**2.1 Brief introduction to AD and PD**

AD is characterized by the gradual decline in the cognitive function, memory loss, and behavior changes [1]. Typical features of the disease are a synaptic deficit in the neocortex and the limbic system, neuronal loss, white matter loss, astrogliosis, microglial cell proliferation, and oxidative stress [2]. The major areas of the human brain affected by AD are schematically represented in **Figure 1**. The pathological hallmarks of AD are the presence of intracellular flame-shaped neurofibrillary tangles and extracellular plaques in the brain. The tangles are especially present in the perinuclear cytoplasm and are prevalently formed by the Tau protein, in a hyperphosphorylated form. The plaques derive from the progressive accumulation of amyloid β-peptide (Aβ) in a filamentous form [3]. The neuritic plaques have a diameter ranging from 10 to more than 120 μm [2]. The methods used for the diagnosis of the pathology have been standardized. They refer to the density and the grade of compactness of the neuritis amyloid plaques and neurofibrillary tangles [4]. AD aggregates can be classified into positive and negative lesions as a function of their localization and level of progression [5]. Typical positive lesions are represented by amyloid plaques and neurofibrillary tangles, neuropil threads, and dystrophic neurites, essentially formed by hyperphosphorylated Tau [6]. The negative lesions provide loss of neurons and neuropil threads [7].

Clinically, PD typically manifests with motor symptoms, such as bradykinesia, rigidity, tremor at rest, and instability. Since there is no definitive test for the diagnosis of PD, the appearance of these clinical manifestations is important for the early treatment of the disease [8]. PD is characterized by the loss of dopaminergic neurons in the *Substantia nigra pars compacta* (**Figure 1**) and by the deposition of

#### **Figure 1.**

*Affected brain regions in AD and PD. Cross-section of human brain showing the principal districts affected by AD (green) and PD (blue). AD typically involves parts of the brain involved in memory, like hippocampus and ventricles, and the cerebral cortex responsible for language. In PD nerve cells of the motor cortex and in part of the basal ganglia (composed by substantia nigra, putamen, caudate nucleus, globus pallidus, and locus coeruleus) degenerate. As a result, the basal ganglia cannot control muscle movement as it normally does, leading to tremor, bradykinesia, and hypokinesia.*

**39**

**Table 1.**

*Polyphenols as Potential Therapeutic Drugs in Neurodegeneration*

intraneuronal proteinaceous aggregates, mainly composed by α -synuclein (Syn), named Lewy bodies and Lewy neurites [9]. Syn was also found in the pathological inclusions of Lewy body variant of both AD and multiple system atrophy. Furthermore, Syn inclusions characterize other neurodegenerative diseases, defined as α-synucleinopathies, including Down's syndrome, progressive autonomic failure, and familial and sporadic AD [10]. In a very recent study, Shahmoradian and coll. have reported that Lewy bodies are not only formed by Syn deposit but also by clusters of lipid vesicles [11]. These important findings further correlate Syn-lipid

AD and PD are generally sporadic and occur in individuals between ages 60 and 70, but the ~20% of patients have a genetically linked familial form. The onset of these forms occurs earlier, and it is associated with mutations in several genes [14]. The main mutations are listed in **Table 1**. The proteins involved in such neurodegenerative diseases, Aβ, tau, and Syn, are completely distinct in terms of structure and putative functions, most of which are not completely clarified. However, the formation of aggregated structures is a common feature among these macromolecules. Fibrils, which originate from the association of monomeric forms of the proteins, pass through intermediate species such as oligomers (**Figure 2**). Generally, they can cross the membrane and spread throughout the brain. Several evidences

**Phenotype Notes Refs**

www.molgen.ua.ac.be/ADMutations [15]

www.molgen.ua.ac.be/ADMutations [16]

>200 mutations [17, 18]

Rare, <40 mutations [19]

>20 mutations [26, 27]

>60 mutations [29]

>10 mutation, deletions [30]

>150 mutations, deletions, insertions [28]

[20–25]

A53T; A30P, E46K, G51D, H50Q, gene duplication and triplication

*DOI: http://dx.doi.org/10.5772/intechopen.89575*

interaction with neurodegeneration [12, 13].

**Disease Mutated** 

**protein**

AD APP Abnormal

production of Aβ

density of Ab plaques High risk of AD, late onset of AD and Down syndrome

Aβ42/Aβ40, and reduced γ-secretase activity

Aβ42/Aβ40, and reduced γ-secretase activity

onset PD

Autosomal dominant PD; mid-to-late onset and slow progress

Early-onset PD and parkinsonism

> early-onset Parkinsonism

recessive PD

ApoE Increase of the

Presenilin1 Increased the

Presenilin2 Increased the

PINK1 Sporadic

DJ-1 Autosomal

*Main mutations involved in familiar forms of AD and PD.*

PD Syn Familiar and early

Leucinerich repeat kinase 2 (LRRK2)

E3 ubiquitin ligase Parkin

#### *Polyphenols as Potential Therapeutic Drugs in Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89575*

*Neuroprotection - New Approaches and Prospects*

order to use them in effective therapies.

**2.1 Brief introduction to AD and PD**

with the Mediterranean diet, such as polyphenols. The unexpected benefits and the wide-range properties of polyphenols suggest deepening the study of these molecules for a more comprehensive understanding of their mechanism of action in

AD is characterized by the gradual decline in the cognitive function, memory loss, and behavior changes [1]. Typical features of the disease are a synaptic deficit in the neocortex and the limbic system, neuronal loss, white matter loss, astrogliosis, microglial cell proliferation, and oxidative stress [2]. The major areas of the human brain affected by AD are schematically represented in **Figure 1**. The pathological hallmarks of AD are the presence of intracellular flame-shaped neurofibrillary tangles and extracellular plaques in the brain. The tangles are especially present in the perinuclear cytoplasm and are prevalently formed by the Tau protein, in a hyperphosphorylated form. The plaques derive from the progressive accumulation of amyloid β-peptide (Aβ) in a filamentous form [3]. The neuritic plaques have a diameter ranging from 10 to more than 120 μm [2]. The methods used for the diagnosis of the pathology have been standardized. They refer to the density and the grade of compactness of the neuritis amyloid plaques and neurofibrillary tangles [4]. AD aggregates can be classified into positive and negative lesions as a function of their localization and level of progression [5]. Typical positive lesions are represented by amyloid plaques and neurofibrillary tangles, neuropil threads, and dystrophic neurites, essentially formed by hyperphosphorylated Tau [6]. The

Clinically, PD typically manifests with motor symptoms, such as bradykinesia,

*Affected brain regions in AD and PD. Cross-section of human brain showing the principal districts affected by AD (green) and PD (blue). AD typically involves parts of the brain involved in memory, like hippocampus and ventricles, and the cerebral cortex responsible for language. In PD nerve cells of the motor cortex and in part of the basal ganglia (composed by substantia nigra, putamen, caudate nucleus, globus pallidus, and locus coeruleus) degenerate. As a result, the basal ganglia cannot control muscle movement as it normally does,* 

rigidity, tremor at rest, and instability. Since there is no definitive test for the diagnosis of PD, the appearance of these clinical manifestations is important for the early treatment of the disease [8]. PD is characterized by the loss of dopaminergic neurons in the *Substantia nigra pars compacta* (**Figure 1**) and by the deposition of

**2. Molecular aspects of Alzheimer's and Parkinson's diseases**

negative lesions provide loss of neurons and neuropil threads [7].

**38**

**Figure 1.**

*leading to tremor, bradykinesia, and hypokinesia.*

intraneuronal proteinaceous aggregates, mainly composed by α -synuclein (Syn), named Lewy bodies and Lewy neurites [9]. Syn was also found in the pathological inclusions of Lewy body variant of both AD and multiple system atrophy. Furthermore, Syn inclusions characterize other neurodegenerative diseases, defined as α-synucleinopathies, including Down's syndrome, progressive autonomic failure, and familial and sporadic AD [10]. In a very recent study, Shahmoradian and coll. have reported that Lewy bodies are not only formed by Syn deposit but also by clusters of lipid vesicles [11]. These important findings further correlate Syn-lipid interaction with neurodegeneration [12, 13].

AD and PD are generally sporadic and occur in individuals between ages 60 and 70, but the ~20% of patients have a genetically linked familial form. The onset of these forms occurs earlier, and it is associated with mutations in several genes [14]. The main mutations are listed in **Table 1**. The proteins involved in such neurodegenerative diseases, Aβ, tau, and Syn, are completely distinct in terms of structure and putative functions, most of which are not completely clarified. However, the formation of aggregated structures is a common feature among these macromolecules. Fibrils, which originate from the association of monomeric forms of the proteins, pass through intermediate species such as oligomers (**Figure 2**). Generally, they can cross the membrane and spread throughout the brain. Several evidences


#### **Table 1.**

*Main mutations involved in familiar forms of AD and PD.*

#### **Figure 2.**

*Scheme of the aggregation process of amyloid proteins. The formation of fibrils occurs through a nucleationdependent pathway starting from the monomeric form of the protein and leading to fibril elongation through intermediates (oligomers and protofibrils). The formation of the nucleus is the rate limiting step, and at this stage, the protein has acquired an aggregation-prone conformation. Fibrils are composed of a β-sheet structure in which hydrogen bonding occurs along the length of the fibril, and the β-strands run perpendicular to the fibril axis.*

suggest that oligomers are the species responsible for the cytotoxicity. There are many proofs in support of this hypothesis, but unfortunately, due to the extreme heterogeneity in oligomer structures and their transient nature, a conclusive view has not been obtained yet [31–33]. The atomic structure of fibrils has been studied by several biophysical techniques. A quite accepted hypothesis agrees with the presence of a common molecular organization independent from the original structure of the involved protein: repetitive β-sheet units parallel to the fibril axis with their strands perpendicular to it [34, 35]. Amyloid fibrils can self-assemble *in vitro* from many structurally different proteins and peptides, not necessarily involved in diseases. It has been postulated that the cross-β structure represents a generic conformation, which represents another folding state for proteins [36, 37]. In addition to these characteristics, there are also some common aspects in the onset of the diseases. Several studies suggest possible interplays and synergistic activities between the involved proteins. Clinton et al. [38] provided evidence that Aβ, tau, and Syn could interact *in vivo* to promote their self-aggregation, thus accelerating the cognitive dysfunction [38]. High levels of Syn were found in patients suffering from AD [39]. Aβ stimulates Syn fibril formation in the transgenic mouse model through a seed mechanism [40]. In another study, Syn seems to inhibit the deposition of Aβ into the amyloid plaques [41].

#### **2.2 Key proteins in neurodegeneration**

#### *2.2.1 Aβ peptide*

The Aβ peptide was found in the amyloid plaques in 1984 [3]. Aβ represents a group of peptides constituted by 37–49 residues (**Figure 3A**), derived from the proteolytic processing of the amyloid precursor protein (APP) [42, 43] (**Figure 4**).

**41**

**Figure 3.**

*binding region.*

*Polyphenols as Potential Therapeutic Drugs in Neurodegeneration*

APP is a single membrane-spanning domain protein, containing a large extracellular glycosylated N-terminus and a shorter cytoplasmic C-terminus. The enzymatic processes responsible for the release of Aβ from APP are to date well elucidated [2]. Specifically, APP undergoes several proteolytic cleavages. The processing by α-secretase results in the release of the large fragment sAPPα in the lumen, and the C-terminal fragment (CTF83) remains in the membrane. Two membrane endoproteases β- and γ-secretase sequentially hydrolyze APP. Firstly, APP releases sAPPβ by the action of β-secretase in the extracellular space. A fragment of 99 amino acids, CTFβ, remains bound to the membrane. CTFβ is successively and rapidly processed by γ-secretase generating Aβ. A precise cleavage site was not defined; therefore, Aβ is characterized by heterogeneity at the C-terminal and the peptide can end at position 40 (Aβ40) with a high frequency of occurrence (~80–90%) or at position 42 (Aβ42, ~5–10%). It is well established that Aβ42 generally generates fibrils more quickly than Aβ40 [44]. The production of Aβ is a normal metabolic event; in fact, these species are found in the cerebrospinal fluid and the plasma in healthy subjects [45]. Their abnormal accumulation, deriving from an imbalance between the production and clearance of these peptides, is associated with the pathogenesis of AD. Monomer, oligomer, and fibril forms of Aβ are differently involved in the onset of AD. The most common hypothesis is the Aβ-amyloid cascade [46]. The overproduction or the reduced clearance of Aβ leads to the deposition of fibrillar Aβ in the

*Sequence and structural domain organization for Aβ (A), tau (B), and Syn (C). For Aβ, the residues 12–24 and 30–40 involved in the formation of a cross-β fibril structure are highlighted and connected by dashed lines. In (B), the longest isoform (441 residues) of tau is shown, where N indicates the possible N-terminal insertion defining other isoform, PRR, the proline-rich region, target of phosphorylation (P), and MTBR, the microtubule binding region that can contain three or four repeats (R), and other phosphorylations (P) occur at the C-terminal. In the case of Syn (C), the N- and C-terminals and NAC domains are shown, as well as the position of the mutations responsible for familiar form of PD. Residues 1–95 form the lipid-*

*DOI: http://dx.doi.org/10.5772/intechopen.89575*

*Polyphenols as Potential Therapeutic Drugs in Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89575*

#### **Figure 3.**

*Neuroprotection - New Approaches and Prospects*

suggest that oligomers are the species responsible for the cytotoxicity. There are many proofs in support of this hypothesis, but unfortunately, due to the extreme heterogeneity in oligomer structures and their transient nature, a conclusive view has not been obtained yet [31–33]. The atomic structure of fibrils has been studied by several biophysical techniques. A quite accepted hypothesis agrees with the presence of a common molecular organization independent from the original structure of the involved protein: repetitive β-sheet units parallel to the fibril axis with their strands perpendicular to it [34, 35]. Amyloid fibrils can self-assemble *in vitro* from many structurally different proteins and peptides, not necessarily involved in diseases. It has been postulated that the cross-β structure represents a generic conformation, which represents another folding state for proteins [36, 37]. In addition to these characteristics, there are also some common aspects in the onset of the diseases. Several studies suggest possible interplays and synergistic activities between the involved proteins. Clinton et al. [38] provided evidence that Aβ, tau, and Syn could interact *in vivo* to promote their self-aggregation, thus accelerating the cognitive dysfunction [38]. High levels of Syn were found in patients suffering from AD [39]. Aβ stimulates Syn fibril formation in the transgenic mouse model through a seed mechanism [40]. In another study, Syn seems to inhibit the

*Scheme of the aggregation process of amyloid proteins. The formation of fibrils occurs through a nucleationdependent pathway starting from the monomeric form of the protein and leading to fibril elongation through intermediates (oligomers and protofibrils). The formation of the nucleus is the rate limiting step, and at this stage, the protein has acquired an aggregation-prone conformation. Fibrils are composed of a β-sheet structure in which hydrogen bonding occurs along the length of the fibril, and the β-strands run perpendicular to the fibril axis.*

The Aβ peptide was found in the amyloid plaques in 1984 [3]. Aβ represents a group of peptides constituted by 37–49 residues (**Figure 3A**), derived from the proteolytic processing of the amyloid precursor protein (APP) [42, 43] (**Figure 4**).

deposition of Aβ into the amyloid plaques [41].

**2.2 Key proteins in neurodegeneration**

**40**

*2.2.1 Aβ peptide*

**Figure 2.**

*Sequence and structural domain organization for Aβ (A), tau (B), and Syn (C). For Aβ, the residues 12–24 and 30–40 involved in the formation of a cross-β fibril structure are highlighted and connected by dashed lines. In (B), the longest isoform (441 residues) of tau is shown, where N indicates the possible N-terminal insertion defining other isoform, PRR, the proline-rich region, target of phosphorylation (P), and MTBR, the microtubule binding region that can contain three or four repeats (R), and other phosphorylations (P) occur at the C-terminal. In the case of Syn (C), the N- and C-terminals and NAC domains are shown, as well as the position of the mutations responsible for familiar form of PD. Residues 1–95 form the lipidbinding region.*

APP is a single membrane-spanning domain protein, containing a large extracellular glycosylated N-terminus and a shorter cytoplasmic C-terminus. The enzymatic processes responsible for the release of Aβ from APP are to date well elucidated [2]. Specifically, APP undergoes several proteolytic cleavages. The processing by α-secretase results in the release of the large fragment sAPPα in the lumen, and the C-terminal fragment (CTF83) remains in the membrane. Two membrane endoproteases β- and γ-secretase sequentially hydrolyze APP. Firstly, APP releases sAPPβ by the action of β-secretase in the extracellular space. A fragment of 99 amino acids, CTFβ, remains bound to the membrane. CTFβ is successively and rapidly processed by γ-secretase generating Aβ. A precise cleavage site was not defined; therefore, Aβ is characterized by heterogeneity at the C-terminal and the peptide can end at position 40 (Aβ40) with a high frequency of occurrence (~80–90%) or at position 42 (Aβ42, ~5–10%). It is well established that Aβ42 generally generates fibrils more quickly than Aβ40 [44]. The production of Aβ is a normal metabolic event; in fact, these species are found in the cerebrospinal fluid and the plasma in healthy subjects [45]. Their abnormal accumulation, deriving from an imbalance between the production and clearance of these peptides, is associated with the pathogenesis of AD. Monomer, oligomer, and fibril forms of Aβ are differently involved in the onset of AD. The most common hypothesis is the Aβ-amyloid cascade [46]. The overproduction or the reduced clearance of Aβ leads to the deposition of fibrillar Aβ in the

#### **Figure 4.**

*Scheme of metabolism of APP and accumulation of the Aβ peptide. Aβ1–40/42 peptides are released from APP by the action of two membrane endoprotease β- and γ-secretases. Firstly, APP releases sAPPβ by the action of β-secretase in the extracellular space, and a fragment of 99 amino acids, CTFβ, remains bound to the membrane. CTFβ is successively and rapidly processed by γ-secretase generating Aβ peptides. Under physiological conditions, Aβ1–40/42 are degraded by enzymatic clearance processes. The proteolytic pathway mediated by α-secretase is also shown.*

brain, determining synaptic and neuronal toxicity and thus neurodegeneration. There are many evidences in support of the so-called Aβ-amyloid oligomer hypothesis [31]. The proteolytic degradation of Aβ is a major route of clearance. Neprilysin (NEP) is considered one of the most important endopeptidase for the control of cerebral Aβ levels [47, 48] and for the degradation of some vasoactive peptides including natriuretic peptides and neuropeptides. Aβ clearance is mediated by other proteolytic enzymes such as apolipoprotein E (apoE) [49] and by autophagy [50]. Reduced activity of the clearance enzymes, which could be caused by aging, can contribute to AD development by promoting Aβ accumulation.

The secondary and tertiary structure of Aβ in solution has been studied by several biophysical techniques. These conformational studies are difficult for the protein high tendency to aggregate in solution. However, Aβ seems to populate distinct states in solution and to adopt a collapsed-coil structure, as deduced by NMR studies [51, 52]. Aβ preferentially binds to negatively charged lipids and acquires α-helical structure in the presence of membranes, membrane-like systems, and fluorinated alcohols [53, 54]. In the presence of phospholipids, Aβ undergoes conformational transition and forms β-sheets [55, 56]. Oligomeric Aβ binds to membranes with high affinity. Upon interaction, a membrane damage can occur as causative of the cellular toxicity [57]. It seems that especially oligomeric Aβ can disrupt the membrane bilayer by a detergent mechanism [58].

#### *2.2.2 Tau*

Tau is a neuronal protein associated with the microtubules [59]. Six Tau isoforms, which differ only in their primary structure, were detected in the human

**43**

*Polyphenols as Potential Therapeutic Drugs in Neurodegeneration*

brain and central nervous system (**Figure 3B**), while in the peripheral nervous system other Tau isoforms were also found [60]. The longest isoform contains 441 residues and the shortest 352 residues [61]. Depending on the isoform, the N-terminal can contain 0, 1, or 2 inserts (N). The protein appears largely posttranslational modified, especially in terms of phosphorylation (P). Other modifications are acetylation, deamidation, methylation, glycosylation, or ubiquitination [59]. Tau proteins are also subjected to proteolytic degradation that seems to be correlated with AD [62]. The region PRR (proline-rich region) contains the main sites of phosphorylation. Although all the post-translational modifications seem to contribute to the physiological and pathological properties of Tau, the signaling cascades and the effect on protein kinases and phosphatases are not completely clarified yet. The region 244–369 (microtubule binding region, MTBR) is responsible for the binding to the microtubule and contains three or four repeats (R1-R4). Physiologically, Tau stabilizes the microtubule through MTBR, and such binding is modulated by the coordinated actions of kinases and phosphatases. Structurally, Tau belongs to the intrinsically disordered proteins, lacking a well-defined secondary and tertiary structure [59] and can interact with several other proteins. Upon aggregation, Tau can form dimers, oligomers, and larger polymers. In such aggregates, cysteine residues may play an important role [63]. Similarly, to other proteins involved in neurodegeneration, the oligomeric forms have a cytotoxic effect and might be involved in the Tau-related pathogeneses [64]. In neurofibrillary tangles, Tau forms the so-called paired helical filaments (PHFs) and straight filaments (SFs) [65, 66]. In PHF, Tau is ∼three to four-fold more hyperphosphorylated than in the normal brain. The Tau filaments exhibit the typical cross-β structure found in other types of fibrils [67].

Syn is a small protein (14.4 kDa) mainly expressed in pre-synaptic nerve terminals of the central nervous system and very abundant in erythrocytes and platelets [68]. Despite the intensive investigation and the discovery that the protein plays a central role in synaptic transmission and vesicle recycling [69], the complete Syn biological function remains still elusive. Syn may control the neurotransmitter release, promoting the formation and assembly of the SNARE complex [70, 71]. Syn structure could be divided into three main domains: N-, central, and C-terminals (**Figure 3C**). The N-terminal region (amino acids 1–60) contains seven imperfect repeats, with a hexameric consensus motif (KTKGEV). All the known missense mutations of Syn, responsible for the familiar forms of PD, are located in this region (**Table 1**). The central hydrophobic domain (amino acids 61–95) is known as the non-amyloid-β component of AD amyloid plaques (NAC). It is responsible for Syn amyloid aggregation [72]. N-terminal and NAC domains together (amino acids 1–95) mediate the interaction of Syn with lipids, membranes, and fatty acids [73]. The C-terminal domain (amino acids 96–140) is an acidic, negatively charged, highly soluble, and disordered tail, target of post-translational modifications. This region plays a series of important roles, modulates Syn binding to membrane and metals, Syn aggregation and its protein-protein interaction properties. The deletion

of this domain increases the aggregation rate of Syn *in vitro* and in cells [74].

Syn is the prototype of the natively unfolded proteins, but adopts a stable secondary structure as a function of the environment [75]. Multiple studies have demonstrated that Syn is more compact than expected for a random coil due to long-range interactions between the C-terminal tail and the NAC domain as well as electrostatic interactions between the N terminus and the C terminus [76]. Syn is supposed to populate different conformers in solution and can undergo conformational transition as a function of the environment and/or upon binding. The extreme Syn conformational

*DOI: http://dx.doi.org/10.5772/intechopen.89575*

*2.2.3 α-Synuclein (Syn)*

#### *Polyphenols as Potential Therapeutic Drugs in Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.89575*

brain and central nervous system (**Figure 3B**), while in the peripheral nervous system other Tau isoforms were also found [60]. The longest isoform contains 441 residues and the shortest 352 residues [61]. Depending on the isoform, the N-terminal can contain 0, 1, or 2 inserts (N). The protein appears largely posttranslational modified, especially in terms of phosphorylation (P). Other modifications are acetylation, deamidation, methylation, glycosylation, or ubiquitination [59]. Tau proteins are also subjected to proteolytic degradation that seems to be correlated with AD [62]. The region PRR (proline-rich region) contains the main sites of phosphorylation. Although all the post-translational modifications seem to contribute to the physiological and pathological properties of Tau, the signaling cascades and the effect on protein kinases and phosphatases are not completely clarified yet. The region 244–369 (microtubule binding region, MTBR) is responsible for the binding to the microtubule and contains three or four repeats (R1-R4). Physiologically, Tau stabilizes the microtubule through MTBR, and such binding is modulated by the coordinated actions of kinases and phosphatases. Structurally, Tau belongs to the intrinsically disordered proteins, lacking a well-defined secondary and tertiary structure [59] and can interact with several other proteins. Upon aggregation, Tau can form dimers, oligomers, and larger polymers. In such aggregates, cysteine residues may play an important role [63]. Similarly, to other proteins involved in neurodegeneration, the oligomeric forms have a cytotoxic effect and might be involved in the Tau-related pathogeneses [64]. In neurofibrillary tangles, Tau forms the so-called paired helical filaments (PHFs) and straight filaments (SFs) [65, 66]. In PHF, Tau is ∼three to four-fold more hyperphosphorylated than in the normal brain. The Tau filaments exhibit the typical cross-β structure found in other types of fibrils [67].

#### *2.2.3 α-Synuclein (Syn)*

*Neuroprotection - New Approaches and Prospects*

brain, determining synaptic and neuronal toxicity and thus neurodegeneration. There are many evidences in support of the so-called Aβ-amyloid oligomer hypothesis [31]. The proteolytic degradation of Aβ is a major route of clearance. Neprilysin (NEP) is considered one of the most important endopeptidase for the control of cerebral Aβ levels [47, 48] and for the degradation of some vasoactive peptides including natriuretic peptides and neuropeptides. Aβ clearance is mediated by other proteolytic enzymes such as apolipoprotein E (apoE) [49] and by autophagy [50]. Reduced activity of the clearance enzymes, which could be caused by aging, can

*Scheme of metabolism of APP and accumulation of the Aβ peptide. Aβ1–40/42 peptides are released from APP by the action of two membrane endoprotease β- and γ-secretases. Firstly, APP releases sAPPβ by the action of β-secretase in the extracellular space, and a fragment of 99 amino acids, CTFβ, remains bound to the membrane. CTFβ is successively and rapidly processed by γ-secretase generating Aβ peptides. Under physiological conditions, Aβ1–40/42 are degraded by enzymatic clearance processes. The proteolytic pathway* 

The secondary and tertiary structure of Aβ in solution has been studied by several biophysical techniques. These conformational studies are difficult for the protein high tendency to aggregate in solution. However, Aβ seems to populate distinct states in solution and to adopt a collapsed-coil structure, as deduced by NMR studies [51, 52]. Aβ preferentially binds to negatively charged lipids and acquires α-helical structure in the presence of membranes, membrane-like systems, and fluorinated alcohols [53, 54]. In the presence of phospholipids, Aβ undergoes conformational transition and forms β-sheets [55, 56]. Oligomeric Aβ binds to membranes with high affinity. Upon interaction, a membrane damage can occur as causative of the cellular toxicity [57]. It seems that especially oligomeric Aβ can

Tau is a neuronal protein associated with the microtubules [59]. Six Tau isoforms, which differ only in their primary structure, were detected in the human

contribute to AD development by promoting Aβ accumulation.

disrupt the membrane bilayer by a detergent mechanism [58].

**42**

*2.2.2 Tau*

**Figure 4.**

*mediated by α-secretase is also shown.*

Syn is a small protein (14.4 kDa) mainly expressed in pre-synaptic nerve terminals of the central nervous system and very abundant in erythrocytes and platelets [68]. Despite the intensive investigation and the discovery that the protein plays a central role in synaptic transmission and vesicle recycling [69], the complete Syn biological function remains still elusive. Syn may control the neurotransmitter release, promoting the formation and assembly of the SNARE complex [70, 71]. Syn structure could be divided into three main domains: N-, central, and C-terminals (**Figure 3C**). The N-terminal region (amino acids 1–60) contains seven imperfect repeats, with a hexameric consensus motif (KTKGEV). All the known missense mutations of Syn, responsible for the familiar forms of PD, are located in this region (**Table 1**). The central hydrophobic domain (amino acids 61–95) is known as the non-amyloid-β component of AD amyloid plaques (NAC). It is responsible for Syn amyloid aggregation [72]. N-terminal and NAC domains together (amino acids 1–95) mediate the interaction of Syn with lipids, membranes, and fatty acids [73]. The C-terminal domain (amino acids 96–140) is an acidic, negatively charged, highly soluble, and disordered tail, target of post-translational modifications. This region plays a series of important roles, modulates Syn binding to membrane and metals, Syn aggregation and its protein-protein interaction properties. The deletion of this domain increases the aggregation rate of Syn *in vitro* and in cells [74].

Syn is the prototype of the natively unfolded proteins, but adopts a stable secondary structure as a function of the environment [75]. Multiple studies have demonstrated that Syn is more compact than expected for a random coil due to long-range interactions between the C-terminal tail and the NAC domain as well as electrostatic interactions between the N terminus and the C terminus [76]. Syn is supposed to populate different conformers in solution and can undergo conformational transition as a function of the environment and/or upon binding. The extreme Syn conformational

flexibility is responsible for its multifunctional properties, its capability to adopt different conformations, and to interact with different systems and other proteins [77]. For example, the interaction of Syn with negatively charged membranes, vesicles, bilayers, and lipids in general has important physiological consequences [78, 79], corroborating the hypothesis that Syn functions are correlated with lipids [80].
