**1. Introduction**

Alzheimer's disease (AD) is a complex and irreversible neurodegenerative disorder character‐ ized by a progressive memory and cognitive impairment. AD patients present a deficiency in short‐term memory and problem‐solving skills, affecting his daily activities and quality of life [1]. According to the World Alzheimer's report, this pathology comprises over the 60% of all causes of dementia, and they estimate that there are around 46.8 million people living with the disease at 2015. Because of their importance in public health, it is necessary to study the causes, diagnosis methods and possible treatments of this pathology [1]. AD is pathologically charac‐ terized by the presence of extracellular deposition of Aβ in the brain called senile plaques and intracellular neurofibrillary tangles (NFTs) containing pathological forms of tau protein [2]. Several studies had shown that these aggregates and its precursors induce neuronal dysfunc‐ tion, leading to the memory and cognitive impairment [3]. Interestingly, in cellular and animal models of AD in which Aβ, tau or both pathological aggregates have been induced, impair‐ ment of mitochondrial function even prior to the characteristic establishment of NFTs and Aβ plaques [4] is shown.

Mitochondria are cellular organelles required for energy and bioenergetics processes and it is also involved in amino acid and lipid metabolism, calcium homeostasis, free radical produc‐ tion and apoptosis [5]. In the brain, mitochondria are involved in energy supply, antioxidant defences, vesicle transport and synaptic communication [4]. These defects could lead to the memory and cognitive impairment seen in AD patients [4].

In this chapter, we discuss the pathways involved in mitochondrial dysfunction observed in different animal and cellular models of AD. These alterations in mitochondrial function include: mitochondrial dynamics, bioenergetics and mitochondrial axonal transport [4]. All these mitochondrial defects lead to an impaired neuronal communication and that could explain the cognitive and memory failure seen in AD [2]. Also in this chapter, we discuss new strategies to diminish mitochondrial injury in AD, in order to ameliorate the pathology progression of this disease.

The references and articles utilized in the development of this chapter were obtained using online compressive search engines like PUBMED and MEDLINE. Scientific articles were obtained from the online subscription services provided by Universidad Autónoma de Chile.

#### **1.1. Defects of mitochondrial dynamics in AD**

Mitochondria is a versatile organelle that forms an intracellular network that undergoes continuous fission and fusion processes named mitochondrial dynamics [6]. This process plays a crucial role in the control of mitochondrial shape, size and number, which influences important mitochondrial properties including bioenergetics and quality control [7]. Mito‐ chondrial fusion serves to unify the mitochondrial compartment, and mitochondrial fission contributes to the removal of damaged organelle via mitophagy and may facilitate apoptosis in conditions of cellular stress [8]. Generally, mitophagy is initiated when mitochondrial membrane potential is compromised [9]. Under this condition, the phosphatase and tensin homolog induced protein kinase 1 (PINK1) and Parkin complex ubiquitinates the mitochon‐ drial outer membrane proteins called, mitofusins, leading to mitochondrial fragmentation and recruitment of optineurin [9]. This process induces recruitment of the autophagy‐related binding protein LC3 (microtubule‐associated protein light chain 3) that promotes nucleation of the autophagosome leading to mitochondrial degradation [9]. Defects in mitochondrial dynamics have been linked to several diseases, and particularly important is the process in neurons [10]. Neurons' requirements are extremely unique, because of their dependence on energy production from mitochondria, which are needed in the synaptic process [8].

**1. Introduction**

350 Update on Dementia

plaques [4] is shown.

progression of this disease.

**1.1. Defects of mitochondrial dynamics in AD**

Alzheimer's disease (AD) is a complex and irreversible neurodegenerative disorder character‐ ized by a progressive memory and cognitive impairment. AD patients present a deficiency in short‐term memory and problem‐solving skills, affecting his daily activities and quality of life [1]. According to the World Alzheimer's report, this pathology comprises over the 60% of all causes of dementia, and they estimate that there are around 46.8 million people living with the disease at 2015. Because of their importance in public health, it is necessary to study the causes, diagnosis methods and possible treatments of this pathology [1]. AD is pathologically charac‐ terized by the presence of extracellular deposition of Aβ in the brain called senile plaques and intracellular neurofibrillary tangles (NFTs) containing pathological forms of tau protein [2]. Several studies had shown that these aggregates and its precursors induce neuronal dysfunc‐ tion, leading to the memory and cognitive impairment [3]. Interestingly, in cellular and animal models of AD in which Aβ, tau or both pathological aggregates have been induced, impair‐ ment of mitochondrial function even prior to the characteristic establishment of NFTs and Aβ

Mitochondria are cellular organelles required for energy and bioenergetics processes and it is also involved in amino acid and lipid metabolism, calcium homeostasis, free radical produc‐ tion and apoptosis [5]. In the brain, mitochondria are involved in energy supply, antioxidant defences, vesicle transport and synaptic communication [4]. These defects could lead to the

In this chapter, we discuss the pathways involved in mitochondrial dysfunction observed in different animal and cellular models of AD. These alterations in mitochondrial function include: mitochondrial dynamics, bioenergetics and mitochondrial axonal transport [4]. All these mitochondrial defects lead to an impaired neuronal communication and that could explain the cognitive and memory failure seen in AD [2]. Also in this chapter, we discuss new strategies to diminish mitochondrial injury in AD, in order to ameliorate the pathology

The references and articles utilized in the development of this chapter were obtained using online compressive search engines like PUBMED and MEDLINE. Scientific articles were obtained from the online subscription services provided by Universidad Autónoma de Chile.

Mitochondria is a versatile organelle that forms an intracellular network that undergoes continuous fission and fusion processes named mitochondrial dynamics [6]. This process plays a crucial role in the control of mitochondrial shape, size and number, which influences important mitochondrial properties including bioenergetics and quality control [7]. Mito‐ chondrial fusion serves to unify the mitochondrial compartment, and mitochondrial fission contributes to the removal of damaged organelle via mitophagy and may facilitate apoptosis in conditions of cellular stress [8]. Generally, mitophagy is initiated when mitochondrial membrane potential is compromised [9]. Under this condition, the phosphatase and tensin

memory and cognitive impairment seen in AD patients [4].

Mitochondrial biogenesis occurs to supply cellular energy through the fission of preexisting mitochondria followed by growth [11]. Little is known about the regulatory mechanisms of mitochondrial biogenesis in mammalian neurons under physiological or pathological condi‐ tions. However, these processes quickly respond to changes due to mitochondrial damage or increased stimulation of PGC‐1 α, Nrf1/2 and TFAM pathways [5]. Interestingly, expression levels of those proteins were significantly decreased in both AD hippocampal tissue and a neuronal cell line with overexpression of Swedish mutant forms of APP protein (APPswe), suggesting that mitochondrial biogenesis was affected during neurodegeneration and contributes to mitochondrial dysfunction in AD [12].

On the other hand, mitochondrial dynamics depends on the interaction of different proteins within the mitochondrial membranes [13, 14]. Mitochondrial fission depends on dynamin‐ related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1) [6]. Drp1 is mainly located in cytoplasm and is recruited by Fis1 that is in the mitochondrial outer membrane [14]. Then Drp1 by its guanosine triphosphatase (GTPase) activity assembles itself constricting mito‐ chondrial membrane until the formation of two daughter mitochondrias [15]. Moreover, fusion of the mitochondria is control by optic atrophy protein (Opa1) and both, mitofusins 1 and 2 (Mfn1 and Mfn2) [16]. This fusion of outer mitochondrial membrane is mediated by the concerted GTPases actions of Mfn 1 and Mfn 2, and fusion of the inner membranes are mediated by Opa1 through its proteolytic processing [4, 7].

Several studies showed that mitochondrial morphological changes are present in AD [17, 18]. Brain‐derived mitochondria from AD patients are smaller and more fragmented compared to age‐matched individuals [19], and reduced mitochondrial density in synaptic structures and shorter mitochondria in brain axons were found in mouse overexpressing APP/Aβ (mAPP transgenic mouse) [20]. In different neuronal cell models treated with Aβ or with overexpres‐ sion of Swedish mutant forms of APP protein, mitochondria present changes in their structure: a fragmented and punctiform form and a reduction of mitochondrial density in neurites [19– 21]. On the other hand, tau also has a role on the Aβ‐induced mitochondrial impairment. In mature neurons, it has been shown that truncated and pseudo‐phosphorylated forms of tau mediates mitochondrial shortening, reducing mitochondrial movement and mitochondrial potential and increasing superoxide levels induced by Aβ [22–24]. All this morphological changes are related to changes in mitochondrial dynamics.

An increase in Fis1 protein expression and a reduced expression of Drp1, Mfn1, Mfn2 and Opa1 in the cytosolic fraction was found in post‐mortem brain tissue and neuroblastoma cell line M17 treated with amyloid‐β‐derived diffusible ligands (ADDLs) [19]. However, Drp1 expression was increased in brain frontal cortex from AD patients [25], suggesting a deregu‐ lation of Drp1 activity associated with mitochondria [25]. Furthermore, it has been shown that oxidative stress‐mediated S‐nitrosylation of Drp1 induced by Aβ triggers mitochondrial fragmentation [26]. Interestingly, in another model of AD, N2a cells that expressed APP Swedish mutation, Aβ accumulation induced a decrease in both Mfn1 and Mfn2 levels, with a subsequent fragmentation of mitochondria [27]. On the other hand, in transgenic mouse models of AD a direct interaction between Drp1 and hyperphosphorylated tau has been found, suggesting a direct effect of tau on the mitochondrial dynamics dysfunction [28].

All these data suggest that tau pathology and Aβ impairs mitochondrial morphology even before the NFTs and senile plaques establishment. These are important features because a regulated fusion‐fission cycle is needed to maintain a healthy mitochondrial pool. In AD, mitochondrial biogenesis is impaired, mitophagy process is reduced and alterations in cycle of mitochondria dynamics generate mitochondrial fragmentation [9, 29]. Overall, these defects could be the cause of an increase in the number of damaged organelles in AD neurons and the source of mitochondrial bioenergetics dysfunction that this disease presents.

#### **1.2. Reduction of mitochondrial bioenergetics performance in AD**

The main function of the mitochondria is generating ATP [30]. In the organelles, the electron transport chain (ETC) is responsible for oxidative phosphorylation, which is the biochemical pathway that produces ATP by consuming oxygen [30]. The electrons pass through the respiratory complexes I–IV of the ETC and as a consequence, a membrane potential is generated for the electrochemical force of a proton gradient [30]. This process generates ATP by complex V, and this energetic molecule would help, among other things, to regulate the intracellular calcium homeostasis [4]. This process normally generates reactive oxygen species (ROS); however, oxidative stress occurs when the balance between the production of oxidants molecules and the endogenous antioxidant defences in cells is deregulated [31].

Bioenergetics damage includes low ATP production, failure in ETC, mitochondria depolari‐ zation, defects in calcium buffering capacity and increase of ROS [10, 18]. Mitochondria are the primary source of oxidative species, and mitochondria‐linked oxidative stress has been found to be a major factor associated with the development and progression of AD [31–33]. In fact, excessive generation of ROS contributes to neuronal dysfunction and bioenergetics failure in AD even before the appearance of Aβ plaques and NFTs [32, 34], thus supporting the hypothesis that mitochondrial failure is an early event in the AD progression.

In animal models of AD, several data suggest that the Aβ pathology is an important participant in mitochondrial bioenergetics dysfunction [35, 36]. Brain slices from APP/Aβ transgenic mice shows Aβ localization in mitochondria and increased levels of oxidative markers, carbonylated proteins and reduced cytochrome c oxidase (CoxIV or Complex IV) activity, suggesting increased oxidative stress and impaired mitochondrial metabolism in this AD model [32]. Besides, several experiments with neuronal cell lines treated with different forms of the Aβ peptide indicated that the treatment generate impairment of ETC, mitochondrial depolariza‐ tion and also, opening of mitochondrial permeability transition pore (mPTP) with the resulting calcium leaking and ROS production [36, 37].

Interestingly, studies have shown that the increased oxidative stress seen in AD could generate a vicious circle in which ROS promotes Aβ generation in in vitro and in vivo models [38]. For example, in brain mitochondria from a variant of APPswe mouse, mitochondrial depolariza‐ tion, low ATP levels and decreased cytochrome c oxidase activity have been found prior to Aβ plaque deposition [39]. Similar results were found in triple Tg (PS1M146V/APPSwe/ TauP301L) mice [40], suggesting that both Aβ and tau pathology present mitochondrial dysfunction prior to the formation of toxic protein aggregates [41, 42].

As we already discussed, neurons are particularly sensitive to mitochondrial dysfunction since they are extremely energy dependent with many cellular activities, such as synaptic trans‐ mission and axonal and dendritic transport [43, 44]. Therefore, it is proposed that mitochon‐ drial bioenergetics defects could be considered as a hallmark in AD, since there is evidence that is an early event in the progression of the disease.

#### **1.3. Mitochondria are not properly transported in AD**

expression was increased in brain frontal cortex from AD patients [25], suggesting a deregu‐ lation of Drp1 activity associated with mitochondria [25]. Furthermore, it has been shown that oxidative stress‐mediated S‐nitrosylation of Drp1 induced by Aβ triggers mitochondrial fragmentation [26]. Interestingly, in another model of AD, N2a cells that expressed APP Swedish mutation, Aβ accumulation induced a decrease in both Mfn1 and Mfn2 levels, with a subsequent fragmentation of mitochondria [27]. On the other hand, in transgenic mouse models of AD a direct interaction between Drp1 and hyperphosphorylated tau has been found,

All these data suggest that tau pathology and Aβ impairs mitochondrial morphology even before the NFTs and senile plaques establishment. These are important features because a regulated fusion‐fission cycle is needed to maintain a healthy mitochondrial pool. In AD, mitochondrial biogenesis is impaired, mitophagy process is reduced and alterations in cycle of mitochondria dynamics generate mitochondrial fragmentation [9, 29]. Overall, these defects could be the cause of an increase in the number of damaged organelles in AD neurons and the

The main function of the mitochondria is generating ATP [30]. In the organelles, the electron transport chain (ETC) is responsible for oxidative phosphorylation, which is the biochemical pathway that produces ATP by consuming oxygen [30]. The electrons pass through the respiratory complexes I–IV of the ETC and as a consequence, a membrane potential is generated for the electrochemical force of a proton gradient [30]. This process generates ATP by complex V, and this energetic molecule would help, among other things, to regulate the intracellular calcium homeostasis [4]. This process normally generates reactive oxygen species (ROS); however, oxidative stress occurs when the balance between the production of oxidants

Bioenergetics damage includes low ATP production, failure in ETC, mitochondria depolari‐ zation, defects in calcium buffering capacity and increase of ROS [10, 18]. Mitochondria are the primary source of oxidative species, and mitochondria‐linked oxidative stress has been found to be a major factor associated with the development and progression of AD [31–33]. In fact, excessive generation of ROS contributes to neuronal dysfunction and bioenergetics failure in AD even before the appearance of Aβ plaques and NFTs [32, 34], thus supporting the

In animal models of AD, several data suggest that the Aβ pathology is an important participant in mitochondrial bioenergetics dysfunction [35, 36]. Brain slices from APP/Aβ transgenic mice shows Aβ localization in mitochondria and increased levels of oxidative markers, carbonylated proteins and reduced cytochrome c oxidase (CoxIV or Complex IV) activity, suggesting increased oxidative stress and impaired mitochondrial metabolism in this AD model [32]. Besides, several experiments with neuronal cell lines treated with different forms of the Aβ peptide indicated that the treatment generate impairment of ETC, mitochondrial depolariza‐ tion and also, opening of mitochondrial permeability transition pore (mPTP) with the resulting

suggesting a direct effect of tau on the mitochondrial dynamics dysfunction [28].

source of mitochondrial bioenergetics dysfunction that this disease presents.

molecules and the endogenous antioxidant defences in cells is deregulated [31].

hypothesis that mitochondrial failure is an early event in the AD progression.

calcium leaking and ROS production [36, 37].

**1.2. Reduction of mitochondrial bioenergetics performance in AD**

352 Update on Dementia

Defects in axonal transport of mitochondria in AD have been reviewed by our group and others [4, 45]. The axonal transport comprises the action of motor proteins that carry organelles, vesicles and other proteins through microtubules [46]. Kinesins family protein commands anterograde transport (from cell body to terminals) and dynein‐dynactin complexes are responsible for the retrograde transport (from terminals to cell body) [46]. Also, each cargo proteins need adaptor proteins to bring specificity to the transport process such the Miro GTPase and trafficking kinesin (TRAK) family of proteins [46]. By the other hand, the docking protein syntaphilin helps mitochondria to stay at zones of higher energy demand, such as synaptic terminals, in a way to modulate the energy requirements of the neurons [47].

Studies on APPswe mice show reduced axonal transport in vivo [48]. Neurons from human APP Tg mice showed reduced moving mitochondria when they were treated with Aβ, and interestingly, knocking down of tau protein prevented this effect [49]. Inversely, neurons of tau knock out mouse transfected with wild‐type tau protein make these cells sensitive to Aβ, showing deficits in axonal transport [49, 50]. Also this group has suggested that GSK‐3β is involved in this mechanism due to its interaction with presenilin 1 (PS1) a transmembrane protein related with Aβ production [50]. Furthermore, in neurons from PS1‐/‐ [51] and PS1M146v, mutation related to familiar AD [52], mice show impaired anterograde axonal transport [53]. Also, in SH‐SY5Y neuroblastoma cells, it has been found that tau directly interacts with dynactin complexes suggesting a potential effect on retrograde axonal transport in tau pathology [54]. Complementary to these studies, the TPR50 transgenic mice that contain a human P301S tau, a tau gene mutant form found in frontotemporal dementia and parkinson‐ ism linked to chromosome 17 (FTDP‐17) [55], exhibited early cognitive impairment, reduced retrograde transport and increased kinesin protein expression [56].

Interestingly, Guo and coworkers found that reduction of cyclophilin D (CycD) prevented axonal transport impairment induced by Aβ [57]. CycD is a component of the mPTP located through the outer and inner mitochondrial membranes [58]. mPTP plays a key role in cell death inducing the release of cytochrome c, collapsing mitochondrial membrane potential and releasing calcium at the cytosol [58]. Furthermore, defects of mitochondrial dynamics and axonal transport induced by Aβ were prevented in CycD‐depleted neurons obtained from CycD knockout mice (Ppif‐/‐). In addition, restoration of mitochondrial dynamics was repli‐ cated using the CycD inhibitor cyclosporin A in the same neuronal model [57].

Overall, defects of mitochondrial transport through axons include the reduced anterograde or/and retrograde movement, increased stationary mitochondria and reduced mitochondrial density in synaptic terminals [45]. These alterations affect neuronal function including autophagy, vesicle transport and energy supply leading to synaptic failure [45].

Mitochondrial defects in dynamics, bioenergetics and transport are tightly related (**Figure 1**). Morphology alterations impair mitochondrial bioenergetics, and this deficiency generates fragmented and dysfunctional mitochondria. Also, defects in both transport and dysfunctional mitochondria could affect the energy and bioavailability of fresh mitochondria in demand zones such as nervous terminals. Together with an increased oxidative stress and reduced mitophagy may affect synaptic communication. Altogether, these alterations in mitochondrial health suggest the possibility that modulating mitochondrial function could be a key strategy to prevent or retard the progression of AD (**Figure 1**).

**Figure 1. Mitochondrial function defects in AD**. (**A**). **Dynamics/morphology**. The regulation of mitochondrial dy‐ namics, such as fusion, fission, biogenesis and mitophagy, represents an important mechanism that control neuronal fate. Mitochondrial morphological alterations are present in all levels in AD, and the consequence is the accumulation of fragmented and dysfunctional mitochondria in all the cell body (**B**). **Transport**. Kinesin and dynein proteins mediate axonal transport of mitochondria. Generally, this movement is bidirectional in an anterograde (kinesin) and retrograde direction (dynein). In several models of AD, a deregulated mitochondrial movement together with an increase of im‐ mobile mitochondria population associated with syntaphilin has been reported. This alteration generates a decrease in the total mitochondria movement and their distribution to the synaptic space (**C**) **Bioenergetics**. Neuronal models of AD present a severe mitochondrial dysfunction with an increase in oxidative stress. This alteration leads to a bioener‐ getic imbalance that affects ATP levels in the presynaptic neuron, with an increase in calcium overload and a conse‐ quent synaptic dysfunction.
