Molecular Basis of Alzheimer’s Disease: Focus on Mitochondria

Mitochondrial cascade hypothesis

The core principal behind research for mitochondria targeted molecules is the mitochondrial cascade hypothesis (MCH). Firstly, it is believed that all persons have a genetically inherited predisposition for mitochondrial function and dysfunction [9]. As shown in Fig. 1, mutant proteins association induce mitochondrial dysfunction. Important to note is that both parents will contribute nuclear DNA (nDNA) (which codes for cyclophilin D in the mitochondrial matrix and Complex II in the mitochondrial inner membrane), but the mitochondrial DNA (mtDNA) believed to be mostly contributed from the mother will contribute greater risk to mitochondrial dysfunction [10–12]. Recently, Luo and colleagues reported that mtDNA inheritance occurs by both parents, indicating that both parents contribute mtDNA to their offspring [13]. These recent findings are in disagreement with original findings of maternal inheritance of mtDNA [11]. However, research is needed to understand the molecular mechanism for this unusual mode of paternal inheritance, how mtDNA is passed on from parent to offspring and may lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

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Fig.1

Neurodegenerative disease and toxic proteins interaction with mitochondria. Mitochondrial damage and subsequent cell death may be attributed to aging and dysfunction resultant of interaction with toxic proteins in Alzheimer’s, Parkinson’s, and Huntington’s diseases, as well as Friedreich ataxia and amyotrophic lateral sclerosis.

Secondly, how these variances in genetics are expressed is determined by interaction between each person’s inherited characteristics and the environment [10]. Overtime, according to the MCH, an observable correlation between mitochondrial age-related dysfunction and brain aging should develop, should the person be genetically susceptible to developing mitochondrial dysfunction [10]. Lastly these interactions are the basis for development of neurodegenerative diseases and determine the variant of the disease, such as early and late onset AD [10]. The MCH thus explains that AD is initiated by mitochondria, and the change in mitochondrial function determines how fast and how severe the disease will be. The MCH provides the principles for researchers to analyze biomarkers that indicate neuronal aging and AD development, and it is with these biomarkers that researchers assure the effectiveness of therapeutics such as antioxidants and mitochondria targeted molecules [10].

Early contributions to the MCH were evidenced by correlation with mtDNA mutation and AD development [10]. Parker detailed how inherited mtDNA guides mitochondrial function and determines risk for AD [10]. Over a person’s lifetime, mtDNA mutations mount up, initiated by toxin induced mitochondrial dysfunction, which expedites the aging process, and initiates AD [10]. The mitochondrial dysfunction impacts the processing of amyloid-β protein precursor (AβPP), which in the presence of toxin-induced mitochondrial dysfunction produces Aβ peptides, and promotes Aβ aggregation [10]. Furthermore, data indicates that AβPP, Aβ, and the γ-secretase complex associate with mitochondria and cause mitochondrial dysfunction [10].

It is important to note that in geriatric populations, more persons have AD than do not have AD, but researchers believe that AD is separate from the normal aging process [10]. AD is not the norm, and everyone will not eventually develop AD should they live long enough [10]. Instead, brain aging may be subdivided into compensated and uncompensated aging [10]. AD would fall under uncompensated aging, where aging effects are not mitigated [10].

Furthermore, research with cytoplasmic hybrid (Cybrid) cell lines contributes a large amount of evidence toward the MCH [10]. Cybrids are eukaryotic cells produced by the fusion of whole cells with cytoplasts without nuclei [10]. Using this technology, with two different neuronal cell lines, SH-SY5Y neuroblastoma and NT2 teratocarcinoma cells, Swerdlow and team members were able to demonstrate significant differences in cytochrome c oxidase (COX, aka ETC complex IV) activity between those of AD and non-AD cells [14]. Since COX is encoded by mtDNA, and not nDNA, a conclusion could be made that mtDNA does play a role in biogenesis of AD cells [14]. Similar differences were noted in cybrid lines with cytoplasm from healthy and mild cognitive impairment (MCI) cells, where molecular differences continued after culturing [15]. MCI is considered an early stage of AD.

Swerdlow and other researchers also noted differences in several other biomarkers that show correlation, but are not necessarily conclusive of AD [10]. These include transcription factor A of mitochondria (TFAM), and AD biomarkers FDG PET, structural MRI, amyloid PET, CSF Aβ, tau, and phosphorylated tau levels [10]. These biomarkers establish that biogenesis, and toxin levels vary with age, and that mitochondrial function plays a central role. These biomarkers also serve as tools for researchers to measure pathogenesis of AD in experimentation, and development of therapeutic drugs [16–18].

Types of mtDNA damage

MtDNA is susceptible to damage, especially from ROS. Alkylation damage is common, and often due to endogenous S-adenosylmethionine (about 30% of which is located in cellular mitochondria) methylation of DNA, and may occur due to exogenous compounds, such as chemotherapeutic agents [11]. Mitochondrial DNA is susceptible to hydrolytic damage which may cause hydrolysis of glycosidic bonds and result in a-basic sites, or may result in hydrolytic deamination of bases, most often with cytosine [11]. Mitochondria may be more susceptible than other portions of the cell to damage from exogenous sources such as tobacco smoke and other chemicals, which may form carcinogen adducts [11]. mtDNA is also susceptible to endogenous sources of adducts such as estrogens [11]. Mismatched bases may result from replication errors from DNA polymerase γ (polymerase responsible for transcription and damage repair of mtDNA in mitochondria) or even oxidation of bases [11]. DNA strand breaks, which may be caused by noxious stimuli, can cause single or double stranded breaks, and determine the type of DNA repair pathway which may lead to additional excision of bases [11]. Oxidative damage, with which most scientific research is done, often is the result of ROS, which are mostly produced by mitochondria [11].

Collectively, strand breaks, a-basic sites, and oxidative DNA adducts are also polymerase-blocking lesions, which more than likely reduce mtDNA copy number [11]. mtDNA is actually more vulnerable to polymerase blocking lesions than nDNA [11].

Some mtDNA base damage is more susceptible to mutation than others. Among the most common is thymine glycol, a product of oxidative thymine base damage, which due to relatively efficient base excision repair excision has low mutagenicity, but temporarily inhibits polymerase-γ’s transcription ability [11]. Another is 7,8-dihydro-8-oxo-2’-deoxyguanosine, or 8-oxodG, which results from oxidative damage, and is mutagenic, causing G:T transversions, though less likely to block polymerase [11]. These differences, along with cytosine’s susceptibility to hydrolytic deamination alludes to differences in oxidative damage leading to mutation between persons with higher versus lower Guanosine-Cytosine base pair content [11].

Oxidative damage of mtDNA

As damage to mtDNA may result from AD pathology, it is very important to understand the mechanism by which mtDNA damage is done. For instance, ROS species are products of nine, or possibly more, enzyme systems in mitochondria, but researchers tend to focus on those in the electron transport chain (ETC) [11]. In addition, oxygen may be converted to hydrogen peroxide via reverse electron transport from complex II to complex I, though this is unlikely [11]. Commonly, electrons may escape the ETC and induce superoxide production, and likely may be exacerbated by mitochondrial permeability transition (MPT) pores induced by Aβ peptides [11]. Normally, superoxide formed outside the mitochondrion prohibits it from crossing its membrane, and damage from superoxide is also limited when its presence inactivates mitochondrial aconitase [11]. When superoxide inactivates mitochondrial aconitase the ETC is slowed down, due to decrease in complex I and III activity [11]. Thus, it may be likely that AD induced reduction in biogenesis may be a healthy reaction to excessive superoxide production.

The superoxide produced by the ETC normally is converted to peroxide, which is the most efficient molecule in inducing oxidative stress because it has a relatively stable structure, and may cross lipid membranes [11]. Peroxide actually does not directly attack mtDNA, but when in contact with a metal cation such as Fe²⁺, may proceed with a Fenton chemical reaction, producing •OH radical, which may rapidly cause mtDNA damage [11]. Researchers have found that damage caused by H₂O₂ was even more frequent in mtDNA than in nDNA, of which the most predominant form of oxidative damage was to the mtDNA sugar-phosphate backbone [11].

Mitochondrial DNA damage repair systems

There are several base repair pathways possible for mtDNA damage that are very similar to those of nDNA. With mtDNA mismatches caused by polymerase-γ slippage error, mismatch repair (MMR) exchanges the mismatched base with the correct base. The repair process depends on the Y-box binding protein (YB-1) and not MSH used in nDNA MMR [11]. It should be noted that in vitro study has shown that polymerase-γ transcription fidelity is reduced when oxidized dGTP content is low (0.06–0.6%), but in vivo mitochondrial oxidized guanine content may be up to 10%, which is indicative of guanine’s susceptibility for oxidation, as a major cause of mtDNA mutation [11].

A DNA strand break can be single stranded, which constitutes loss of one nucleotide, and warrants single stranded base repair, or double stranded, which constitutes a break in the phosphate backbone in both strands and often accompanied by the loss of one or more nucleotide warranting double stranded base repair. In both types of repair, there is damage at either the 5’ terminus or 3’ terminus [11]. In single strand breaks (SSB), H₂O₂ oxidative damage can cause the abortion of topoisomerase I activity, even more often than with double strand breaks (DSB) [11]. SSB repair is very similar to base excision repair and differs with enzymes such as aprataxin, TDP1, and APE, and does not use DNA glycosylases [11].

In repair of DSB, there may be homologous recombination (HR) or non-homologous end joining (NHEJ). Recombination of mtDNA is infrequent as it may result in mutations, and the resultant mutant mtDNA must then be isolated [11]. HR is dependent on mitochondrial copy number, which would allow for a template for a successful repair of the DSB. HR is also able to be conducted between one individual mitochondrion and another, due to the network of mitochondria which at times may have multiple mitochondrial recombination junctions and catenation [11]. Mammalian mtDNA may even use recombination dependent initiation for replication, which is evidence that the balance mitochondrial dynamics (fission/fusion/mitophagy) greatly affects mtDNA copy number, especially non-mutated mtDNA copy number [11]. Notable as well is the fact that there is a lack of extensive recombination junctions in human infant mtDNA [11]. This suggests that recombination likely increases with age, and that the success of a healthy network of mitochondria is dependent on this process [11]. How Aβ peptides and phosphorylated tau fibrils affect this process is likely key for the healthy mitochondrial bioenergetics and dynamics.

With NHEJ, mitochondrial enzymes may join blunt-end DNA fragments, and the accuracy of preserving functionality of the genes is low [11]. Restriction enzymes that cut around the DSB of the damaged mtDNA target preferential end points, and sometimes the majority of the damaged mtDNA is degraded altogether [11]. If excessive damage is done to mtDNA within a mitochondrial network, resultant cells may lack mtDNA altogether, which denotes and means by which AD pathology may lead to degeneracy of a mitochondrial network within a neuron [11].

The leading mtDNA damage repair system is base excision repair (BER) and is the most studied process thus far [11]. Most oxidative damage of mtDNA is repaired via BER, where damaged base(s) are excised and replaced with proper base sequence [11]. This is important as the most common product of mtDNA damage is 8-oxodG, which suggests that malfunctioning of BER systems in mitochondria with high Guanine mtDNA content may be more susceptible to mutation, fragmentation, and/or mitophagy [11]. Short-patch BER is initiated by DNA glycosylase which recognizes the damaged base, whereas Long-patch BER is initiated by an AP site (absence of purine or pyrimidine) [11]. In BER, once a damaged base is removed, polymerase-γ and DNA ligase IIIα (Lig 3) fill the gap with the correct base and seal the gap between the phosphate backbones [11]. It is important to note that experimental knockout of Ligase 3 has been shown to result in lower mtDNA count, and that researchers have successfully introduced exogenous mitochondrial targeted Ligase 3 to cells, which was shown to restore viability [11]. Thus, where AD pathology induces damage of endogenous Ligase 3, introduction of exogenous mitochondrial targeted Ligase 3 may be a plausible means for future restoration of mitochondrial viability in AD cells.

Degradation of mtDNA

Extensive research has been conducted on the efficacy of BER systems, and due to the excessive copies of mtDNA, research has strayed away from determining how mtDNA damage, leading to loss of mtDNA copy number may affect mitochondrial health and dynamics [11]. Loss of mtDNA copy number may be prevented by antioxidants such as coenzyme Q, vitamin E, melatonin, and mtDNA re-synthesis processes [11]. In an experiment with intra-gastric introduction of ethanol, approximately half of mtDNA copies in all organs sampled were lost, including heart, liver, brain, and skeletal muscles of mice; meanwhile antioxidant administration was able to prevent and loss [20]. This indicates that given the right toxic substance, loss of mtDNA copy number may be a legitimate threat to mitochondrial and cell viability, though antioxidants may succeed in restoration. Crawford et al. were also able to show that oxidative stress initiated by H₂O₂, propagated the influx of Ca²⁺ and resulted in subsequent degradation of mtDNA, reducing copy number [21]. These studies highlight two needed areas of future study, especially longitudinal study, analyzing the effects of oxidative stress induced by AD pathology, and mitochondrial degeneracy due to mtDNA degradation and reduction of copy number.

In experiments conducted by Alexeyev et al. when BER system was inhibited, excessive damaged mtDNA which exceeded a particular threshold level were degraded [11]. Their conformation in fact was altered to become linear, which likely exposed them to exonucleolytic degradation [11].

Mitochondrial DNA peroxidation damage in Alzheimer’s disease

Aging

In AD, in addition to mtDNA damage, nDNA damage results from oxidative stress. Considerable changes include the acceleration of the aging process, which manifests in several ways. Increased somatic mtDNA mutations, gene regulation, via methylation, which may result from oxidative stress, as well as resultant altered microRNA production, can affect the course of disease in AD [11, 25].

Experts estimate that more than 70% of miRNA transcribed in the body are in the brain, and under stressful conditions, these miRNA may upregulate p53 production [25]. The p53 protein is not only responsible for tumor suppression, but also expedition of the aging process which consequently provides the environment for AD development [25]. In addition, miR-26a regulates GSK-3β, which also expedites the aging process via gene regulation [25]. When mitochondrial antioxidants and cellular antioxidants are unable to fully detox their environment from ROS, oxidative stress results, and this is followed by aging and mitochondrial dysfunction [25].

Aging is a part of the normal human life cycle and is often coupled with gradual age-related disability [9]. These age-related disabilities include worsening muscle, skeletal, cardiovascular, and neuronal conditions, and diseases such as diabetes, cancer, and neurodegenerative disease [9]. The two most common neurodegenerative diseases, AD and Parkinson’s disease, occur mostly sporadically and susceptibility increases with age [9]. Thus, age is a strong correlative factor when considering the development of late-onset AD [26]. It is hypothesized that a main reason for late-onset AD is the dysfunction in ability for clearance of ROS species, which increases with age, damages mitochondria, reduces bioenergetics, and has been shown to reduce lifespan across many species [26]. Furthermore, phosphorylation of tau particles is known to increase with age, and thus age is strongly correlated with the development of debilitating neurofibrillary tangles in the brain [1]. It should also be noted that DNA damage repair systems’ efficiency wanes with age, and thus possibility of mutation of key genes, especially the AβPP gene, increases with age [9].

Gender

AD is two times more prevalent in women than men [27]. Menopause could be a factor and studies have shown a correlation between decline in estrogen levels and memory decline [27]. In laboratory studies estrogen reduces Aβ formation, inhibits phosphorylation of tau protein, and increases apolipoprotein expression, which suggests that postmenopausal estrogen replacement therapy may be beneficial for treatment of cognitive impairment [27].

Low testosterone levels also show a strong correlation with the development of AD [28, 29]. With men, andropause, a condition where there is a decline in testosterone most often with age, likely increases the risk for AD [28, 29]. In many cases, men with AD, have been documented to have metabolic syndrome up to a decade after first signs of low-testosterone [28, 29]. Even more so, a progression to T2D increases the loss of testosterone, which then in a cyclical fashion intensifies the effects of T2D [28, 29]. Studies have shown that testosterone therapy may alleviate and improve conditions stemming from T2D, metabolic syndrome, especially insulin resistance, excessive adipose tissue, and high cholesterol [28, 29]. Meanwhile, testosterone depletion therapy, in response to prostate cancer, has been shown to multiply the incidence and prevalence of T2D [28].

Amyloid-β association with mitochondria

The Reddy laboratory [35] and others [36–38] have established an Aβ association with mitochondria. This abnormal association induces free radical production, reduces mitochondrial ATP and mitochondrial enzyme activities, and increases in ROS production [35, 40].

Shi Du Yan’s group reported Aβ binds to the mitochondrial enzyme amyloid binding alcohol dehydrogenase (ABAD), inducing a perturbation of the enzyme, prohibiting ABAD from binding the coenzyme NAD⁺ and effectively inhibiting its enzymatic activity [37, 41]. The loss of enzymatic activity of ABAD leads to the loss of ability to remove toxic aldehydes, and alters mitochondrial membrane structural integrity, changing its permeability and compromising respiratory enzymes [2, 37].

Furthermore, it was found that Aβ interaction with mitochondrial matrix protein cyclophilin D (CypD) interferes with several mitochondrial pathways, including components of the ETC, and are directly responsible for reduction in bioenergetics [42]. CypD is an enzyme with peptidyl-prolyl isomerase (PPI) activity, able to change the conformation of peptide bonds around the rigid amino acid proline between cis and trans conformations [12]. These PPIs are able to augment phosphorylation of proteins as well, which likely affects tau function and structure [12]. The AβPP contains proline, but Aβ does not. It is believed that PPI phosphorylation at the Thr668 site of AβPP, and increases the production of Aβ [12]. CypD, aka Cyp40, encoded by PPID gene on chromosome 4 and located in the matrices of mitochondria, is believed to regulate the MPT pore opening and closing [12, 43]. When CypD, which associates strongly with Aβ, forms a CypD-Aβ complex, there is an increase in cellular ROS [12]. The resultant CypD-Aβ initiated oxidative damage is believed to strongly contribute to mitochondrial dysfunction in AD [12].

Aβ also exhibits its toxic effects by physically preventing the cytochrome c from affixing itself to cytochrome c oxidase [2, 45]. This prevents the cristae of mitochondria from folding to contain the complexes, and inhibits proper electron transport in the ETC [2, 45].

Phosphorylated tau interaction with mitochondria

Recently, the Reddy laboratory reported abnormal interaction of phosphorylated tau with Drp1, leading to increased Drp1 GTPase enzymatic activity and excessive mitochondrial fragmentation in AD [46].

Tau particles associate with and bind to microtubules within the cytosol of the cell [40]. These microtubules are necessary to give the cytoskeleton and the cell its structure, and are vehicles for mitochondria and other organelles within the cell to move about [40]. The tau particles normally provide stability to the microtubules but are regularly phosphorylated via oxidative phosphorylation during neural activity [3]. When they are hyperphosphorylated, they help to break up and depolymerize microtubules [8, 40]. There is a strong correlation between this process and neuron degeneration in AD [40]. Interaction between neurons of the hippocampus and glutamate result in sustained increases in Ca²⁺ which cause hyperphosphorylation of tau, and damage mitochondria [8]. Lipid peroxidation and ROS from mitochondria also phosphorylate tau, which may be exacerbated by SOD deficiency [8].

Mitochondrial dynamics

Fission enables mitochondria to remain a favorably small size for better mobility within the cell [49] (Fig. 5). Fusion enables mitochondria to attach to intracellular structures such as the endoplasmic reticulum [49]. Mitochondrial fusion also allows the mixture of the mitochondrial contents that dilute possible damaged components. Fission increases the surface area of the mitochondrial network, effectively augmenting the capability of aerobic glycolysis, while fusion provides more intracellular surface area for oxidative phosphorylation and fatty acid oxidation with the addition of more cristae on the inner mitochondrial membrane (IMM) [49].

Mitochondria are not static organelles. They have motility within the cell, powered by GTPases, and may change their structure and shape as well, via mitofusin proteins. These morphology changes are summarized as either fusion or fission [48]. Fission is the ability for a mitochondrion to divide into two or more independent mitochondria, while fusion is the ability for two or more mitochondria to fuse and become one [50, 51]. Fission and fusion are likely vestigial processes of the mitochondrion when its ancestral form was a separate organism, and these processes hold a similar purpose of reproduction of mitochondria today [11]. Throughout the lifespan of the mitochondrion, there is a build-up of damage. The network of mitochondria within a cell work together to salvage viable portions of a mitochondrion to form a separate independent mitochondrion via fission or fusion [48]. Meanwhile, the damaged portion is engulfed and discarded via mitochondrial autophagy, or mitophagy. Dysfunction in mitochondrial fusion or fission is also strongly connected to cancer, cardiovascular diseases, and neurodegenerative disease, such as AD [48].

The structure and function the IMM and outer mitochondrial membrane (OMM) of the mitochondrion are very different and require different mechanics and pathways for fission and fusion [1, 50]. Included among the proteins responsible for OMM fission are: the cytosolic GTPase Dynamin-related protein-1 (Drp1; also known as dynamin-like protein 1 or DLP1), Fission-1 (Fis1), Mitochondrial fission factor (MFF), two homologous proteins Mid49 and Mid51 (aka MIEF1) function as Drp1 receptors which recruit and assemble Drp1 on the OMM and TBC1D15 [1, 50]. Less is known about IMM fission factors [50]. Mitochondrial Protein 18 (MTP 18), is an 18kDa protein that likely interacts with Drp1, which produced in excess causes mitochondrial fragmentation, but diminution results in fusion [50, 51]. The short optic dominant atrophy 1 (S-OPA 1), protein, produced from the proteolysis of long OPA1 (L-OPA1) supports IMM fusion, also is regulated for IMM fission [1].

Inclusive of the fusion proteins are the outer mitochondrial membrane’s GTPase mitofusins (Mfn1 and Mfn2) and the optic atrophy 1 (OPA1) protein of the inner mitochondrial membrane, all these proteins are involved in mitochondrial fusion machinery [52]. Mfn1 and Mfn2 have catalytic domains that bind GTP at their N-terminals and transmembrane domains at the C terminals [52]. Loss of either Mfn1 or Mfn2 causes the mitochondrion to fragment [52]. The GTPase OPA1 may be spliced to form L-OPA1, and evidence shows that the regulation of the balance between L-OPA1 and OPA1 is used to maintain the balance between fission and fusion [52]. vThere are other proteins in addition to the aforementioned proteins necessary for fission/fusion, though many are still not fully understood [1].

When AD impairs mitochondrial dynamics, there is dysfunction in glucose utilization, ATP production, Ca²⁺ regulation, growth and differentiation of neuronal axons, and frequency of cellular apoptosis [8]. This dysregulation results in upregulation of fission factors, downregulation of fusion factors, as well as reduction in mitophagy [8].

Aβ and phosphorylated tau interaction with Drp1: impaired mitochondrial dynamics

Recently, the Reddy laboratory reported abnormal interaction of phosphorylated tau with Drp1, leads to increased Drp1 GTPase enzymatic activity and excessive mitochondrial fragmentation in AD [46] (Fig. 5). Reddy’s group also reported that Aβ and phosphorylated tau interaction with Drp1, causes additional mitochondrial dysfunction and synaptic damage in AD neurons [46, 47]. These mechanistic insights are well received and much appreciated in AD and mitochondrial fields.

The excessive fission disrupts the inter-mitochondrial connectivity, and disrupts axonal transport of mitochondria. The successive bioenergetics reduction due to this fragmentation then leads to synapse inhibition, neuronal damage, and cognitive deficiency in AD [2, 3]. It is thus strongly believed that the essential source of mitochondrial dysfunction in AD is the interaction between Aβ and Drp1 [3]. Aβ particles also stimulate release of ROS species, which in turn damage mtDNA, which induces the creation of additional Aβ particles [45].

Mitochondrial fragmentation

Fragmentation has numerous causes, including: 1) ROS interaction with fission proteins, especially GTPase Drp1 enzyme inducing mitochondrial fission; () interaction between Drp1 and mutant Htt, Aβ or DJ1/LRKK2 proteins which induce upsurge of GTPase Drp1 activities leading to fragmentation; 3) S-Nitrosylation of nitric oxide to cysteine thiol changes conformation of Drp1 enzyme to improve GTPase Drp1 activity, inducing mitochondrial fission; and 4) phosphorylation of Serine amino acids at 616, 585, and 637 sites within Drp1 changes GTPase activity, resulting in fission dysfunction [46, 53]. In AD, there is upregulation of Drp1, Fis1, MiD51, MiD49, and Mff which causes excessive division of mitochondria into fragments [8].

Coupled with the upregulation of fission factors is the downregulation of fusion factors such as Mfn1, Mfn2, and OPA-1 [8]. This inhibits mitochondria from forming larger mitochondria, creating bio-energetically inefficient mitochondria, and rendering the mitochondrial network incapable of recovering from fragmentation [8].

Mitophagy and Alzheimer’s disease

In addition to fission and fusion, damaged mitochondrial dynamics are mitigated with mitophagy. It is believed to occur at fertilization, throughout gestation, through the infant stage and the entirety of human life [8]. Mitophagy, which is autophagy of the mitochondrion, involves the engulfment by a vesicle and fusion with lysosomes for destruction of the mitochondrion. [8, 53]. The autophagy process is regulated by highly evolutionary conserved encoding and may be categorized as macroautophagy, chaperone-mediated autophagy, or microautophagy [8]. Macroautophagy is the autophagy of focus in mitophagy [8].

Mitophagy is governed by PTEN-induced putative kinase 1 or PINK1 and the ubiquitin ligase parkin [8, 53]. When damaged mitochondria experience depolarization of the IMM, the mitochondrial surface receptor protein PTEN-induced kinase 1 (PINK1) is triggered to change into its stable conformation, which then adheres to the OMM [8]. PINK1 recruits parkin to the OMM, and indicates to parkin to begin E3 ligase activity in the cytoplasm [8, 53]. Parkin in turn induces ubiquitination conformational changes of proteins in the OMM [8, 53]. These proteins are marked for recognition and bound by optineurin (OPTN), p62, NDP52, and NBR1 that usher the mitochondrion towards the mitophagy pathway [8]. Finally, this pathway recruits an autophagosome to engulf the mitochondrion, which will then fuse membranes with a lysosome, effectively destroying it and its contents [8, 53].

Neuroprotective behaviors, such as fasting caloric restriction and exercise, which also increase bioenergetics increase autophagy and mitophagy, whereas the converse, such as poor diet, excessive eating, and lack of exercise, reduce mitophagy [8]. When resources such as ATP are low, a mitochondrion is less likely to be targeted for mitophagy, and mitochondrial fusion is upregulated [8, 50]. In fact, reduction in mitophagy is a sign of aging, and combined with Aβ amyloidosis and phosphorylated tau particles, reduced mitophagy is a sign for AD [8].

Mitophagy is further inhibited by Aβ and phosphorylated tau, which induce oxidative damage and cellular deficits that result in reduction of Parkin and PINK1 proteins [8]. The reduction of Parkin and PINK1 reduces the number of completed mitophagy processes, which in turn contributes to increased Aβ and tau particles [8]. This AD induced impairment of mitophagy is also characterized with accumulation of dysfunctional lysosomes and autophagosomal vacuoles unable to recruit proteins to complete mitophagy, and accumulation of dysfunctional mitochondria in the neuronal soma, worsened by hyperphosphorylation of tau which inhibits mitochondrial axonal transport [8].

Mitochondrial membrane and membrane potential

The OMM contains many pores for proteins and ions to pass through. It surrounds the IMM, and is separated from it via the intermembrane space. The IMM contains high levels of cardiolipin, but lacks cholesterol, unlike other membranes, and thus is very rigid and difficult to permeate. Its surface contains many folds, compressed closely to form cristae [1, 54]. Within the cristae are the many transmembrane, peripheral, and integral proteins, which are particularly necessary for the ETC and oxidative phosphorylation [1, 54]. Contained within the IMM is the matrix, where the Kreb’s cycle takes place [1, 54].

The mitochondrion is the site for two important processes, the Krebs’s cycle (Citric Acid Cycle), and the ETC, coupled with oxidative phosphorylation by complex V or ATP synthase, to create ATP [1]. In the mitochondrial matrix, the Kreb’s cycle churns acetyl co-enzyme A (produced from pyruvate product of glycolysis in the cytosol) coupled with H₂O, and oxaloacetate through a process of addition and removal of carbon backbones to phosphorylate GDP (guanosine diphosphate) to GTP (guanosine triphosphate); and reduce NAD⁺ (nicotinamide adenine dinucleotide) to NADH, ubiquinone to ubiquinol, and flavin adenine dinucleotide (FAD) to the semiquinone, FADH₂ [49]. The aforementioned biomolecules are then oxidized by complexes I, III, and IV, of IMM [49, 54]. The complexes, along with cytochrome c, and quinone, operate the ETC along the IMM. Complex I or Complex II transfers two electrons to quinone, reducing it to ubiquinol, which in turn transfers electrons to complex III [26].

Amid transferring of electrons, the complexes pump the hydrogen ions (or protons) into the intermembrane space, creating a high proton gradient between the matrix and the intermembrane space [49]. This gradient, creates a strong negative potential across the IMM from 150 to 180 mV, and is the driving force for transmembrane enzyme ATP synthase to phosphorylate ADP to ATP [26]. The phosphorylation of ADP to ATP by ATP synthase is known as oxidative phosphorylation, and it is coupled by the reduction of O₂ to H₂O [26]. The following diagrams provide a general schematic for the Krebs’s cycle, ETC, and oxidative phosphorylation [49]. A side effect of mitochondria’s role to generate energy is the production of ROS in the process, which in excess have the potential to damage mitochondria and the cell [1]. Mitochondrial dysfunction from ROS damage is followed up with loss of membrane potential [54]. UCP2 reduces mitochondrial ROS by shifting the movement of protons into the matrix rather than the intermembrane space, effectively behaving like an antioxidant [49, 55]. Antioxidants such as superoxide dismutase, coenzyme Q, and vitamin E also aid to reduce damage by ROS [11].

Mitochondrial dysfunction

At the heart of disruption of mitochondrial respiration is the opening of the mitochondrial permeability transition pore [55]. The translocase of the OMM permits the Aβ peptide to enter the mitochondria, where it then travels to the IMM [56] (Fig. 3). Aβ polypeptide induces MPT pore widening when oxidized cardiolipin (oxidized by mitochondrial hydrogen peroxide, which is enhanced by the presence of cytochrome c) synergizes with calcium ion [55]. As the MPT pore widens, it disrupts the permeability barrier of the IMM and leads to drop in intermembrane proton gradient, intermembrane potential, and uncoupling of oxidative phosphorylation [55]. This MPT pore also allows cytochrome c and other pro-apoptotic proteins to escape into the cytosol and trigger the caspase cascade and cell death by apoptosis [8, 55]. Aβ manipulates enzyme activity of complexes I, II, and IV [55] (Fig. 4). Thus, disruption of mitochondrial membrane by Aβ interaction disrupts Ca²⁺ levels, mitochondrial dynamics, leading to fragmentation, dysfunction oxidative damage, and thus degradation of AD neuron structure and function [3, 8].

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Fig.3

Aβ interaction with cellular membrane. The change in Ca²⁺ gradient caused by the amyloid beta pore formation inhibits neurotransmission, and subsequently reduced cognition. The influx of Ca²⁺ also causes mitochondrial dysfunction, inducing the production of ROS, creating mPTP, disrupting the ETC, causing the outflow of ROS, which leads to neuronal apoptosis and cell death [2, 25].

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Fig.4

Alzheimer’s disease mitochondrion. AD initiation within the mitochondrion may be attributed to toxic protein interaction (Aβ and phosphorylated tau), chemical imbalance (reactive oxygen species overproduction, calcium ion imbalance) leading to membrane potential loss, and structural damage (lipid peroxidation, mitochondrial DNA damage, and cardiolipin loss).

Studies show that the loss of phospholipids in the IMM may be the starting point for defective complex IV, and reduction of mitochondrial bioenergetics [55]. Experimentally adding phospholipids to the IMM, after there has been a reduction in cytochrome C, reverses defectiveness of complex IV [55]. Out of the three main phospholipids in the IMM (phosphatidylethanolamine, phosphatidylcholine, and cardiolipin), cardiolipin is the only one not present in the other layers of the mitochondria [55]. Due to cardiolipin’s conical shape, it does not form a bilayer, but instead interacts with other lipids on the membrane, causing bends in the membrane’s shape, curving into cristae [55].

Cardiolipin plays a role in the management of the supercomplexes on the IMM, which are essential components of the ETC. If there is disorder in the supercomplex formation, then there will likely be an increase in ROS production from complex I [53, 55]. Cardiolipin is especially vulnerable to oxidative damage, and release of ROS from complex I would only lead to more reduction in cardiolipin in the IMM [53, 55]. Cardiolipin also helps to affix cytochrome c to the IMM, and aids electron transport between complexes III and IV [55].

Restoration of bioenergetics of mitochondria (particularly restoration of cytochrome c, and complexes I, III, IV; structure of mitochondrial cristae; flow of electrons through the ETC) will depend on restoration of cardiolipin in the IMM [55].

ATP levels

Neurons have a high demand for ATP, and a dormant neuron alone requires about 4.7 billion ATP molecules per second [1]. Glycolysis alone would not suffice for ATP production, so the network of mitochondria within neuronal cells is the main powerhouse for neuronal activities, including the synapses and maintenance of healthy cognition [1].

ATP production is a sign of mitochondrial health. ROS species not only damage mtDNA and alter mitochondrial membrane potential, but also may decrease ATP levels due to mutation of the ATP6 gene resulting in a dysfunctional Complex V or ATP synthase [49, 57].

As the functionality of the mitochondrion deteriorates, the efficiency at which it may produce ATP, the main biomolecule for storage and delivery of energy will decline. There are several causes, including lipid peroxidation of membranes, which causes pores to open up in the IMM, reducing the proton gradient, and ROS that manipulate mtDNA coding for ATP synthase [54]. Reduced ATP production leads to inadequate supply of energy to sustain the cell [54, 57]. A substantial reduction in ATP production is a tell-tale sign of AD pathology [54, 57].

Essential to ATP production is gene expression of cellular and mitochondrial DNA. The amino acid lysine (Lys or K) is an essential amino acid, and a component of many proteins in the body. Lysine’s positioning on histones of cellular DNA, sets it up for acetylation and/or deacetylation, a biomolecular mechanism for gene expression control [58]. The acetylation, addition of a negatively charged acetyl group to lysine’s amine moiety at the end of its R-group (ɛ, Epsilon amino group) effectively loosens up tightly wound, also negatively charged DNA via repulsion, readying it for transcription [58]. This is an essential step for the transcription of proteins functionality for metabolic processes, cellular transport, control of gene transcription, and signal transduction [58]. The lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) work together to conceal the ɛ-amine’s positive charge (acetylation) or expose it (deacetylation) [58]. The overall effect on the protein is a slight conformational change that reduces or increases the protein’s activity, affinity, structural integrity, or interaction with other proteins [58].

The most recognizable KAT/KDAC combination is of histone acetyltransferases (HAT) and histone deacetylases (HDACs) [58]. Sirtuins comprise a class of KDACs, are abbreviated SIRT and are numbered 1 through 7. Sirtuins 4 and 6 regulate apoptosis and DNA repair via catabolism of the coenzyme nicotinamide adenine dinucleotide (NAD⁺) into nicotinamide and 2’-O-acetyl-ADP-ribose and/or 3’-O-acetyl-ADP-ribose and function as ADP-ribosyl transferases to proteins [58]. SIRT4 and SIRT5 function mostly in the mitochondria of the cell, and it should be noted that balance between NAD⁺ and NADH during bioenergetics pathways affects SIRT4 activity [56]. SIRT4 and SIRT5 present minimal deacetylase activity [59]. SIRT5, performs demalonylation (particularly of lysine malonylation), desuccinylation (leading to activation of Cu/Zn SOD1), and glutarylation of mitochondrial proteins 1000 times greater of a ratio to its deacetylation activity [59]. Its desuccinylation activity in particular moderates mitochondrial physiology and biogenesis via promotion of respiration and ATP synthesis and its desuccinylation of SOD1 eliminates ROS [59]. SIRT4 monitors ATP balance and mitochondrial biogenesis [60] (Fig. 7).

Sirtuins 1, 2, 3, 5, and 7 function as deacetylases and can serve as transcription regulators that may cause histones to adhere tightly to DNA [58]. When ROS damage leads to DNA DSBs, SIRT1 and SIRT6 are activated for DNA repair [59]. SIRT1 is present mostly in the cytosol of cells in the brain, and functions to deacetylate PGC-1α, conjugating it into its active form [58]. PGC-1α, a regulator of mitochondrial biogenesis, is activated by brain-derived neurotrophic factor (BDNF), and operates as a coactivator varying signals with biological responses, allowing the cell to adapt to adjustments in energy demand [8, 58]. PGC-1α, also via coactivation, regulates nuclear respiratory factors (NRFs) which moderate transcription of nuclear-encoded mitochondrial structural protein, which are transported to a mitochondrion, and processed from their pre-protein form to final protein form within the mitochondrion [58]. These nuclear respiratory factors coactivated by PGC-1α also moderate the transcription of TFAM which is necessary for replication, transcription, and maintenance of health mtDNA [58]. BDNF is responsible for hippocampal synaptic plasticity, learning, memory, and neuronal stress resistance and impairment of its function is a marker of AD [8]. Studies have shown that dysfunction in BDNF signaling affects a cascade of dysfunction culminating in the synapse and causing neuronal degeneration in AD [8].

Aβ and sirtuin interaction

Limited interaction with transient Aβ results in the transcription of mitochondrial SIRT4 and SIRT5 [59]. Nicotinamide functions as an inhibitor of all sirtuins, while EX527 and AGK2 are inhibitors of SIRT1 and SIRT2, respectively [58]. Exercise, however, which stimulates neuronal activity may upregulate SIRT3, which induces neuroplasticity and cellular stress resistance [8] (Fig. 6).

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Fig.5

Mitochondrial dynamics. Fusion, fission, and mitophagy sustain a healthy network of intracellular mitochondria. These processes maintain mtDNA copy number, mtDNA fidelity, mitochondrial size, and bioenergetics.

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Fig.6

SIRT4 ADP-ribosyl transfer and inhibition of glutamine dehydrogenase (GDH). A) Multiple transaminase convert α-ketoglutarate to glutamate and GDH converts glutamate to α-ketoglutarate, while reducing NAD⁺ to NADH. B) Aβ upregulates SIRT4 activity, which converts NAD⁺ via ADP ribosylation to nicotinamide and 2-O-acetyl-ADP ribose and 3-O-acetyl-ADP ribose, reducing NAD⁺ availability for GDH. C) ADP ribosylation inhibits GDH, and reduces α-ketoglutarate necessary for citric acid cycle and bioenergetics of mitochondria.

Researchers have found that Aβ peptides may induce overexpression of PARP1 and cause neuronal death [59]. The PARP1 overexpression in particular leads to NAD⁺ depletion, which inhibits activity of SIRT1, particularly its ADP-ribosyltransferase activity [59].

It has been shown that inhibition of PARP1 may conversely lead to expression of SIRT1, which has been shown to promote expression of α-secretase, downregulation of BACE1, and reduced Aβ generation [59]. Furthermore, γ-secretase may also be regulated by SIRTs [59].

Free radical production

ROS species must be kept in balance, and their byproducts are often used as a sign of a healthy functioning cell [23]. Mitochondrial derived ROS initiate propagation of cytokines for inflammatory response and aid the abstraction of bacteria [47]. Hydrogen peroxide (H₂O₂) derived from the mitochondria suppresses M1 expression of macrophages and upregulates M2 activity of macrophages, supporting macrophage polarization [9, 57]. Mitochondrial ROS are needed for initiating NLRP3 inflammasome innate immunity response, particularly propagation of cytokines interleukin-1β and –18 [49]. Mitochondrial ROS from complex III of the ETC activates CD4⁺ of T Cells [48]. ROS from mitochondrial glycerol-3-phosphate dehydrogenase 2 form ubiquinol from ubiquinone in the IMM, which in turn propagates the production of ROS from complex I in the ETC, which also is important for T Cell stimulation [49, 53]. Mitochondrial ROS also augments B Cell activity through heme amalgamation, changing immunoglobulin function [49].

Nonetheless, imbalances in ROS caused by mitochondrial damage, and/or reduction in antioxidants or their related enzymes can cause overproduction of ROS [23, 57]. Overproduction of ROS and their byproducts is a sign of AD and is often coupled by energy deficiency [23, 57]. This reduction in bioenergetics is exacerbated by reduced levels of mitochondrial complex V, or ATP synthase in brain regions affected by phosphorylated tau [8].

As discussed, complexes I and III of the ETC are a normal source of ROS production, but with aging of the body, and deficiency in the ETC, there is an increase in electron leaking and ROS generation [49, 55]. Damage from ROS is difficult to avoid, as they are necessary for signaling within the body and reacting with biomolecules, but also damage living tissue, and thus are a catch-22 [23, 53]. Their reactiveness allows them to oxidize biomolecules, especially nucleic acids, proteins, and lipids [23]. The brain is especially susceptible, given the high demand on energy, high demand on oxygen, high supply of peroxidizable polyunsaturated fatty acids (the brain also being 60% fat), high concentration of iron capable of ROS generation, and scarcity of antioxidants and enzymes with antioxidant functionality [23]. The mitochondrion is a source of ROS, and is also highly susceptible to damage from ROS [23]. This is strong evidence for the compound damaging effects that lead to AD [23, 57].

Furthermore, a mitochondrion in a neuron typically has a longer half-life than one of another bodily tissue, and is more susceptible to long-term nitroxidative damage due to this and neurons’ low concentrations of glutathione which serves as an antioxidant [1]. This sustained mitochondrial dysfunction is worsened by reduction in mitophagy in AD cells, where damaged mitochondria persist much longer than they would in healthy cells [8].

ROS induction of AD

Energy deficiency is a common denominator with aging and is strongly associated with age related diseases, such as AD. The mitochondrion, as the energy powerhouse of the cells, producing 90% of cellular energy, takes center stage in research regarding the onset of AD following decline in bioenergetics [55, 61]. Decline in energy in critical organs and tissues in the CNS, muscle, heart, and kidney have critical impact [55]. Thus, current research focuses on compounds which may target mitochondrial ROS and induce mitochondrial autophagy of dysfunctional mitochondria [55].

Several researchers have proven the damage caused by lipid peroxidation and H₂O₂. In a study conducted by Reddy’s group in 2010, mouse neuroblastoma (N2a) cells incubated with Aβ treatment there was greater H₂O₂ production and lipid peroxidation, reduced ATP production, cytochrome oxidase activity, and cell viability, along with impaired mitochondrial membrane potential [44]. In fact, the IMM of cells incubated with Aβ were seldom visible or highly damaged, and was coupled by reduction in neurite outgrowth [44]. Similar effects were noted by Reddy’s group again with N2a cells transfected with mutant cDNA expressing mutant AβPP, where the N2a cells demonstrated AD pathology with greater H₂O₂ and lipid peroxidation production, reduced ATP production and cytochrome oxidase activity [62]. In addition, there was reduced cell viability and a reduction in mtDNA copy number, as well as increased GTPase Drp1 enzymatic activity [62]. In another study by Reddy’s group, N2a were inoculated with Aβ toxicity to induce AD pathology, which resulted in increased H₂O₂ and lipid peroxidation, reduced mitochondrial ATP and cytochrome oxidase activity [63]. These effects also markedly reduced cell viability [63].

In vivo studies with mice grown to exhibit mutant AβPP AD pathology, greater lipid peroxidation and H₂O₂ production was exhibited, combined with evidence of reduced mitochondrial fusion, cytochrome oxidase activity and ATP production [64]. These damaging effects were proven to be reversed by SS-31 administration as well [64] (Fig. 8).

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Fig.7

Mitochondrial biogenesis and reactive oxygen species production. Mitochondrial biogenesis plays a critical role in cellular metabolism. Processes include the tricarboxylic acid (TCA) cycle, the electron transport chain inclusive of complexes I, II, III, and IV, ATP synthase, quinone (Q) and cytochrome c (C), and balancing of electrolytes (Ca²⁺) and reactive oxygen species (H₂O₂ and O₂^–).

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Fig.8

Mitochondrial axonal transport. Loss of mitochondrial axonal transport is the major cause for cognitive decline in AD. The AD neuron has greatly reduced neurotransmission at the synapse in comparison to healthy neurons.

In another transgenic tau mice study, Kandimalla and colleagues [65] found mitochondrial dysfunction, including increased H₂O₂ and lipid peroxidation levels, reduced cytochrome oxidase activity, and mitochondrial ATP levels, primarily from phosphorylated tau impairment [65]. Furthermore, fusion proteins were decreased, mitochondrial length was reduced and mitochondrial number was increased in these mice [65]. These findings indicate that phosphorylated tau induces mitochondrial abnormalities [65].

Furthermore, it should be noted that in postmortem human AD brains, mitochondria have a notable deficit of oxidative phosphorylation, especially a great reduction in cytochrome oxidase activity [66]. The reduction in cytochrome oxidase activity was most profound in the hippocampus and temporal cortex of the AD brain, which is believed to be the main contribution to the reduction in bioenergetics in the AD brain [66]. This is likely owed to oxidative stress noted above.

Mitochondrial oxidative stress

Oxidative stress is one of the major sources of initial pathogenesis of AD, and the mitochondria plays a critical role as the source of 90% of endogenous ROS [23]. Via the ETC, there is inevitable leakage of electrons from the IMM, which despite checks and balances from antioxidant enzymes, mitochondrial ROS provides a constant source of superoxide anion [23, 61]. The ROS species produced also aid to inhibit productivity of the mitochondria, and evidence shows that dysfunctional mitochondria are inefficient at generating ATP, and simultaneously are excessive producers of ROS [23, 61]. In this light, the mitochondrion of AD cells becomes self-destructive. The majority, if not all mitochondrial functionality is impaired in AD tissue [23].

There is evidence that superoxide and the radicals formed via lipid peroxidation are products of the same oxidative damage pathway which activate uncoupling proteins [67]. It should thus be duly noted that ROS are an agent of damage and degeneration in cells, particularly when it comes to nucleotide damage, propagation of free radicals, membrane damage, and protein denaturation. However, the cell maintains a balance between ROS produced and mitigating pathways, including those conducted by enzymes. When ROS levels are too low in a cell, cells are vulnerable to promoting mutation proliferation, cancer and tumor growth, since phagocytosis and apoptosis of damaged cells and organelles, and processes such as mitophagy which excise damaged mitochondria, are necessary for healthy cells and tissues [51, 68].