sends information down an axon, away from the cell body. The action potential is an explosion of electrical activity that is created by a depolarizing current. This means that a stimulus causes the rest-

1.When the depolarization near the axon hillock reaches about −55 mv as a result of summation of EPSPs, a neuron will fire an action potential. This is the threshold. If the neuron does not reach this critical threshold level, then no

2.Also, when the threshold level is reached, an action potential of a fixed sized will always fire for any given neuron; the size of the action potential is always

There are no big or small action potentials in one nerve cell. All action potentials are the same in size in a particular neuron type (it can differ between different types of neurons). Therefore, either the neuron does not reach the threshold or a full

action potential is fired—this is the "all or none" principle.

izing currents resulting in EPSP or IPSP, respectively (**Figure 3**).

**14**

**Figure 3.**

*EPSP and IPSP on a neuron.*

A neuron encodes the intensity of a stimulus in the frequency of firing and not in the size of a single impulse. Neurotransmitters may have inhibitory effects if they help to drive the membrane away from threshold. An excitatory postsynaptic potential (EPSP) is a summation of signals that brings the membrane closer to the threshold (depolarizing effect). An inhibitory postsynaptic potential (IPSP) drives the membrane away from threshold by a hyperpolarizing effect.

#### *1.1.1 Excitatory neurotransmitters*

An excitatory postsynaptic potential (EPSP) is a temporary increase in postsynaptic membrane potential within dendrites or cell bodies caused by the flow of sodium ions into the postsynaptic cell. EPSPs are additive. Larger EPSPs result in greater membrane depolarization and thus increase the likelihood that the postsynaptic cell reaches the threshold for firing an action potential. When an active presynaptic cell releases neurotransmitters into the synapse, some of them bind to receptors on the postsynaptic cell. Many of these receptors contain an ion channel capable of passing positively charged ions either into or out of the cell. At excitatory synapses, the ion channel typically allows sodium into the cell, generating an excitatory postsynaptic current.

#### *1.1.2 Inhibitory neurotransmitters*

GABA and glycine are inhibitory, both instead of depolarizing the postsynaptic membrane and producing an EPSP; they hyperpolarize the postsynaptic membrane and produce IPSP. IPSP is the change in membrane voltage of a postsynaptic neuron which results from synaptic activation of inhibitory neurotransmitter receptors. The most common inhibitory neurotransmitters in the nervous system are γ-aminobutyric acid (GABA) and glycine. At a typical inhibitory synapse, the postsynaptic neural membrane permeability increases for K+ ions and Cl<sup>−</sup> ions but not for Na<sup>+</sup> ions.

This generally causes an influx of chloride ions and efflux of K<sup>+</sup> ions, thereby bringing the membrane potential closer to the equilibrium potential of these ions.

## **2. Amino acid neurotransmitters**

Amino acid transmitters provide the majority of excitatory and inhibitory neurotransmission in the nervous system. Amino acids used for synaptic transmission are compartmentalized (e.g., glutamate, compartmentalized from metabolic glutamate used for protein synthesis by packaging the transmitter into synaptic vesicles for subsequent Ca2+-dependent release). Amino acid neurotransmitters are all products of intermediary metabolism with the exception of GABA. Unlike all the other amino acid neurotransmitters, GABA is not used in protein synthesis and is produced by an enzyme (glutamic acid decarboxylase; GAD) uniquely located in neurons. Antibodies to GAD can be used to identify neurons that release GABA.

#### **2.1 Glutamate**

Glutamate is used at the great majority of fast excitatory synapses in the brain and spinal cord. Glutamatergic neurons are particularly prominent in the cerebral cortex. They project to a variety of subcortical structures like the hippocampus, the basolateral complex of the amygdala, the substantia nigra, the nucleus accumbens, the superior colliculus, the caudate nucleus (nucleus ruber), and the pons. At glutamatergic synapses, NMDA receptors (NMDARs) are localized with other ionotropic glutamate receptors [AMPA receptors (AMPARs) and kainate receptors] and with metabotropic glutamate receptors. Glutamate receptors are necessary for neuronal development, synaptic plasticity, excitotoxicity, pain perception, and learning and memory [5]. Among these EPSP-producing glutamate receptors, which could occur as homomeric or heteromeric structures, are classified according to the binding of the most common agonist [6].

Four subtypes can be distinguished, out of which three are ionotropic receptors and one metabotropic receptor, activated by quisqualate. These are named according to the molecules (other than glutamate) that they bind and include:


#### *2.1.1 NMDA receptors*

NMDA receptor is very important for controlling developmental synaptic plasticity and learning and memory function. NMDARs have critical roles in excitatory synaptic transmission, plasticity, and excitotoxicity in the CNS (**Figure 5**). The NR1 subunit is evenly expressed in most of the brain, but the NR2 subunit (NR2A, NR2B, NR2C, and NR2D) shows distinct regional distributions [6, 7]. NMDA receptors show three specific properties by which they differ from other types of ionotropic receptors:


**17**

*Synaptic Transmission and Amino Acid Neurotransmitters*

c.The response of NMDA receptor to neurotransmitter like glutamate and glycine under physiological conditions is modified by certain extracellular molecules

Glutamate binds to the S1 and S2 regions of NR2 subunit, whereas glycine binds to the S1 and S2 regions of NR1 subunit. Individual NR1 or NR2 subunits contain an extracellular N terminus which forms S1, an intracellular C terminus, and an extracellular loop between M3 and M4 that constitutes S2. The channel lining domain is formed by a reentrant pore loop called as M2 loop that enters the channel from the cytoplasmic side and forms a narrow constriction at that channel. The critical asparagine residue located within M2 loop determines the selectivity of NMDAR

The function of NMDA receptors is totally dependent upon AMPA receptors. In the absence of AMPA, NMDA is initially expressed, and it forms the silent synapse. The NMDA receptors are not activated unless the postsynaptic region is depolarized by AMPA

AMPA receptors are ionotropic and belong to the group of non-NMDA receptors and associated with a cation-selective ion channel which is permeable for monova-

Kainate receptors can be activated by kainite and glutamate. Like AMPA receptors, the kainite receptors are associated with an ionic channel which is permeable

in modulating the release of excitatory amino acids and additional neurotransmit-

The metabotropic receptors are activated by glutamate and quisqualate and

GABA is the most ubiquitous inhibitory neurotransmitter in the brain. GABA was discovered in 1883, and its inhibitory function was described in the late 1950s by Bazemore et al. [8]. It was the first amino acid to be established as a neurotransmitter in vertebrate and invertebrate nervous systems. GABA is synthesized in nervous tissue exclusively from glutamate by the alpha decarboxylation of glutamic

. Under certain combinatorial conditions of the receptor

and for Ca2+. These receptors are mainly involved

heteromeric assemblies, composed of two NR1 and two NR2 subunits.

, Zn2+, and polyamines. Most of the NMDA receptors function only in

*DOI: http://dx.doi.org/10.5772/intechopen.82121*

channel for Mg2+ block and Ca2+ permeability.

and K<sup>+</sup>

resistant to activation by NMDA, AMPA, or kainate.

K+

subunits, it also becomes permeable to Ca2+.

like H+

**Figure 5.** *NMDA receptor.*

receptors.

**2.2 GABA**

lent cations, like Na<sup>+</sup>

for the monovalent cations Na+

ters or neuromodulators.

*Synaptic Transmission and Amino Acid Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.82121*

**Figure 5.** *NMDA receptor.*

*Neurochemical Basis of Brain Function and Dysfunction*

Amino acid transmitters provide the majority of excitatory and inhibitory neurotransmission in the nervous system. Amino acids used for synaptic transmission are compartmentalized (e.g., glutamate, compartmentalized from metabolic glutamate used for protein synthesis by packaging the transmitter into synaptic vesicles for subsequent Ca2+-dependent release). Amino acid neurotransmitters are all products of intermediary metabolism with the exception of GABA. Unlike all the other amino acid neurotransmitters, GABA is not used in protein synthesis and is produced by an enzyme (glutamic acid decarboxylase; GAD) uniquely located in neurons. Antibodies to GAD can be used to identify neurons that release GABA.

Glutamate is used at the great majority of fast excitatory synapses in the brain and spinal cord. Glutamatergic neurons are particularly prominent in the cerebral cortex. They project to a variety of subcortical structures like the hippocampus, the basolateral complex of the amygdala, the substantia nigra, the nucleus accumbens, the superior colliculus, the caudate nucleus (nucleus ruber), and the pons. At glutamatergic synapses, NMDA receptors (NMDARs) are localized with other ionotropic glutamate receptors [AMPA receptors (AMPARs) and kainate receptors] and with metabotropic glutamate receptors. Glutamate receptors are necessary for neuronal development, synaptic plasticity, excitotoxicity, pain perception, and learning and memory [5]. Among these EPSP-producing glutamate receptors, which could occur as homomeric or heteromeric structures, are classified according to the binding of the most common agonist [6].

Four subtypes can be distinguished, out of which three are ionotropic receptors and one metabotropic receptor, activated by quisqualate. These are named accord-

2.AMPA receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)

NMDA receptor is very important for controlling developmental synaptic plasticity and learning and memory function. NMDARs have critical roles in excitatory synaptic transmission, plasticity, and excitotoxicity in the CNS (**Figure 5**). The NR1 subunit is evenly expressed in most of the brain, but the NR2 subunit (NR2A, NR2B, NR2C, and NR2D) shows distinct regional distributions [6, 7]. NMDA receptors show three specific properties by which they differ from other types of

a.NMDA receptor ion channel is subjected to voltage-dependent block by the

b.They display a high permeability to Ca2+ ions. Ca2+ influx through NMDA receptor channel leads to a cascade of intracellular events triggering long-term potentiation (LTP) and long-term depression (LTD) of synaptic currents.

ing to the molecules (other than glutamate) that they bind and include:

1.NMDA receptors (named for N-methyl-D-aspartate)

4.Receptors which are activated by quisqualate

**2. Amino acid neurotransmitters**

**2.1 Glutamate**

3.Kainate receptors

*2.1.1 NMDA receptors*

ionotropic receptors:

extracellular Mg2+ ion.

**16**

c.The response of NMDA receptor to neurotransmitter like glutamate and glycine under physiological conditions is modified by certain extracellular molecules like H+ , Zn2+, and polyamines. Most of the NMDA receptors function only in heteromeric assemblies, composed of two NR1 and two NR2 subunits.

Glutamate binds to the S1 and S2 regions of NR2 subunit, whereas glycine binds to the S1 and S2 regions of NR1 subunit. Individual NR1 or NR2 subunits contain an extracellular N terminus which forms S1, an intracellular C terminus, and an extracellular loop between M3 and M4 that constitutes S2. The channel lining domain is formed by a reentrant pore loop called as M2 loop that enters the channel from the cytoplasmic side and forms a narrow constriction at that channel. The critical asparagine residue located within M2 loop determines the selectivity of NMDAR channel for Mg2+ block and Ca2+ permeability.

The function of NMDA receptors is totally dependent upon AMPA receptors. In the absence of AMPA, NMDA is initially expressed, and it forms the silent synapse. The NMDA receptors are not activated unless the postsynaptic region is depolarized by AMPA receptors.

AMPA receptors are ionotropic and belong to the group of non-NMDA receptors and associated with a cation-selective ion channel which is permeable for monovalent cations, like Na<sup>+</sup> and K<sup>+</sup> . Under certain combinatorial conditions of the receptor subunits, it also becomes permeable to Ca2+.

Kainate receptors can be activated by kainite and glutamate. Like AMPA receptors, the kainite receptors are associated with an ionic channel which is permeable for the monovalent cations Na+ K+ and for Ca2+. These receptors are mainly involved in modulating the release of excitatory amino acids and additional neurotransmitters or neuromodulators.

The metabotropic receptors are activated by glutamate and quisqualate and resistant to activation by NMDA, AMPA, or kainate.

#### **2.2 GABA**

GABA is the most ubiquitous inhibitory neurotransmitter in the brain. GABA was discovered in 1883, and its inhibitory function was described in the late 1950s by Bazemore et al. [8]. It was the first amino acid to be established as a neurotransmitter in vertebrate and invertebrate nervous systems. GABA is synthesized in nervous tissue exclusively from glutamate by the alpha decarboxylation of glutamic acid in the presence of glutamic acid decarboxylase (GAD). The apparent prominent role of GAD in modulation of GABA levels becomes obvious under pathological conditions, where GAD concentration can differ significantly from normal levels.

Striatum contained nearly 95% of the cells which are GABAergic. GABA is also suspected to operate as an inhibitory neurotransmitter in the cerebral cortex, lateral vestibular nucleus, and spinal cord.

#### *2.2.1 GABA receptors*

GABA exerts its effects via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAA receptors show a ubiquitous distribution throughout the CNS and have been identified on both neuron and glia. GABA can act on both rapid and slow inhibitory receptors (the GABAA and GABAB), respectively. GABAA receptors are chloride channels that in response to GABA binding increases chloride influx into the neuron. The agonist of these receptors includes GABA and muscimol. The GABAB receptors are potassium channels that when activated by GABA leads to potassium efflux from the cell. GABAA receptors are ionotropic receptors leading to increased Cl<sup>−</sup> ion conductance, whereas GABAB receptors are metabotropic receptors which are coupled to G proteins and thereby indirectly alter membrane ion permeability and neuronal excitability [4].

#### **2.3 Glycine**

Glycine is the simplest of amino acids, consisting of an amino group and a carboxyl (acidic) group attached to a carbon atom. In mammals, glycine belongs to the nonessential amino acids [9]. Until the early 1960s, glycine was of minor importance in synaptic transmission because of its simple structure and its ubiquitous distribution as a member of protein and nucleotide metabolism. Glycine's function is a potent neurotransmitter in the spinal cord and brain. Glycine is a constituent of glutathione, an antioxidant tripeptide found in high concentrations in intestinal epithelial cells. The availability of glycine has the potential to control the cellular levels of glutathione in enterocytes. This amino acid functions as an excitatory transmitter during embryonic development and is an essential coagonist at glutamatergic synapses containing the NMDA subtype of glutamate receptors. Hydroxymethyl transferase converts the amino acid serine to glycine. More recently, glycine has been found to play a role in the functional modulation of NMDA receptors.

#### *2.3.1 Glycine receptor*

Glycine receptors are ligand-gated ion channels that increase Cl<sup>−</sup> influx. Glycine molecules may be taken back into the presynaptic cell by two highaffinity glycine transporters (Glyt-1 and Glyt-2). Glyt-1 is found primarily in glial cells, whereas Glty-2 is found primarily in neuronal cells. The transport of glycine via Glyt-1 is coupled to the movement of Na<sup>+</sup> and Cl<sup>−</sup>, with a Na<sup>+</sup> :Cl<sup>−</sup>:glycine stoichiometry of 2:1:1.

The glycine receptor GlyR belongs to the superfamily of ligand-gated ion channels, like GABAA, and is primarily found in the ventral spinal cord. Strychnine is a glycine antagonist which can bind to the glycine receptor without opening the chloride ion channel (i.e., it inhibits inhibition). GlyR is a strychnine-sensitive glycoprotein which is composed of five subunits. The receptor has a pentameric structure with three ligand-binding α subunits and two β subunits forming an ion channel. This heterogenicity is responsible for the distinct pharmaceutical and

**19**

*Synaptic Transmission and Amino Acid Neurotransmitters*

functional properties displayed by the various receptor configurations that are

The glycine receptor is presently considered to form a complex consisting of a glycine recognition site and an associated chloride channel. Hyperekplexia, or startle disease, is a rare neurological disorder characterized by an exaggerated response to unexpected stimuli. The response is typically accompanied by a tran-

Glutamate and aspartate are nonessential amino acids that do not cross the blood-brain barrier and, therefore, are synthesized from glucose and a variety of other precursors. The synthetic and metabolic enzymes for glutamate and aspartate have been localized to the two main compartments of the brain, neurons and glial cells. Aspartate is the most abundant excitatory neurotransmitter in the CNS. Like glycine, aspartate is primarily localized to the ventral spinal cord. Like glycine, aspartate opens an ion channel and is inactivated by reabsorption into the presynaptic membrane. Unlike glycine, however, aspartate is an excitatory neurotransmitter, which increases the likelihood of depolarization in the postsynaptic membrane [9, 10]. Aspartate is a highly selective agonist for NMDAR-type glutamate receptors and does not activate AMPA-type glutamate receptors. Hence, synapses only releasing aspartate should therefore generate only NMDAR currents despite a full

Aspartate and glycine form an excitatory/inhibitory pair in the ventral spinal cord comparable to the excitatory/inhibitory pair formed by glutamate and GABA in the brain. Interestingly, the two excitatory amino acids, glutamic acid and aspartic acid, are the two acidic amino acids found in proteins, insofar as both have two carboxyl groups rather than one. Thus, variation in the vesicular content of glutamate and aspartate might have a profound effect on the relative contribution of

Neurotransmitters are the brain chemicals that communicate information throughout our brain and body. They relay signals between neurons. Amino acid neurotransmitters can be subdivided into the excitatory amino acids aspartate and glutamate and the inhibitory amino acids GABA and glycine. Common inhibitory neurotransmitters such as GABA and glycine calm the brain and help create balance, whereas excitatory neurotransmitters such as glutamate and aspartate stimu-

The author is thankful to the support by funding from the Project DAE-BRNS, Mumbai, No. 37(1)14/27/2015/ BRNS and DRDO, New Delhi, No. O/o DG

The authors declare that they have no conflict of interest.

differentially expressed and assembled during development [10].

sient but complete muscular rigidity (stiff baby syndrome).

*DOI: http://dx.doi.org/10.5772/intechopen.82121*

postsynaptic complement of AMPARs [11].

NMDARs and AMPARs to synaptic transmission [12, 13].

**2.4 Aspartate**

**3. Conclusion**

late the brain.

**Acknowledgements**

**Conflict of interest**

(TM)/81/48222/LSRB-294/PEE&BS/2017.

#### *Synaptic Transmission and Amino Acid Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.82121*

functional properties displayed by the various receptor configurations that are differentially expressed and assembled during development [10].

The glycine receptor is presently considered to form a complex consisting of a glycine recognition site and an associated chloride channel. Hyperekplexia, or startle disease, is a rare neurological disorder characterized by an exaggerated response to unexpected stimuli. The response is typically accompanied by a transient but complete muscular rigidity (stiff baby syndrome).

#### **2.4 Aspartate**

*Neurochemical Basis of Brain Function and Dysfunction*

vestibular nucleus, and spinal cord.

permeability and neuronal excitability [4].

via Glyt-1 is coupled to the movement of Na<sup>+</sup>

*2.2.1 GABA receptors*

**2.3 Glycine**

*2.3.1 Glycine receptor*

stoichiometry of 2:1:1.

levels.

acid in the presence of glutamic acid decarboxylase (GAD). The apparent prominent role of GAD in modulation of GABA levels becomes obvious under pathological conditions, where GAD concentration can differ significantly from normal

Striatum contained nearly 95% of the cells which are GABAergic. GABA is also suspected to operate as an inhibitory neurotransmitter in the cerebral cortex, lateral

GABA exerts its effects via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAA receptors show a ubiquitous distribution throughout the CNS and have been identified on both neuron and glia. GABA can act on both rapid and slow inhibitory receptors (the GABAA and GABAB), respectively. GABAA receptors are chloride channels that in response to GABA binding increases chloride influx into the neuron. The agonist of these receptors includes GABA and muscimol. The GABAB receptors are potassium channels that when activated by GABA leads to potassium efflux from the cell. GABAA receptors are ionotropic receptors leading to increased Cl<sup>−</sup> ion conductance, whereas GABAB receptors are metabotropic receptors which are coupled to G proteins and thereby indirectly alter membrane ion

Glycine is the simplest of amino acids, consisting of an amino group and a carboxyl (acidic) group attached to a carbon atom. In mammals, glycine belongs to the nonessential amino acids [9]. Until the early 1960s, glycine was of minor importance in synaptic transmission because of its simple structure and its ubiquitous distribution as a member of protein and nucleotide metabolism. Glycine's function is a potent neurotransmitter in the spinal cord and brain. Glycine is a constituent of glutathione, an antioxidant tripeptide found in high concentrations in intestinal epithelial cells. The availability of glycine has the potential to control the cellular levels of glutathione in enterocytes. This amino acid functions as an excitatory transmitter during embryonic development and is an essential coagonist at glutamatergic synapses containing the NMDA subtype of glutamate receptors. Hydroxymethyl transferase converts the amino acid serine to glycine. More recently, glycine has been

found to play a role in the functional modulation of NMDA receptors.

Glycine receptors are ligand-gated ion channels that increase Cl<sup>−</sup> influx. Glycine molecules may be taken back into the presynaptic cell by two high-

The glycine receptor GlyR belongs to the superfamily of ligand-gated ion channels, like GABAA, and is primarily found in the ventral spinal cord. Strychnine is a glycine antagonist which can bind to the glycine receptor without opening the chloride ion channel (i.e., it inhibits inhibition). GlyR is a strychnine-sensitive glycoprotein which is composed of five subunits. The receptor has a pentameric structure with three ligand-binding α subunits and two β subunits forming an ion channel. This heterogenicity is responsible for the distinct pharmaceutical and

affinity glycine transporters (Glyt-1 and Glyt-2). Glyt-1 is found primarily in glial cells, whereas Glty-2 is found primarily in neuronal cells. The transport of glycine

and Cl<sup>−</sup>, with a Na<sup>+</sup>

:Cl<sup>−</sup>:glycine

**18**

Glutamate and aspartate are nonessential amino acids that do not cross the blood-brain barrier and, therefore, are synthesized from glucose and a variety of other precursors. The synthetic and metabolic enzymes for glutamate and aspartate have been localized to the two main compartments of the brain, neurons and glial cells. Aspartate is the most abundant excitatory neurotransmitter in the CNS. Like glycine, aspartate is primarily localized to the ventral spinal cord. Like glycine, aspartate opens an ion channel and is inactivated by reabsorption into the presynaptic membrane. Unlike glycine, however, aspartate is an excitatory neurotransmitter, which increases the likelihood of depolarization in the postsynaptic membrane [9, 10]. Aspartate is a highly selective agonist for NMDAR-type glutamate receptors and does not activate AMPA-type glutamate receptors. Hence, synapses only releasing aspartate should therefore generate only NMDAR currents despite a full postsynaptic complement of AMPARs [11].

Aspartate and glycine form an excitatory/inhibitory pair in the ventral spinal cord comparable to the excitatory/inhibitory pair formed by glutamate and GABA in the brain. Interestingly, the two excitatory amino acids, glutamic acid and aspartic acid, are the two acidic amino acids found in proteins, insofar as both have two carboxyl groups rather than one. Thus, variation in the vesicular content of glutamate and aspartate might have a profound effect on the relative contribution of NMDARs and AMPARs to synaptic transmission [12, 13].

### **3. Conclusion**

Neurotransmitters are the brain chemicals that communicate information throughout our brain and body. They relay signals between neurons. Amino acid neurotransmitters can be subdivided into the excitatory amino acids aspartate and glutamate and the inhibitory amino acids GABA and glycine. Common inhibitory neurotransmitters such as GABA and glycine calm the brain and help create balance, whereas excitatory neurotransmitters such as glutamate and aspartate stimulate the brain.

### **Acknowledgements**

The author is thankful to the support by funding from the Project DAE-BRNS, Mumbai, No. 37(1)14/27/2015/ BRNS and DRDO, New Delhi, No. O/o DG (TM)/81/48222/LSRB-294/PEE&BS/2017.

## **Conflict of interest**

The authors declare that they have no conflict of interest.

*Neurochemical Basis of Brain Function and Dysfunction*

## **Author details**

Manorama Patri Neurobiology Laboratory, Department of Zoology, School of Life Sciences, Ravenshaw University, Cuttack, Odisha, India

\*Address all correspondence to: mpatri@ravenshawuniversity.ac.in

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**21**

*Synaptic Transmission and Amino Acid Neurotransmitters*

[11] Aprison MH, Daly EC. Biochemical aspects of transmission at inhibitory synapses: The role of glycine. Advances in Neurochemistry. 1978;**3**:203-294

[12] Li B et al. Differential regulation of synaptic and extrasynaptic NMDARs. Nature Neuroscience. 2002;**5**:833

[13] Kohr G. NMDAR function: Subunit composition versus spatial distribution. Cell and Tissue Research. 2006;**326**:439

*DOI: http://dx.doi.org/10.5772/intechopen.82121*

[1] Lodish H, Berk A, Zipursky SL. Molecular Cell Biology: Section 21.4 Neurotransmitters, Synapses, and Impulse Transmission. 4th ed. New York: W. H. Freeman; 2000

[2] Ayano G. Common

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[4] Stein V, Nicoll RA. GABA generates excitement. Neuron. 2003;**37**:375-378

[6] Jansen M, Dannhardt G. Antagonists and agonists at the glycine site of the NMDA receptors for therapeutic interventions. European Journal of Medicinal Chemistry. 2003;**38**:661-670

[7] Nicoll RA. Expression mechanisms underlying long-term potentiation: A postsynaptic view. Philosophical Transactions of the Royal Society, B: Biological Sciences. 2003;**358**:721-726

[8] Bazemore A, Elliott KA. Florey e factor i and gamma-aminobutyric acid.

[9] Vannier C, Triller A. Biology of the postsynaptic receptor. International Review of Cytology. 1997;**176**:201-244

[10] Rajendra S, Lynch JW, Schofield PR. The glycine receptor. Pharmacology &

Nature. 1956;**178**:1052-1053

Therapeutics. 1997;**73**:121-146

[5] Jonas P, Monyer H, editors. Handbook Ionotrophic Glutamate receptors in the CNS. Berlin, Hedelberg: *Synaptic Transmission and Amino Acid Neurotransmitters DOI: http://dx.doi.org/10.5772/intechopen.82121*

## **References**

*Neurochemical Basis of Brain Function and Dysfunction*

**20**

**Author details**

Manorama Patri

provided the original work is properly cited.

Ravenshaw University, Cuttack, Odisha, India

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Neurobiology Laboratory, Department of Zoology, School of Life Sciences,

\*Address all correspondence to: mpatri@ravenshawuniversity.ac.in

[1] Lodish H, Berk A, Zipursky SL. Molecular Cell Biology: Section 21.4 Neurotransmitters, Synapses, and Impulse Transmission. 4th ed. New York: W. H. Freeman; 2000

[2] Ayano G. Common Neurotransmitters: Criteria for neurotransmitters, key locations, classifications and functions. Advances in Psychology and Neuroscience. 2016;**4**(6):91-95

[3] Oliver VB, Halbach DR. Neurotransmitters and Neuromodulators: Handbook of Receptors and Biological Effects. 2nd ed. Weinheim: Wiley-VCH Verlag GmbH and Co. KGaA; 2006. ISBN: 3-527-31307-9

[4] Stein V, Nicoll RA. GABA generates excitement. Neuron. 2003;**37**:375-378

[5] Jonas P, Monyer H, editors. Handbook Ionotrophic Glutamate receptors in the CNS. Berlin, Hedelberg: Springer; 1999

[6] Jansen M, Dannhardt G. Antagonists and agonists at the glycine site of the NMDA receptors for therapeutic interventions. European Journal of Medicinal Chemistry. 2003;**38**:661-670

[7] Nicoll RA. Expression mechanisms underlying long-term potentiation: A postsynaptic view. Philosophical Transactions of the Royal Society, B: Biological Sciences. 2003;**358**:721-726

[8] Bazemore A, Elliott KA. Florey e factor i and gamma-aminobutyric acid. Nature. 1956;**178**:1052-1053

[9] Vannier C, Triller A. Biology of the postsynaptic receptor. International Review of Cytology. 1997;**176**:201-244

[10] Rajendra S, Lynch JW, Schofield PR. The glycine receptor. Pharmacology & Therapeutics. 1997;**73**:121-146

[11] Aprison MH, Daly EC. Biochemical aspects of transmission at inhibitory synapses: The role of glycine. Advances in Neurochemistry. 1978;**3**:203-294

[12] Li B et al. Differential regulation of synaptic and extrasynaptic NMDARs. Nature Neuroscience. 2002;**5**:833

[13] Kohr G. NMDAR function: Subunit composition versus spatial distribution. Cell and Tissue Research. 2006;**326**:439

Chapter 3

Abstract

Abdulbaki Agbas

studies including our new findings.

aging, proteinopathy, amyloid plaque

1. Introduction

Huntington's disease, etc.

23

1.1 An overview for protein-folding

Trends of Protein Aggregation in

Protein aggregation trends in neurodegenerative diseases are largely unmapped

due to the complex nature of protein-protein interactions and their regulatory machineries such as protein proteolytic systems. Since the protein aggregation process in humans is a slow process, early determination of the patients that will develop neurodegenerative diseases later in life is critical in terms of starting effective treatment, which will reduce the expensive health care. In this chapter, I will discuss the nature of protein aggregation of signature proteins and the status of protein proteolytic systems such as proteasome and autophagosome in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Huntington's disease, and prion disease under the light of recent

Keywords: protein aggregation, protein misfolding, neurodegenerative disease,

Extracellular deposits of protein aggregates are often relevant to human diseases in general. Protein aggregates are the product of misfolded proteins that escape from protein quality checkpoints such as the chaperon/chaperonin system, heat shock proteins (Hs90, Hs70, etc.), proteasomes, and the autophagosome system. They are mostly insoluble and tend to form amyloid plaques over time. In this chapter, I will review trends of protein aggregation in the most studied neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), frontotemporal lobar degeneration (FTLD), prion,

Biological self-assembly of proteins in a compact three-dimensional (3D) structure is the universal example of how the functional proteins can be separated from other biomolecules. This feature provides a functional advantage for proteins. 3D folding brings functional groups to close proximity creating a space where chemical reactions can occur; hence, the protein molecule becomes functional. Properly folded proteins need to maintain their stability which requires naturally interacting partners during their life term [1]. Failure of this native environment-protein interaction can lead to a wide variety of pathological conditions called proteinopathy.

Neurodegenerative Diseases

## Chapter 3

## Trends of Protein Aggregation in Neurodegenerative Diseases

Abdulbaki Agbas

### Abstract

Protein aggregation trends in neurodegenerative diseases are largely unmapped due to the complex nature of protein-protein interactions and their regulatory machineries such as protein proteolytic systems. Since the protein aggregation process in humans is a slow process, early determination of the patients that will develop neurodegenerative diseases later in life is critical in terms of starting effective treatment, which will reduce the expensive health care. In this chapter, I will discuss the nature of protein aggregation of signature proteins and the status of protein proteolytic systems such as proteasome and autophagosome in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Huntington's disease, and prion disease under the light of recent studies including our new findings.

Keywords: protein aggregation, protein misfolding, neurodegenerative disease, aging, proteinopathy, amyloid plaque

## 1. Introduction

Extracellular deposits of protein aggregates are often relevant to human diseases in general. Protein aggregates are the product of misfolded proteins that escape from protein quality checkpoints such as the chaperon/chaperonin system, heat shock proteins (Hs90, Hs70, etc.), proteasomes, and the autophagosome system. They are mostly insoluble and tend to form amyloid plaques over time. In this chapter, I will review trends of protein aggregation in the most studied neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), frontotemporal lobar degeneration (FTLD), prion, Huntington's disease, etc.

#### 1.1 An overview for protein-folding

Biological self-assembly of proteins in a compact three-dimensional (3D) structure is the universal example of how the functional proteins can be separated from other biomolecules. This feature provides a functional advantage for proteins. 3D folding brings functional groups to close proximity creating a space where chemical reactions can occur; hence, the protein molecule becomes functional. Properly folded proteins need to maintain their stability which requires naturally interacting partners during their life term [1]. Failure of this native environment-protein interaction can lead to a wide variety of pathological conditions called proteinopathy. Approximately 30 or more structurally different proteins have the potential to form an amyloid structure. Although there is no obvious homology in their primary structure, they all share a beta-pleated sheet (β-structure) as a polymer scaffold [3].

1.3 Protein misfolding in the cell

DOI: http://dx.doi.org/10.5772/intechopen.81224

Trends of Protein Aggregation in Neurodegenerative Diseases

neurodegenerative diseases [17].

sary environmental conditions are achieved.

25

Although protein-folding principles are universal, the protein-folding environment needs to be taken into consideration in order to comprehend the proteinmisfolding event. Some protein-folding is co-translational; they are initiated before leaving the ribosomal machinery upon completion of primary structure [6]. Most proteins undergo proper folding process in the cytoplasm after they leave the ribosome "quality control checkpoints" and began to interact with chaperones and heat shock proteins (HSPs). Recent studies reveal that molecular chaperones are essential not only in preventing misfolding but also in rescuing misfolded proteins even in their early stage of aggregation enabling them to have a "second chance" to fold correctly; this process requires ATP [7, 8]. Increased concentration of chaperone molecules and HSPs during cellular stress supports the notion that ATP is required [9]. Chaperonins, a subclass of chaperones, are the preferred molecules participating in the protein-folding process [10–12]. A possible chaperonin-naïve protein adverse interaction may very well initiate protein misfolding that will lead to protein aggregation. There are other proteins that complete their folding process in certain organelles such as the endoplasmic reticulum (ER) and mitochondria after being translocated into these organelles [7, 8]. The ER contains a large repertoire of molecular chaperons and folding catalysts [13, 14], making this organelle a major folding site and also the source of misfolded protein-related diseases [15]. Such organelles may utilize internal signals that allow certain proteins to penetrate into cell organelles to complete their folding. Wang et al. [16] recently demonstrated that a nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) penetrates into mitochondria using such internal signals and binds, preferably mitochondria-transcribed mRNA that encodes respiratory complex-I subunits (ND3 and ND6). This subsequently interferes with the proper assembly of complex-I and mitochondrial functions causing them to be impaired. The mitochondrial Hsp60/Hsp10 chaperonin system is essential for proper folding of proteins that are transported from cytosol to the inside of mitochondria via porins, and any mutation on this mitochondrial chaperonin system could be associated with

Many misfolded proteins that escaped the "quality control checkpoints" have exposed regions that are normally buried in the hydrophobic core of the protein. Such regions could inappropriately interact with other macromolecules within the crowded bioenvironment of the cytosol [18]. This leads to the initiation of protein aggregation that may be the foundation of protein-relevant disease, proteinopathy. The readers should have a broad perspective of diverse process like translocation across the membranes, trafficking, secretion, the immune response, and regulation of the cell cycle that are dependent on the protein-folding mechanism [19]. Any failure of proper folding or the escape from quality control checkpoints gives rise to cell malfunctioning and, hence, to development of a proteinopathy [20, 21]. Protein propensity can determine the probability of misfolded protein that has a relatively higher extracellular milieu. Such protein propensity can be analyzed by employing Predictors of Naturally Disordered Regions (PONDR) analysis. A few representatives of neurodegenerative disease hallmark proteins' PONDR® analysis were performed based on their primary amino acid sequence, and the results were shown in Figure 2. All four proteins possess reasonably high levels of disorder region that makes the protein a good candidate to undergo aggregation process once the neces-

Protein misfolding is likely to initiate the formation of the seed for aggregation. Therefore, researchers have studied the protein-folding chaperone machinery and HSPs in the context of neurodegenerative disease [22]. Mutant Cu/Zn superoxide

#### 1.2 Energy landscape in protein-folding

Proteins in their native state, under the physiological conditions, are in a low energy state which provides thermodynamic stability [4]. With a large number of permutations, a systematic search for a stable polypeptide chain requires an enormous length of time (1.6 <sup>10</sup><sup>15</sup> trillion years). This makes it clear that the proteinfolding process does not involve sequential steps. Cyrus Levinthal's calculation known as Levinthal's paradox reveals that proteins do not follow a folding process by trying every possible conformation; instead, they follow a partially defined pathway consisting of intermediates between fully denatured protein and its native structure (Figure 1) [1]. Two basic questions have not yet been answered: (i) what determines the correct folding state from the intermediate stage and (ii) how is the energy landscape unique to a specific protein-folding? Folding characteristics of small proteins (100 amino acid residue) provide invaluable information about the amino acid sequence and energy landscape. A specific mutation in an amino acid sequence may provide critical information about the folding and unfolding kinetics [5]. Therefore, the energy landscape of certain signature proteins in neurodegenerative diseases may provide some critical information about the trends of such proteins that misfold and form aggregate. The problem lies on how to study the specific energy landscape of such proteins that are obtained from the patients (AD, ALS, PD, Creutzfeldt-Jakob disease, and Huntington's disease), which will predict the aggregate formation of the proteins. This will help in designing new drugs that either postpone or eliminate such aggregate formations; consequently, treatment options for neurodegenerative diseases may be possible.

#### Figure 1.

Components of a partially denatured protein solution. In a half-unfolded protein solution, half of the protein species are fully folded and the other half are unfolded. This is an experimental condition; therefore, it is not known whether same or similar condition is existing in biology. The image is redrawn from 6th Edition of Biochemistry [1].

## 1.3 Protein misfolding in the cell

Approximately 30 or more structurally different proteins have the potential to form an amyloid structure. Although there is no obvious homology in their primary structure, they all share a beta-pleated sheet (β-structure) as a polymer

Proteins in their native state, under the physiological conditions, are in a low energy state which provides thermodynamic stability [4]. With a large number of permutations, a systematic search for a stable polypeptide chain requires an enormous length of time (1.6 <sup>10</sup><sup>15</sup> trillion years). This makes it clear that the proteinfolding process does not involve sequential steps. Cyrus Levinthal's calculation known as Levinthal's paradox reveals that proteins do not follow a folding process by trying every possible conformation; instead, they follow a partially defined pathway consisting of intermediates between fully denatured protein and its native structure (Figure 1) [1]. Two basic questions have not yet been answered: (i) what determines the correct folding state from the intermediate stage and (ii) how is the energy landscape unique to a specific protein-folding? Folding characteristics of small proteins (100 amino acid residue) provide invaluable information about the amino acid sequence and energy landscape. A specific mutation in an amino acid sequence may provide critical information about the folding and unfolding kinetics [5]. Therefore, the energy landscape of certain signature proteins in neurodegenerative diseases may provide some critical information about the trends of such proteins that misfold and form aggregate. The problem lies on how to study the specific energy landscape of such proteins that are obtained from the patients (AD, ALS, PD, Creutzfeldt-Jakob disease, and Huntington's disease), which will predict the aggregate formation of the proteins. This will help in designing new drugs that either postpone or eliminate such aggregate formations; consequently, treatment

Components of a partially denatured protein solution. In a half-unfolded protein solution, half of the protein species are fully folded and the other half are unfolded. This is an experimental condition; therefore, it is not known whether same or similar condition is existing in biology. The image is redrawn from 6th Edition of

scaffold [3].

Figure 1.

24

Biochemistry [1].

1.2 Energy landscape in protein-folding

Neurochemical Basis of Brain Function and Dysfunction

options for neurodegenerative diseases may be possible.

Although protein-folding principles are universal, the protein-folding environment needs to be taken into consideration in order to comprehend the proteinmisfolding event. Some protein-folding is co-translational; they are initiated before leaving the ribosomal machinery upon completion of primary structure [6]. Most proteins undergo proper folding process in the cytoplasm after they leave the ribosome "quality control checkpoints" and began to interact with chaperones and heat shock proteins (HSPs). Recent studies reveal that molecular chaperones are essential not only in preventing misfolding but also in rescuing misfolded proteins even in their early stage of aggregation enabling them to have a "second chance" to fold correctly; this process requires ATP [7, 8]. Increased concentration of chaperone molecules and HSPs during cellular stress supports the notion that ATP is required [9]. Chaperonins, a subclass of chaperones, are the preferred molecules participating in the protein-folding process [10–12]. A possible chaperonin-naïve protein adverse interaction may very well initiate protein misfolding that will lead to protein aggregation. There are other proteins that complete their folding process in certain organelles such as the endoplasmic reticulum (ER) and mitochondria after being translocated into these organelles [7, 8]. The ER contains a large repertoire of molecular chaperons and folding catalysts [13, 14], making this organelle a major folding site and also the source of misfolded protein-related diseases [15]. Such organelles may utilize internal signals that allow certain proteins to penetrate into cell organelles to complete their folding. Wang et al. [16] recently demonstrated that a nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) penetrates into mitochondria using such internal signals and binds, preferably mitochondria-transcribed mRNA that encodes respiratory complex-I subunits (ND3 and ND6). This subsequently interferes with the proper assembly of complex-I and mitochondrial functions causing them to be impaired. The mitochondrial Hsp60/Hsp10 chaperonin system is essential for proper folding of proteins that are transported from cytosol to the inside of mitochondria via porins, and any mutation on this mitochondrial chaperonin system could be associated with neurodegenerative diseases [17].

Many misfolded proteins that escaped the "quality control checkpoints" have exposed regions that are normally buried in the hydrophobic core of the protein. Such regions could inappropriately interact with other macromolecules within the crowded bioenvironment of the cytosol [18]. This leads to the initiation of protein aggregation that may be the foundation of protein-relevant disease, proteinopathy. The readers should have a broad perspective of diverse process like translocation across the membranes, trafficking, secretion, the immune response, and regulation of the cell cycle that are dependent on the protein-folding mechanism [19]. Any failure of proper folding or the escape from quality control checkpoints gives rise to cell malfunctioning and, hence, to development of a proteinopathy [20, 21]. Protein propensity can determine the probability of misfolded protein that has a relatively higher extracellular milieu. Such protein propensity can be analyzed by employing Predictors of Naturally Disordered Regions (PONDR) analysis. A few representatives of neurodegenerative disease hallmark proteins' PONDR® analysis were performed based on their primary amino acid sequence, and the results were shown in Figure 2. All four proteins possess reasonably high levels of disorder region that makes the protein a good candidate to undergo aggregation process once the necessary environmental conditions are achieved.

Protein misfolding is likely to initiate the formation of the seed for aggregation. Therefore, researchers have studied the protein-folding chaperone machinery and HSPs in the context of neurodegenerative disease [22]. Mutant Cu/Zn superoxide

(amyloidosis: abnormal proteins called amyloids buildup in the tissue). Although there is no consensus homology in their amino acid sequence and molecular details of amyloid fibrils have some commonalities, among them are as follows: (i) all share β-sheet as a polymer scaffold; (ii) all show specific optical behavior on binding dye molecule Congo red, displaying long-unbranched and often twisted structures; and (iii) a characteristic cross-beta X-ray fiber differentiation pattern [3, 30]. Sequence characteristics of certain regions, especially at either N- or C-terminal, may predict the protein propensity to form amyloid fibrils. PONDR® (Figure 2) analysis shows known neurodegenerative disease protein's tendency to form amyloid fibers. The idea that the relative aggregate rates for a wide range of polypeptides and proteins correlate with the physicochemical features of the molecules such as charge, secondary structure propensities, and hydrophobicity [31] was experimentally

Trends of Protein Aggregation in Neurodegenerative Diseases

DOI: http://dx.doi.org/10.5772/intechopen.81224

It is now known that polypeptides or proteins that have propensity for β-pleated structure have a tendency to form amyloid plaques. These β-pleated-enriched proteins fall to the lowest energy level in the energy landscape (Figure 3), and they are more hydrophobic. Consider a globular protein; the main polypeptide chain and hydrophobic regions are buried in the core of the protein. When these regions are exposed to more hydrophilic environment due to partial unfolding caused by low pH, proteolytic fragmentation, etc., conversion to amyloid fibrillation becomes possible [9]. Amyloid fibril formation takes years before it reveals clinical manifestation, and the fibril formation follows a lag phase followed by a period of rapid growth [32, 33]. The fibril structure is measurable and determinable by laboratory techniques; however, it requires postmortem tissues. It is now critical to develop some approaches that utilize less invasively obtained biosamples (e.g., blood) so that protein fibrillation may be monitored and fibril formation can be restrained

Energy landscape of protein folding and aggregation. The purple surface shows possibility of the conformations leading to the thermodynamically balanced state (native state). Cyan-colored area of the landscape indicates the conformations moving toward to amorphous aggregates of insoluble amyloid fibrils (adopted and redrawn,

with at early stages as part of early treatment option.

supported.

Figure 3.

27

Vabulas et al. [34].

#### Figure 2.

Prediction analysis of some of the signature proteins for neurodegenerative diseases. PONDR® score predicts the disorder probability for a given protein or polypeptide based on the amino acid sequence. Disordered regions are defined as the entire proteins or regions of proteins that lack a fixed tertiary structure. These figures represent disordered regions of major proteins in neurodegenerative diseases. The black rectangle on the 0.5 line indicates the region that was visible in the crystal structure with this protein bound to its binding partner (www. molecularkinetics.com; main@molecularkinetics.com) under license from the WSU Research Foundation. PONDR® is copyright C\_1999 by the WSU Research Foundation, all rights reserved).

dismutase (SOD1G93A) abundant in motor neurons and HSP interactions was studied. The proposal was made and experimentally demonstrated that mutant SOD1 binding to HSPs (Hsp70 and Hsp25) makes this chaperone unavailable for their anti-apoptotic functions and eventually leads to motor neuron death [23]. Our laboratory has also demonstrated both in vitro and in vivo that mutant SOD1 failed to bind calcineurin (CaN) in a fashion that CaN lost its activity [24]. This failed interaction may yield the accumulation of hyper-phosphorylated protein aggregations [25] since CaN is one of the Ser-/Thr-specific phosphatase that removes the phosphate from proteins [26]. Under the light of existing literature, it is now known that a number of diseases such as AD, PD, prion disease, and typ. 2 diabetes are directly relevant to aberrant proteins that escaped from chaperone quality check system and form insoluble aggregates [21, 27–29].

#### 1.3.1 Amyloid formation

Filament-like (fibrous) protein aggregates are generally referred to as amyloid. The word amyloid indicates a starch-like compound. It is an accepted term for a group of conformational disorders. About 30 or so proteins have the tendency to form amyloid structure, and they are involved in the well-defined amyloidosis

Trends of Protein Aggregation in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.81224

(amyloidosis: abnormal proteins called amyloids buildup in the tissue). Although there is no consensus homology in their amino acid sequence and molecular details of amyloid fibrils have some commonalities, among them are as follows: (i) all share β-sheet as a polymer scaffold; (ii) all show specific optical behavior on binding dye molecule Congo red, displaying long-unbranched and often twisted structures; and (iii) a characteristic cross-beta X-ray fiber differentiation pattern [3, 30]. Sequence characteristics of certain regions, especially at either N- or C-terminal, may predict the protein propensity to form amyloid fibrils. PONDR® (Figure 2) analysis shows known neurodegenerative disease protein's tendency to form amyloid fibers. The idea that the relative aggregate rates for a wide range of polypeptides and proteins correlate with the physicochemical features of the molecules such as charge, secondary structure propensities, and hydrophobicity [31] was experimentally supported.

It is now known that polypeptides or proteins that have propensity for β-pleated structure have a tendency to form amyloid plaques. These β-pleated-enriched proteins fall to the lowest energy level in the energy landscape (Figure 3), and they are more hydrophobic. Consider a globular protein; the main polypeptide chain and hydrophobic regions are buried in the core of the protein. When these regions are exposed to more hydrophilic environment due to partial unfolding caused by low pH, proteolytic fragmentation, etc., conversion to amyloid fibrillation becomes possible [9]. Amyloid fibril formation takes years before it reveals clinical manifestation, and the fibril formation follows a lag phase followed by a period of rapid growth [32, 33]. The fibril structure is measurable and determinable by laboratory techniques; however, it requires postmortem tissues. It is now critical to develop some approaches that utilize less invasively obtained biosamples (e.g., blood) so that protein fibrillation may be monitored and fibril formation can be restrained with at early stages as part of early treatment option.

#### Figure 3.

dismutase (SOD1G93A) abundant in motor neurons and HSP interactions was studied. The proposal was made and experimentally demonstrated that mutant SOD1 binding to HSPs (Hsp70 and Hsp25) makes this chaperone unavailable for their anti-apoptotic functions and eventually leads to motor neuron death [23]. Our laboratory has also demonstrated both in vitro and in vivo that mutant SOD1 failed to bind calcineurin (CaN) in a fashion that CaN lost its activity [24]. This failed interaction may yield the accumulation of hyper-phosphorylated protein aggregations [25] since CaN is one of the Ser-/Thr-specific phosphatase that removes the phosphate from proteins [26]. Under the light of existing literature, it is now known that a number of diseases such as AD, PD, prion disease, and typ. 2 diabetes are directly relevant to aberrant proteins that escaped from chaperone quality check

PONDR® is copyright C\_1999 by the WSU Research Foundation, all rights reserved).

Prediction analysis of some of the signature proteins for neurodegenerative diseases. PONDR® score predicts the disorder probability for a given protein or polypeptide based on the amino acid sequence. Disordered regions are defined as the entire proteins or regions of proteins that lack a fixed tertiary structure. These figures represent disordered regions of major proteins in neurodegenerative diseases. The black rectangle on the 0.5 line indicates the region that was visible in the crystal structure with this protein bound to its binding partner (www. molecularkinetics.com; main@molecularkinetics.com) under license from the WSU Research Foundation.

Filament-like (fibrous) protein aggregates are generally referred to as amyloid. The word amyloid indicates a starch-like compound. It is an accepted term for a group of conformational disorders. About 30 or so proteins have the tendency to form amyloid structure, and they are involved in the well-defined amyloidosis

system and form insoluble aggregates [21, 27–29].

Neurochemical Basis of Brain Function and Dysfunction

1.3.1 Amyloid formation

26

Figure 2.

Energy landscape of protein folding and aggregation. The purple surface shows possibility of the conformations leading to the thermodynamically balanced state (native state). Cyan-colored area of the landscape indicates the conformations moving toward to amorphous aggregates of insoluble amyloid fibrils (adopted and redrawn, Vabulas et al. [34].

#### 1.3.2 Proteolysis-generated toxic protein species

Thermodynamic stability of a protein and its conformational kinetic determines the state of proper folding. Amyloid fibrils maintain the thermodynamically stable conformation in a highly organized hydrogen-bonded structure that is insoluble in aqueous media. This structure takes many years to progressively build up in tissues. Cellular homeostasis recognizes this event as toxic event and begins to encapsulate the amyloid fibrils in a plaque formation as part of the cellular defense mechanism. This plaque formation slows down and can eliminate further growth of the subsequent conversion of additional quantities of the same protein into amyloid fibrils [9]. However, readers should be aware that there are some naturally occurring nonpathogenic amyloid-like fibril formations such as the nanostructure of certain bacteria [35] and the mammalian melanocyte integral membrane protein [36]. The pathogenesis of amyloidogenic proteinopathy may be initiated with amyloidogenic peptide fragments by one or more proteases [37]. Human amyloid pathologies known to require proteolytic processing of a precursor protein include AD where Aβ peptide fragments are liberated from a large APP precursor protein by β- and γsecretases [38]. A new potential biomarker for AD TDP-43 [39] may be involved in activating β-secretase that will generate Aβ peptide fragments [2]. Figure 4 illustrates a simplified diagram of APP processing [2]. Modulation of Aβ generation by bio-metals was studied in both cell-free and cell-based assays. It was found that zinc (Zn2+) ion induces APP-C99 dimerization, which prevents APP cleavage by γsecretase and Aβ production [40]. The same group reported that copper (Cu2+) ion was a γ-secretase inhibitor affecting APP processing [40]. These findings may suggest that the metal dyshomeostasis is a critical issue in generation of toxic protein species.

#### 1.3.3 Proteasome malfunctioning

The β-sheet structure-enriched amyloid fibril formation relevant to protein aggregation is tightly controlled by molecular chaperones and the proteasome machinery. The proteasome is a large multisubunit complex that can be analogous to a food waste disposer. The proper function of such system is absolutely necessary for maintaining cell homeostasis [41].

It is expected that any proteasomal abnormalities may contribute to misfolding and protein aggregation diseases [42, 43]. In a pilot study, we observed that proteasome activity levels were reduced in plasma/platelet obtained from AD and ALS patients (Figure 5), while TDP-43 protein levels were increased in platelets

obtained from AD patients (Figure 6). This suggests that proteasome machinery was either malfunctioning or overwhelmed due to massive protein aggregation. Proteasome activity measurements in human plasma were successfully performed as a useful potential marker for various malignant and nonmalignant diseases [44]. Amyloid fibril formation that leads to abnormal protein aggregation may result in two functional consequences: (i) a toxic gain of function and (ii) a loss of function of the protein in question. Although the mature and organized protein fibrils are usually benign [32, 45], it is not well documented how disordered amyloid fibrils are being converted to malfunctioned protein species. One thought would be that the nonnative hydrophobic surfaces of the aberrant protein's interaction with cell

Platelet lysate TDP-43 profile. TDP-43 protein levels were determined in platelet lysate by Western blotting method with a TDP-43 Ab (1:1000 dilution). Blood samples were obtained from AD patients (n = 3) and agematched healthy subject (n = 3). Approximately <60% increase were observed in TDP-43 levels in AD patients

Proteasome activity measurements in plasma. Extracellular proteasome activity levels were measured by proteasome 20S assay fluorogenic system (Enzo Biochem Inc. cat#BML-AK740-0001) designed to measure chymotrypsin-like protease activity of purified 20S proteasome. The detection of proteolytic activity is based on the release of three fluorogenic peptides. About 50% decrease in proteasome activity were observed in AD and

membrane or other cellular components may initiate cell death [46].

Figure 5.

Figure 6.

29

ALS patient plasma (n = 3) (unpublished data).

Trends of Protein Aggregation in Neurodegenerative Diseases

DOI: http://dx.doi.org/10.5772/intechopen.81224

(the figure was reproduced by permission [39]).

#### Figure 4.

Simplified diagram of APP structure and processing. APP undergoes sequential proteolysis by β-secretase, αsecretase, and γ-secretase for the release of Aβ from the neuronal plasma membrane. TDP-43 has been shown to increase intraneuronal Aβ accumulation via increased β-secretase activation (adapted and modified from Ref. [2]).

Trends of Protein Aggregation in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.81224

#### Figure 5.

1.3.2 Proteolysis-generated toxic protein species

Neurochemical Basis of Brain Function and Dysfunction

protein species.

Figure 4.

Ref. [2]).

28

1.3.3 Proteasome malfunctioning

for maintaining cell homeostasis [41].

Thermodynamic stability of a protein and its conformational kinetic determines the state of proper folding. Amyloid fibrils maintain the thermodynamically stable conformation in a highly organized hydrogen-bonded structure that is insoluble in aqueous media. This structure takes many years to progressively build up in tissues. Cellular homeostasis recognizes this event as toxic event and begins to encapsulate the amyloid fibrils in a plaque formation as part of the cellular defense mechanism. This plaque formation slows down and can eliminate further growth of the subsequent conversion of additional quantities of the same protein into amyloid fibrils [9]. However, readers should be aware that there are some naturally occurring nonpathogenic amyloid-like fibril formations such as the nanostructure of certain bacteria [35] and the mammalian melanocyte integral membrane protein [36]. The pathogenesis of amyloidogenic proteinopathy may be initiated with amyloidogenic peptide fragments by one or more proteases [37]. Human amyloid pathologies known to require proteolytic processing of a precursor protein include AD where Aβ peptide fragments are liberated from a large APP precursor protein by β- and γsecretases [38]. A new potential biomarker for AD TDP-43 [39] may be involved in activating β-secretase that will generate Aβ peptide fragments [2]. Figure 4 illustrates a simplified diagram of APP processing [2]. Modulation of Aβ generation by bio-metals was studied in both cell-free and cell-based assays. It was found that zinc (Zn2+) ion induces APP-C99 dimerization, which prevents APP cleavage by γsecretase and Aβ production [40]. The same group reported that copper (Cu2+) ion was a γ-secretase inhibitor affecting APP processing [40]. These findings may suggest that the metal dyshomeostasis is a critical issue in generation of toxic

The β-sheet structure-enriched amyloid fibril formation relevant to protein aggregation is tightly controlled by molecular chaperones and the proteasome machinery. The proteasome is a large multisubunit complex that can be analogous to a food waste disposer. The proper function of such system is absolutely necessary

It is expected that any proteasomal abnormalities may contribute to misfolding

Simplified diagram of APP structure and processing. APP undergoes sequential proteolysis by β-secretase, αsecretase, and γ-secretase for the release of Aβ from the neuronal plasma membrane. TDP-43 has been shown to increase intraneuronal Aβ accumulation via increased β-secretase activation (adapted and modified from

and protein aggregation diseases [42, 43]. In a pilot study, we observed that proteasome activity levels were reduced in plasma/platelet obtained from AD and ALS patients (Figure 5), while TDP-43 protein levels were increased in platelets

Proteasome activity measurements in plasma. Extracellular proteasome activity levels were measured by proteasome 20S assay fluorogenic system (Enzo Biochem Inc. cat#BML-AK740-0001) designed to measure chymotrypsin-like protease activity of purified 20S proteasome. The detection of proteolytic activity is based on the release of three fluorogenic peptides. About 50% decrease in proteasome activity were observed in AD and ALS patient plasma (n = 3) (unpublished data).

#### Figure 6.

Platelet lysate TDP-43 profile. TDP-43 protein levels were determined in platelet lysate by Western blotting method with a TDP-43 Ab (1:1000 dilution). Blood samples were obtained from AD patients (n = 3) and agematched healthy subject (n = 3). Approximately <60% increase were observed in TDP-43 levels in AD patients (the figure was reproduced by permission [39]).

obtained from AD patients (Figure 6). This suggests that proteasome machinery was either malfunctioning or overwhelmed due to massive protein aggregation. Proteasome activity measurements in human plasma were successfully performed as a useful potential marker for various malignant and nonmalignant diseases [44]. Amyloid fibril formation that leads to abnormal protein aggregation may result in two functional consequences: (i) a toxic gain of function and (ii) a loss of function of the protein in question. Although the mature and organized protein fibrils are usually benign [32, 45], it is not well documented how disordered amyloid fibrils are being converted to malfunctioned protein species. One thought would be that the nonnative hydrophobic surfaces of the aberrant protein's interaction with cell membrane or other cellular components may initiate cell death [46].

## 2. Trends for misfolded proteins lead to neurodegeneration

Insoluble extracellular protein deposits in various human diseases have been recognized for a long time. Many proteins that have a tendency to be misfolded do form aggregates that initiate cellular dysfunction [47]. This section discusses how the proteins involved in neurodegenerative disease have a tendency to misfold. If we comprehend the biochemical and biophysical behavior of the proteins and their misfolding features, we will have a better understanding of proteinopathy and relevance to neurodegenerative diseases.

Therefore, the growth of the protein aggregate seed is kinetically unfavorable; hence, recruitment of new aberrant proteins for aggregate formation takes a longer period of time [54, 55]. Once the nucleation achieves a critical mass, the fibril formation and subsequent plaque formation become accelerated. This is why clinical manifestation of amyloidoses relevant to neurodegenerative disease mostly appears in old age. This is mostly true for sporadic AD; however, in familial AD, which represents only 5–10% of the total AD population, the disease onset tends to occur at the middle age (50 and above). In Down syndrome, β-amyloid precursor protein (APP), is encoded on chromosome 21 [47]. Patients with trisomy 21 develop abundant Aβ aggregates in the brain at younger age. Therefore, the lifelong aggregate formation is inevitable in Down syndrome patients, which supports the notion that Down syndrome patients are at high risk in developing AD. In the USA, it is estimated that more than a quarter million individuals live with Down syndrome and all will develop AD pathology as early as in their 30s [56]. In summary, protein aggregate formation starts at an early stage of life. This process is quicker in individuals with genetic conditions. Others display clinical signs at an old age as part of

Trends of Protein Aggregation in Neurodegenerative Diseases

DOI: http://dx.doi.org/10.5772/intechopen.81224

Cellular misfolded proteins are inclined to accumulate in nearby organs, in a preferred cell type in a particular tissue [57], and in a particular cell organelle [58]. Although these proteins are distributed in systemic circulation, high concentrations can be maintained in organs. For example, Aβ fragments deposition in brain regions of AD patient, SOD1 and TDP-43 accumulation in spinal cord of ALS patients, and α-synuclein plaques in brain regions (neocortex, hippocampus, substantia nigra, thalamus, and cerebellum) of PD patients. Appearance of signature proteins (i.e., SOD1, TDP-43, α-synuclein, Aβ fragments, etc.) in systemic circulation supports the development of a surrogate biomarker in the blood when tissue sampling is not

It is interesting to note that misfolded aberrant proteins interact with apoptotic proteins in organ-specific organelle. Pasinelli et al. have demonstrated that antiapoptotic protein Bcl-2 binds to detergent-insoluble mutant SOD1 (SOD1G93A) protein aggregates that are present in mitochondria from the spinal cord but not in the liver in both mice and humans [58]. This observation suggests that misfolded aberrant protein functions are location specific. Valentine has reviewed studies on mutant SOD1 fragmentation in the Golgi apparatus, which may reveal early molecular signals before the onset of ALS symptoms [59]. There are more emerging studies in which emphasizing the region-specific protein aggregation can be con-

The circulating proteins that have the potential to form extracellular amyloid deposits in multiple organs have been recently reviewed [47]. Local production of amyloid and non-amyloid protein species achieves the critical concentration for oligomerization and fibrillogenesis in specific organs. For example, Aβ deposition appears specifically in the brain tissue in AD. The Cu/Zn superoxide dismutase (SOD1) and TDP-43 deposition are measurable in brain and spinal cord tissues in ALS. These signature proteins can also be measurable in systemic circulation

We have recently published a paper describing platelet TDP-43 measurements as a proxy for brain tissue TDP-43 levels in AD patients [39]. This approach will aid

sidered a discriminatory signature in neurodegenerative diseases [60].

aging process.

accessible [39].

2.4 Systemic amyloidosis

(Table 1).

31

2.3 Regional protein aggregation

#### 2.1 Common protein behavior in aggregation

In the last 25 years or so, many diseases have been linked to protein-misfolding cases, although their etiology of such diseases is different. In this section, the focus will be on neurodegenerative diseases because of the following reasons: (i) they are progressive, (ii) early diagnosis for these diseases are not available yet, (iii) they are not effectively treatable, and (iv) they inflict enormous personal, societal, and economic burdens. Some of them are aging relevant such as AD and PD; some of them are not like ALS, Creutzfeldt-Jakob disease (mad cow disease), and other human prion diseases (e.g., variant Creutzfeldt-Jakob diseases, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru).

Specific polypeptides that go into aggregation are different in each amyloidosis; however, there is a common feature in the behavior of these proteins; they all present enriched β-sheet structure. Such proteins are normally soluble in cytosol and in extracellular environment; however, somehow they progress into β-sheetenriched insoluble filamentous polymers [47]. Not all β-structure-enriched insoluble filaments are an amyloid in nature. For example, some forms of SOD1-ALS are a conformational disease which involves amorphous aggregation of misfolded SOD1 [16, 48]. That is to say, the common structural motif in all amyloid fibers consists of cross-β-sheets. It is not uncommon that normally soluble proteins can undergo β-sheet-enriched conformational rearrangement and they tend to be more insoluble in nature. The concept of the β-sheet-enriched protein aggregation now becomes a common trend for polypeptide chains regardless of amino acid sequence [27]. The causes for the initiation of protein aggregation are not well documented; however, oxidative stress-induced reactive oxygen species (ROS) may be involved due to the role of ROS in several pathological disorders and aging [49, 50]. For example, glycine (Gly) residues are particularly susceptible for loss of a hydrogen ion, which results in the formation of Gly radical on the protein backbone which destabilizes protein structure [51]. Consequently, buried hydrophobic regions of the protein are exposed to the aqueous environment, and β-sheet structure formation is enhanced. These newly formed β-sheet structures link with that of neighboring structure which leads to the formation of a "seed" that eventually produces an aggregate [1].

#### 2.2 Milestones for aggregate formation

Another characteristic in protein-folding disorders is a prolonged period in aggregate/plaque formation before clinical manifestation becomes obvious [47]. In aging-dependent neurodegenerative diseases such as AD and PD, aggregate/plaque formation is a lengthy process, while in ALS, SOD1, and TDP-43, aggregates are formed in the middle [52] or even younger ages [53]. One explanation for a lengthy process would be that the initial nucleation of a misfolded protein is very small and energetically stays in the upper level of the energy landscape (see Figure 3).

Trends of Protein Aggregation in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.81224

Therefore, the growth of the protein aggregate seed is kinetically unfavorable; hence, recruitment of new aberrant proteins for aggregate formation takes a longer period of time [54, 55]. Once the nucleation achieves a critical mass, the fibril formation and subsequent plaque formation become accelerated. This is why clinical manifestation of amyloidoses relevant to neurodegenerative disease mostly appears in old age. This is mostly true for sporadic AD; however, in familial AD, which represents only 5–10% of the total AD population, the disease onset tends to occur at the middle age (50 and above). In Down syndrome, β-amyloid precursor protein (APP), is encoded on chromosome 21 [47]. Patients with trisomy 21 develop abundant Aβ aggregates in the brain at younger age. Therefore, the lifelong aggregate formation is inevitable in Down syndrome patients, which supports the notion that Down syndrome patients are at high risk in developing AD. In the USA, it is estimated that more than a quarter million individuals live with Down syndrome and all will develop AD pathology as early as in their 30s [56]. In summary, protein aggregate formation starts at an early stage of life. This process is quicker in individuals with genetic conditions. Others display clinical signs at an old age as part of aging process.

#### 2.3 Regional protein aggregation

2. Trends for misfolded proteins lead to neurodegeneration

Straussler-Scheinker syndrome, fatal familial insomnia, and kuru).

relevance to neurodegenerative diseases.

2.2 Milestones for aggregate formation

30

2.1 Common protein behavior in aggregation

Neurochemical Basis of Brain Function and Dysfunction

Insoluble extracellular protein deposits in various human diseases have been recognized for a long time. Many proteins that have a tendency to be misfolded do form aggregates that initiate cellular dysfunction [47]. This section discusses how the proteins involved in neurodegenerative disease have a tendency to misfold. If we comprehend the biochemical and biophysical behavior of the proteins and their misfolding features, we will have a better understanding of proteinopathy and

In the last 25 years or so, many diseases have been linked to protein-misfolding cases, although their etiology of such diseases is different. In this section, the focus will be on neurodegenerative diseases because of the following reasons: (i) they are progressive, (ii) early diagnosis for these diseases are not available yet, (iii) they are not effectively treatable, and (iv) they inflict enormous personal, societal, and economic burdens. Some of them are aging relevant such as AD and PD; some of them are not like ALS, Creutzfeldt-Jakob disease (mad cow disease), and other human prion diseases (e.g., variant Creutzfeldt-Jakob diseases, Gerstmann-

Specific polypeptides that go into aggregation are different in each amyloidosis;

however, there is a common feature in the behavior of these proteins; they all present enriched β-sheet structure. Such proteins are normally soluble in cytosol and in extracellular environment; however, somehow they progress into β-sheetenriched insoluble filamentous polymers [47]. Not all β-structure-enriched insoluble filaments are an amyloid in nature. For example, some forms of SOD1-ALS are a conformational disease which involves amorphous aggregation of misfolded SOD1 [16, 48]. That is to say, the common structural motif in all amyloid fibers consists of cross-β-sheets. It is not uncommon that normally soluble proteins can undergo β-sheet-enriched conformational rearrangement and they tend to be more insoluble in nature. The concept of the β-sheet-enriched protein aggregation now becomes a common trend for polypeptide chains regardless of amino acid sequence [27]. The causes for the initiation of protein aggregation are not well documented; however, oxidative stress-induced reactive oxygen species (ROS) may be involved due to the role of ROS in several pathological disorders and aging [49, 50]. For example, glycine (Gly) residues are particularly susceptible for loss of a hydrogen ion, which results in the formation of Gly radical on the protein backbone which destabilizes protein structure [51]. Consequently, buried hydrophobic regions of the protein are exposed to the aqueous environment, and β-sheet structure formation is enhanced. These newly formed β-sheet structures link with that of neighboring structure which leads to the formation of a "seed" that eventually produces an aggregate [1].

Another characteristic in protein-folding disorders is a prolonged period in aggregate/plaque formation before clinical manifestation becomes obvious [47]. In aging-dependent neurodegenerative diseases such as AD and PD, aggregate/plaque formation is a lengthy process, while in ALS, SOD1, and TDP-43, aggregates are formed in the middle [52] or even younger ages [53]. One explanation for a lengthy process would be that the initial nucleation of a misfolded protein is very small and energetically stays in the upper level of the energy landscape (see Figure 3).

Cellular misfolded proteins are inclined to accumulate in nearby organs, in a preferred cell type in a particular tissue [57], and in a particular cell organelle [58]. Although these proteins are distributed in systemic circulation, high concentrations can be maintained in organs. For example, Aβ fragments deposition in brain regions of AD patient, SOD1 and TDP-43 accumulation in spinal cord of ALS patients, and α-synuclein plaques in brain regions (neocortex, hippocampus, substantia nigra, thalamus, and cerebellum) of PD patients. Appearance of signature proteins (i.e., SOD1, TDP-43, α-synuclein, Aβ fragments, etc.) in systemic circulation supports the development of a surrogate biomarker in the blood when tissue sampling is not accessible [39].

It is interesting to note that misfolded aberrant proteins interact with apoptotic proteins in organ-specific organelle. Pasinelli et al. have demonstrated that antiapoptotic protein Bcl-2 binds to detergent-insoluble mutant SOD1 (SOD1G93A) protein aggregates that are present in mitochondria from the spinal cord but not in the liver in both mice and humans [58]. This observation suggests that misfolded aberrant protein functions are location specific. Valentine has reviewed studies on mutant SOD1 fragmentation in the Golgi apparatus, which may reveal early molecular signals before the onset of ALS symptoms [59]. There are more emerging studies in which emphasizing the region-specific protein aggregation can be considered a discriminatory signature in neurodegenerative diseases [60].

#### 2.4 Systemic amyloidosis

The circulating proteins that have the potential to form extracellular amyloid deposits in multiple organs have been recently reviewed [47]. Local production of amyloid and non-amyloid protein species achieves the critical concentration for oligomerization and fibrillogenesis in specific organs. For example, Aβ deposition appears specifically in the brain tissue in AD. The Cu/Zn superoxide dismutase (SOD1) and TDP-43 deposition are measurable in brain and spinal cord tissues in ALS. These signature proteins can also be measurable in systemic circulation (Table 1).

We have recently published a paper describing platelet TDP-43 measurements as a proxy for brain tissue TDP-43 levels in AD patients [39]. This approach will aid


Levels of γ- and β-secretase activities are greater in brain tissue samples from AD patients than non-demented control subjects [68, 69]. Experimental studies

Trends of Protein Aggregation in Neurodegenerative Diseases

DOI: http://dx.doi.org/10.5772/intechopen.81224

diseases so that early treatment options could be available [39].

2.5 Aging and protein aggregation

33

conducted on 3xTg-AD (swAPP, PS1-M146V, tau-P301L) demonstrate that BACE-1 activity levels were elevated in the brain tissue and γ-secretase inhibitors reduced the BACE-1 activity, suggesting that γ-secretase mediates oxidative stress-induced expression of BACE-1 resulting in excessive Aβ production in AD [70]. Extracellular cleavage of APP by BACE-1 creates a soluble extracellular fragment and a cell membrane-bound fragment referred to as C99. Cleavage of C99 within its transmembrane domain by γ-secretase releases the intracellular domain of APP and produces Aβ. Since γ-secretase cleaves APP closer to the cell membrane than BACE1 does, it removes a fragment of the Aβ peptide. Initial cleavage of APP by α-secretase rather than BACE-1 prevents eventual generation of Aβ [71]. It is clear that the most notable neurodegenerative diseases (i.e., AD, ALS, FTLD, and PD) share a common prominent pathological feature, TDP-43 proteinopathy. This issue has been recently reviewed, and possibility of targeting TDP-43 as a common therapeutic approach to formulate a treatment for neurodegenerative diseases was discussed [72]. In our laboratory, we are also working on an assay methodology that uses peripheral blood-derived platelet TDP-43 profile that may help for early diagnosis of such

Aging is the normal biological process that includes increased protein misfolding and aggregation process due to either reduced levels of quality control checkpoints such as chaperone system or proteasome complex. The proteins that form orderly (amyloid fibers) and disorderly (amorphous aggregates and plaques) show some common and essential biochemical and biophysical features that were discussed earlier. Therefore, such age-related neurodegenerative diseases may be considered a special form of amyloidoses [47]. The biochemical processes in aging are contributing to free radical-induced protein oxidation; hence, unnatural disulfide bridge formations can contribute oxidized protein aggregation as well [73] . Dismutase metalloenzyme (e.g., SOD1 and SOD2) activity levels are also reduced in aging [74, 75]. This contributes the inefficient removal of reactive oxygen ions; hence, more protein oxidation events take place, and subsequently protein aggregation occurs. Principal unanswered questions about these neurodegenerative disorders remain: how preciously native soluble proteins undergo partial unfolding, and does aberrant refolding produce highly stable polypeptide polymers? We can still make some predictions about the propensity of a given protein through PONDR® analysis. This publicly available online prediction program (www.pondr.com) can be utilized for predicting which regions of the protein will be more susceptible for disorderliness which may increase the chance of aberrant protein refolding [76, 77]. It is clear that time and supraphysiological concentrations of predictable proaggregate proteins are two important parameters in aggregation process. Other factors such as oxidizable amino acids (e.g., cysteine and methionine), population, local pH, and higher hydrophobic propensity of the protein help the oligomerization process. Equilibrium between natively folded protein and aberrant-folded protein can last for a long period of time. One approach would be that molten globule-like intermediates have persistent structure in unstable α-structure. Stable β-structure of the protein provides a template (seed) for the recruitment of additional peptide chains through physical interaction of those two structural regions of the protein. Finally, new hydrogen bonds form and stabilize the protein in an insoluble amyloid fibril [78, 79]. On the other hand, fibril deposition is not a necessary feature in prion disease. NMR structure of a domain of prion protein (PrR(121–231)) indicates that

#### Table 1.

Extracellular fibril types in disease

to monitor the progress of aberrant protein aggregation in neurodegenerative diseases.

In age-related neurodegenerative diseases, it is quite common to observe β-sheet-enriched protein aggregates that are mostly detergent-insoluble. These insoluble protein aggregates tend to accumulate inside the cell; however, ultrastructure analysis of these aggregates may not be the same as that of extracellular amyloid fibrils. The commonality of these aggregate-forming proteins was discussed earlier (Section 1.3.1). Therefore, it is reasonably acceptable to classify the aberrant protein aggregation-related neurodegenerative diseases as a special form of amyloidosis [47].

Protein misfolding and subsequent aggregation are central in neurodegenerative diseases; however, the protein behavior in forming aggregates is somehow disease specific. In case of the α-synuclein, this protein is natively folded and normally water soluble in the cell. In normal health conditions, α-synuclein participates in the maintenance of synaptic vesicle supplies at the presynaptic terminals [61]. In PD, this protein misfolds and accumulates in spherical filamentous structures called Lewy bodies. This encapsulated structure forms particularly in dopaminergic and noradrenergic brainstem neurons and causes premature cell death [62, 63]. Therefore, Lewy bodies become a signature pattern for PD. Polyglutamine repeat of corresponding proteins that are produced as the result of different mutant genes becomes a distinct pattern in Huntington's disease and several forms of familial spinocerebellar ataxia [64]. AD is the only brain disorder that displays the accumulation of amyloid forming proteins both extracellularly (Aβ fragments) and intracellularly (hyper-phosphorylated tau) [47]. The question would be whether hyperphosphorylated tau neurofibrillary tangles or Aβ accumulation initiate AD. The amyloid cascade hypothesis has been the most studied model of molecular pathogenesis in AD. This is a long-debated issue in the scientific community which has polarized into two schools of thought: "Baptists" that believe Aβ accumulation is the starting event or "Tauist" that believes that tau-relevant neurofibrillary tangles are the initiators for AD [65]. However, inherited mutations in tau protein do not directly lead to AD; yet, it causes another devastating disorder, FTLD with PD [66]. It is now more probable that inherited mutations in APP or in one of the APP cleaving proteases (e.g., presenilin/γ-secretase) cause aggressive early onset forms of AD [67].

What are the initiation factors in sporadic AD which makes about 5–10% of all AD cases? Not much is known so far. A new player in AD field is TDP-43 protein which induces intramural Aβ accumulation via increasing β-secretase (BACE-1) [2]. As it can be seen in Figure 4, TDP-43 acts on an upstream in the APP structure and may induce β-secretase. A ripple effect may induce to generate toxic Aβ fragments.

Trends of Protein Aggregation in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.81224

Levels of γ- and β-secretase activities are greater in brain tissue samples from AD patients than non-demented control subjects [68, 69]. Experimental studies conducted on 3xTg-AD (swAPP, PS1-M146V, tau-P301L) demonstrate that BACE-1 activity levels were elevated in the brain tissue and γ-secretase inhibitors reduced the BACE-1 activity, suggesting that γ-secretase mediates oxidative stress-induced expression of BACE-1 resulting in excessive Aβ production in AD [70]. Extracellular cleavage of APP by BACE-1 creates a soluble extracellular fragment and a cell membrane-bound fragment referred to as C99. Cleavage of C99 within its transmembrane domain by γ-secretase releases the intracellular domain of APP and produces Aβ. Since γ-secretase cleaves APP closer to the cell membrane than BACE1 does, it removes a fragment of the Aβ peptide. Initial cleavage of APP by α-secretase rather than BACE-1 prevents eventual generation of Aβ [71]. It is clear that the most notable neurodegenerative diseases (i.e., AD, ALS, FTLD, and PD) share a common prominent pathological feature, TDP-43 proteinopathy. This issue has been recently reviewed, and possibility of targeting TDP-43 as a common therapeutic approach to formulate a treatment for neurodegenerative diseases was discussed [72]. In our laboratory, we are also working on an assay methodology that uses peripheral blood-derived platelet TDP-43 profile that may help for early diagnosis of such diseases so that early treatment options could be available [39].

#### 2.5 Aging and protein aggregation

to monitor the progress of aberrant protein aggregation in neurodegenerative

In age-related neurodegenerative diseases, it is quite common to observe β-sheet-enriched protein aggregates that are mostly detergent-insoluble. These insoluble protein aggregates tend to accumulate inside the cell; however, ultrastructure analysis of these aggregates may not be the same as that of extracellular amyloid fibrils. The commonality of these aggregate-forming proteins was

discussed earlier (Section 1.3.1). Therefore, it is reasonably acceptable to classify the aberrant protein aggregation-related neurodegenerative diseases as a special form of

Protein misfolding and subsequent aggregation are central in neurodegenerative diseases; however, the protein behavior in forming aggregates is somehow disease specific. In case of the α-synuclein, this protein is natively folded and normally water soluble in the cell. In normal health conditions, α-synuclein participates in the maintenance of synaptic vesicle supplies at the presynaptic terminals [61]. In PD, this protein misfolds and accumulates in spherical filamentous structures called Lewy bodies. This encapsulated structure forms particularly in dopaminergic and noradrenergic brainstem neurons and causes premature cell death [62, 63]. Therefore, Lewy bodies become a signature pattern for PD. Polyglutamine repeat of corresponding proteins that are produced as the result of different mutant genes becomes a distinct pattern in Huntington's disease and several forms of familial spinocerebellar ataxia [64]. AD is the only brain disorder that displays the accumulation of amyloid forming proteins both extracellularly (Aβ fragments) and intracellularly (hyper-phosphorylated tau) [47]. The question would be whether hyperphosphorylated tau neurofibrillary tangles or Aβ accumulation initiate AD. The amyloid cascade hypothesis has been the most studied model of molecular pathogenesis in AD. This is a long-debated issue in the scientific community which has polarized into two schools of thought: "Baptists" that believe Aβ accumulation is the starting event or "Tauist" that believes that tau-relevant neurofibrillary tangles are the initiators for AD [65]. However, inherited mutations in tau protein do not directly lead to AD; yet, it causes another devastating disorder, FTLD with PD [66]. It is now more probable that inherited mutations in APP or in one of the APP cleaving proteases (e.g., presenilin/γ-secretase) cause aggressive early onset forms

What are the initiation factors in sporadic AD which makes about 5–10% of all AD cases? Not much is known so far. A new player in AD field is TDP-43 protein which induces intramural Aβ accumulation via increasing β-secretase (BACE-1) [2]. As it can be seen in Figure 4, TDP-43 acts on an upstream in the APP structure and may induce β-secretase. A ripple effect may induce to generate toxic Aβ fragments.

diseases.

Table 1.

Extracellular fibril types in disease

Neurochemical Basis of Brain Function and Dysfunction

amyloidosis [47].

of AD [67].

32

Aging is the normal biological process that includes increased protein misfolding and aggregation process due to either reduced levels of quality control checkpoints such as chaperone system or proteasome complex. The proteins that form orderly (amyloid fibers) and disorderly (amorphous aggregates and plaques) show some common and essential biochemical and biophysical features that were discussed earlier. Therefore, such age-related neurodegenerative diseases may be considered a special form of amyloidoses [47]. The biochemical processes in aging are contributing to free radical-induced protein oxidation; hence, unnatural disulfide bridge formations can contribute oxidized protein aggregation as well [73] . Dismutase metalloenzyme (e.g., SOD1 and SOD2) activity levels are also reduced in aging [74, 75]. This contributes the inefficient removal of reactive oxygen ions; hence, more protein oxidation events take place, and subsequently protein aggregation occurs. Principal unanswered questions about these neurodegenerative disorders remain: how preciously native soluble proteins undergo partial unfolding, and does aberrant refolding produce highly stable polypeptide polymers? We can still make some predictions about the propensity of a given protein through PONDR® analysis. This publicly available online prediction program (www.pondr.com) can be utilized for predicting which regions of the protein will be more susceptible for disorderliness which may increase the chance of aberrant protein refolding [76, 77]. It is clear that time and supraphysiological concentrations of predictable proaggregate proteins are two important parameters in aggregation process. Other factors such as oxidizable amino acids (e.g., cysteine and methionine), population, local pH, and higher hydrophobic propensity of the protein help the oligomerization process. Equilibrium between natively folded protein and aberrant-folded protein can last for a long period of time. One approach would be that molten globule-like intermediates have persistent structure in unstable α-structure. Stable β-structure of the protein provides a template (seed) for the recruitment of additional peptide chains through physical interaction of those two structural regions of the protein. Finally, new hydrogen bonds form and stabilize the protein in an insoluble amyloid fibril [78, 79]. On the other hand, fibril deposition is not a necessary feature in prion disease. NMR structure of a domain of prion protein (PrR(121–231)) indicates that

mutated amino acids in prion protein are involved in the maintenance of the hydrophobic core [80]. Exactly, how prion conversion propagates? The disease is currently under study [46]. As mentioned in Section 1.3, the ER contains a large repertoire of molecular chaperones and folding catalysts [13, 14]. The ER proteinfolding system is also affected by aging process, and less fold-assisting proteins would be available; hence, unfolded and misfolded protein levels would be expected to be high. Two of the unconventional ER chaperone molecules are calnexin (Cnx) and calreticulin (Crt) [81–83] as cited in [84]. In a pilot study, we demonstrated that Cnx levels were reduced in aging rat brain as well as in neuronal cell culture (Figure 7). This observation supports the other works published in literature stating that protein-folding mechanisms are less efficient; therefore, aberrant-misfolded proteins rise and form aggregates.

taken into consideration as well. Many mitochondrial proteins are nuclear-coded and transported into mitochondria [86]. These proteins are being transferred into mitochondria via outer membrane pores. Although these pores are specific, such small toxic protein species can nonspecifically bind the pores and slow down the protein entries into mitochondria if not completely block. Consequently, energy production mechanism of mitochondria may be compromised. Such hypothetical ideas need to be experimentally tested and should provide more convincing data about the cytotoxic effects of such protein species. Misfolded protein species get involved in apoptosis induction. Pasinelli et al. have reported that anti-apoptotic protein Bcl-2 interacts with both wt and mutant SOD1 (SOD1G93A) [58]. This interaction induces apoptotic cascade because SOD1G93A mitochondria triggers

Proper protein-folding in the cell occurs either in the cytoplasm or within the secretory pathway. Dobson has reviewed this concept in detail [9].The readers are referred to this review to attain more in-depth understanding of the protein-folding relevant issues. The trend of misfolding protein increases when part of the polypeptide chain does not participate in a proper folding process. However, there are mechanisms that are available for aiding protein-folding. Chaperones are the molecules that collaborate with misfolded proteins to give a polypeptide chain several opportunities to fold. ATP-dependent chaperone molecules are critical for ensuring accuracy in proper folding [88]. The lumen of the ER also participates in proteinfolding process by modifying the secretory proteins while they are still associated with the ER [89]. Any of these mechanistic failures contribute misfolded protein accumulation. Despite the attempt to rescue misfolded proteins that are destined to form insoluble aggregates, proteasomes, protein aggregate removal machinery, degrade such proteins from the cell so that cell homeostasis be maintained.

Several other protein misfolding-relevant diseases are caused by conformational modifications in extracellular milieu. Protein quality control-check systems of the cell (chaperones and heat shock protein family) cannot be linked to such misfolded protein population because such aggregation formation does not take place in cytosol. However, recent studies demonstrate the presence of extracellular proteasome machinery [92–94]. It is not clear yet whether these extracellular proteasomes are in the same categories in that of cytosolic since a recent report demonstrated that such extracellular proteasomes structurally differ from their cytosolic counterparts [94]. The major representatives of such disorders are the amyloidoses, in which protein aggregation in the extracellular space is associated with the presence of malfunctioned protein molecules [37, 90]. The chaperone-like small molecules may have the potential to be included in the treatment options for amyloidoses [91]. The more knowledge we attain on how chaperones and heat shock proteins interact with protein-folding process the better design for small molecules would be feasible. Folding process of proteins is an environment-dependent physicochemical process. Some proteins have a folding issue where protein-folding takes place (i.e., lysosomal enzymes) while the others are efficiently folded in the ER but misfolded and misassembled at the destination (i.e., amyloidogenic proteins). This knowledge is helping industry-academia partnership for developing pharmacological intervention that reduces the mutant protein production, increases the rate of clearance of misfolded/mildly aggregated proteins, and increases the native stability of the

apoptosis more strongly than the cytosolic mutant SOD1 [87].

Trends of Protein Aggregation in Neurodegenerative Diseases

DOI: http://dx.doi.org/10.5772/intechopen.81224

3. Therapeutic approach to protein-misfolding disease

proteins.

35

#### 2.6 Misfolded aberrant proteins cause cell dysfunction

Dynamic equilibrium between misfolded and natively folded proteins may be shifted in favor of protein misfolding and oligomerization in proteinopathy. No definite amyloid fibrils are seen in diseases, suggesting that smaller diffusible toxic protein species consisting of dimers, trimers, tetramers, and large oligomers may be involved in cell cytotoxicity [47]. Therefore, forming a plaque or aggregate may be considered a defense mechanism of cell against recruitment of more toxic protein species. The aggregate in plaque no longer poses toxicity for cell life; however, having such foreign structure in the cytosol or in extracellular milieu brings some serious problems in cell homeostasis. For example, the plaques are insoluble and indestructible by ubiquitinated proteasome system. The increased population of such aggregates may overwhelm or even block the proteasome machinery. Therefore, proteasome either slows down or becomes less functional. We have observed such reduced proteasome activity in AD and ALS cases (Figure 5).

Another interference of such toxic protein species is that they nonspecifically bind to receptors and channel proteins on the plasma membrane, thus interfering with numerous cell-signaling events [16, 85]. Mitochondrion homeostasis should be

#### Figure 7.

Calnexin protein levels in aging. Calnexin protein levels were analyzed by Western blotting. Cnx levels were reduced during the aging process in total brain homogenate, synaptic plasma membrane, and neuronal cell culture. All samples were prepared from rat (n = 3) (unpublished data).

Trends of Protein Aggregation in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.81224

mutated amino acids in prion protein are involved in the maintenance of the hydrophobic core [80]. Exactly, how prion conversion propagates? The disease is currently under study [46]. As mentioned in Section 1.3, the ER contains a large repertoire of molecular chaperones and folding catalysts [13, 14]. The ER proteinfolding system is also affected by aging process, and less fold-assisting proteins would be available; hence, unfolded and misfolded protein levels would be expected to be high. Two of the unconventional ER chaperone molecules are calnexin (Cnx) and calreticulin (Crt) [81–83] as cited in [84]. In a pilot study, we demonstrated that Cnx levels were reduced in aging rat brain as well as in neuronal cell culture (Figure 7). This observation supports the other works published in literature stating that protein-folding mechanisms are less efficient; therefore, aberrant-misfolded

Dynamic equilibrium between misfolded and natively folded proteins may be shifted in favor of protein misfolding and oligomerization in proteinopathy. No definite amyloid fibrils are seen in diseases, suggesting that smaller diffusible toxic protein species consisting of dimers, trimers, tetramers, and large oligomers may be involved in cell cytotoxicity [47]. Therefore, forming a plaque or aggregate may be considered a defense mechanism of cell against recruitment of more toxic protein species. The aggregate in plaque no longer poses toxicity for cell life; however, having such foreign structure in the cytosol or in extracellular milieu brings some serious problems in cell homeostasis. For example, the plaques are insoluble and indestructible by ubiquitinated proteasome system. The increased population of such aggregates may overwhelm or even block the proteasome machinery. Therefore, proteasome either slows down or becomes less functional. We have observed

Another interference of such toxic protein species is that they nonspecifically bind to receptors and channel proteins on the plasma membrane, thus interfering with numerous cell-signaling events [16, 85]. Mitochondrion homeostasis should be

Calnexin protein levels in aging. Calnexin protein levels were analyzed by Western blotting. Cnx levels were reduced during the aging process in total brain homogenate, synaptic plasma membrane, and neuronal cell

culture. All samples were prepared from rat (n = 3) (unpublished data).

proteins rise and form aggregates.

Figure 7.

34

2.6 Misfolded aberrant proteins cause cell dysfunction

Neurochemical Basis of Brain Function and Dysfunction

such reduced proteasome activity in AD and ALS cases (Figure 5).

taken into consideration as well. Many mitochondrial proteins are nuclear-coded and transported into mitochondria [86]. These proteins are being transferred into mitochondria via outer membrane pores. Although these pores are specific, such small toxic protein species can nonspecifically bind the pores and slow down the protein entries into mitochondria if not completely block. Consequently, energy production mechanism of mitochondria may be compromised. Such hypothetical ideas need to be experimentally tested and should provide more convincing data about the cytotoxic effects of such protein species. Misfolded protein species get involved in apoptosis induction. Pasinelli et al. have reported that anti-apoptotic protein Bcl-2 interacts with both wt and mutant SOD1 (SOD1G93A) [58]. This interaction induces apoptotic cascade because SOD1G93A mitochondria triggers apoptosis more strongly than the cytosolic mutant SOD1 [87].

### 3. Therapeutic approach to protein-misfolding disease

Proper protein-folding in the cell occurs either in the cytoplasm or within the secretory pathway. Dobson has reviewed this concept in detail [9].The readers are referred to this review to attain more in-depth understanding of the protein-folding relevant issues. The trend of misfolding protein increases when part of the polypeptide chain does not participate in a proper folding process. However, there are mechanisms that are available for aiding protein-folding. Chaperones are the molecules that collaborate with misfolded proteins to give a polypeptide chain several opportunities to fold. ATP-dependent chaperone molecules are critical for ensuring accuracy in proper folding [88]. The lumen of the ER also participates in proteinfolding process by modifying the secretory proteins while they are still associated with the ER [89]. Any of these mechanistic failures contribute misfolded protein accumulation. Despite the attempt to rescue misfolded proteins that are destined to form insoluble aggregates, proteasomes, protein aggregate removal machinery, degrade such proteins from the cell so that cell homeostasis be maintained.

Several other protein misfolding-relevant diseases are caused by conformational modifications in extracellular milieu. Protein quality control-check systems of the cell (chaperones and heat shock protein family) cannot be linked to such misfolded protein population because such aggregation formation does not take place in cytosol. However, recent studies demonstrate the presence of extracellular proteasome machinery [92–94]. It is not clear yet whether these extracellular proteasomes are in the same categories in that of cytosolic since a recent report demonstrated that such extracellular proteasomes structurally differ from their cytosolic counterparts [94].

The major representatives of such disorders are the amyloidoses, in which protein aggregation in the extracellular space is associated with the presence of malfunctioned protein molecules [37, 90]. The chaperone-like small molecules may have the potential to be included in the treatment options for amyloidoses [91]. The more knowledge we attain on how chaperones and heat shock proteins interact with protein-folding process the better design for small molecules would be feasible.

Folding process of proteins is an environment-dependent physicochemical process. Some proteins have a folding issue where protein-folding takes place (i.e., lysosomal enzymes) while the others are efficiently folded in the ER but misfolded and misassembled at the destination (i.e., amyloidogenic proteins). This knowledge is helping industry-academia partnership for developing pharmacological intervention that reduces the mutant protein production, increases the rate of clearance of misfolded/mildly aggregated proteins, and increases the native stability of the proteins.

## 4. Conclusions

The majority of non-treatable neurodegenerative diseases are related to misfolding protein-induced aggregation and insoluble plaque formation. This process is very slow which makes early diagnosis of neurodegenerative diseases almost impossible at this time. However, some predictive studies may help to identify the proteins that have a tendency to form amyloid plaques. To know protein behavior in various physiological conditions and environmental factors will contribute to designing disease-specific drugs that interfere the aggregation formation in neurodegenerative diseases.

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## Acknowledgements

This work is partially supported by an intramural grant provided by the Office of Research and Sponsored Programs (ORSP) at Kansas City University of Medicine and Biosciences. Cultured neuronal cells were provided by Dr. Asma Zaidi. I would like to thank Dr. E. Dora Krizsan-Agbas for her constructive criticism and Tuba Agbas for proofreading the manuscript.

## Conflict of interest

The author declares no conflict of interest.

## Author details

Abdulbaki Agbas Department of Basic Sciences, Kansas City University of Medicine and Biosciences, Kansas City, MO, USA

\*Address all correspondence to: aagbas@kcumb.edu

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Trends of Protein Aggregation in Neurodegenerative Diseases DOI: http://dx.doi.org/10.5772/intechopen.81224

## References

4. Conclusions

neurodegenerative diseases.

Agbas for proofreading the manuscript.

The author declares no conflict of interest.

Neurochemical Basis of Brain Function and Dysfunction

\*Address all correspondence to: aagbas@kcumb.edu

provided the original work is properly cited.

Acknowledgements

Conflict of interest

Author details

Abdulbaki Agbas

36

Kansas City, MO, USA

The majority of non-treatable neurodegenerative diseases are related to misfolding protein-induced aggregation and insoluble plaque formation. This process is very slow which makes early diagnosis of neurodegenerative diseases almost impossible at this time. However, some predictive studies may help to identify the proteins that have a tendency to form amyloid plaques. To know protein behavior in various physiological conditions and environmental factors will contribute to designing disease-specific drugs that interfere the aggregation formation in

This work is partially supported by an intramural grant provided by the Office of Research and Sponsored Programs (ORSP) at Kansas City University of Medicine and Biosciences. Cultured neuronal cells were provided by Dr. Asma Zaidi. I would like to thank Dr. E. Dora Krizsan-Agbas for her constructive criticism and Tuba

Department of Basic Sciences, Kansas City University of Medicine and Biosciences,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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**43**

**Chapter 4**

**Abstract**

*and Ottavio Arancio*

Targeting the NO/cGMP/CREB

Pathway in Alzheimer's Disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common form of senile dementia. Recently, scientists have put significant effort into exploring the molecular mechanisms involved in the pathological processes leading to the disease. A vast number of studies have focused on understanding the nitric oxide (NO) signaling pathway, which culminates with the phosphorylation of the transcription factor cAMP-responsive element-binding protein (CREB) through the increase of the second messenger cyclic guanosine monophosphate (cGMP) and activation of cGMP-dependent protein kinase. This book chapter provides an overview of the progress being made in modulating the hippocampal synaptic transmissions, which are critical for learning and memory, by targeting the different components of the NO/cGMP/CREB phosphorylation signaling pathway. Furthermore, a description of recent research on this pathway

Phosphorylation Signaling

*Jole Fiorito, Shi-Xian Deng, Donald W. Landry* 

through the use of phosphodiesterase inhibitors is emphasized.

cAMP-regulatory element-binding protein

**1. Introduction**

**Keywords:** Alzheimer's disease, nitric oxide, cyclic guanosine monophosphate, cGMP-dependent protein kinase, phosphodiesterases, phosphodiesterase inhibitors,

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that involves cognitive impairment, such as loss of memory and reasoning and decline in mental ability. The AD brain is characterized by cell death and intra- and extracellular accumulation of amyloid-beta (Aβ) and tau proteins that form senile plaques and neurofibrillary tangles, respectively. Nowadays, medical treatments available on the market comprise two classes of drugs, acetylcholinesterase (AchE) inhibitors (i.e. donepezil, galantamine, and rivastigmine) and *N*-methyl-D-aspartate (NMDA) receptor antagonist (i.e. memantine). Based on the AD cholinergic hypothesis, acetylcholine-containing neurons project diffusely to the cortex and modulate cognitive processing. Damage of these projections has been associated with learning and memory impairment. Thus, AchE inhibitors block the acetylcholine-degrading enzyme consequently raising the levels of the acetylcholine neurotransmitter in the brain [1]. Differently from AchE inhibitors, memantine antagonizes the NMDA receptors, modulating dysfunctions in the glutamatergic

## **Chapter 4**
