Macroautophagy And Mitophagy in Neurodegenerative Disorders: Focus On Therapeutic Interventions Part 2

3.1.2. Mitophagy in AD

The accumulation of damaged mitochondria is a hallmark of AD. Mitochondrial dysfunction and the associated bioenergetic deficits and oxidative stress contribute to Aβ aggregation and hyperphosphorylation of tau [154,155], which, in turn, are mediators of mitochondrial defects [156]. 

Mitochondria are considered to be the energy factories within cells, and damaged mitochondria may cause a series of health problems, including memory loss. But there are also positive aspects. As long as we take the right measures, we can effectively improve the state of mitochondria and improve our memory.

The main function of mitochondria is to produce energy, which can meet the various needs of cells. But in some cases, mitochondria may be damaged, resulting in reduced energy production, which may lead to health problems such as memory loss. In addition, mitochondria may also cause inflammation, further affecting physical health.

However, in the context of the development of modern medicine, we can use some methods to help mitochondria restore their health. For example, aerobic exercise, a balanced diet, and physical exercise can promote the health of mitochondria, thereby improving our memory. In addition, some natural foods such as natural herbs and nuts also have good mitochondrial repair and protection effects, to maintain the normal state of the body.

In short, although mitochondrial damage may cause health problems, we should continue to actively explore various ways to seek ways to improve our mitochondrial health to better maintain physical health and intelligence. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The effect of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in various ways.

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Accordingly, Aβ peptides were not toxic to cells without functional mitochondria (mitochondrial DNA-depleted cells) [157]. Exposure of primary cortical neurons to Aβ1-42 peptides induces the opening of the mitochondrial membrane transition pore [156]. 

Moreover, co-exposure of cortical neurons with Aβ and NMDA-mediated activation of NMDAR further induces mitochondrial depolarization and increased mitochondrial calcium retention [158]. Additionally, synaptic terminals exposed to Aβ aggregates displayed mitochondrial dysfunction, higher levels of oxidative stress, and impaired glutamate and glucose transport [159]. 

Furthermore, samples from the brain tissue of tau mutant mice exhibited mitochondrial respiratory defects and higher production of ROS [160]. Accumulation of an N-terminal tau fragment was associated with reduced mitochondrial cytochrome c oxidase activity in human AD brains [161]. 

Moreover, the depletion in ATP caused by mitochondrial defects activates AMPK and consequently autophagy [162], linking mitochondrial function with autophagy regulation. 

The clearance of unhealthy mitochondria by mitophagy is compromised in AD although the mechanism is not fully characterized. Previous studies using post-mortem hippocampal tissues from AD patients and AD iPSC-derived neurons showed lower levels of mitophagy-related proteins, lower levels of PINK1 and BNIP3L/NIX, and inactivation of mitophagy initiation proteins, such as phospho-ULK1 [163]. 

The impairment in mitophagy was also described in APP and tau-overexpression models [164]. The role of defective mitophagy in AD pathogenesis was further supported by an improvement of memory in APP/PS1 mouse models after the correction of neuronal mitophagy [163]. Studies with AD models showed tau insertion into the mitochondrial membrane abrogating Parkin-mediated mitophagy [165]. 

Moreover, overexpression of Parkin-restored mitophagy in Aβ-treated cells and ameliorated mitochondrial function [166]. Despite an increased translocation of Parkin to the mitochondria, the presence of undigested mitochondria inside lysosomes demonstrates a deficient lysosomal efficiency in AD at early stages [167]. 

The levels of proteins involved in mitochondrial fission, a step that is essential to isolate damaged mitochondria before its engulfment by the autophagosome, are elevated in AD brains [168]. 

Consistently, altered mitochondrial morphology towards fragmentation was observed in AD, which is aggravated when the levels of Aβ and p-tau increase and these proteins interact with Drp1 fission-related protein [169]. 

An impairment in anterograde movement contributes to mitochondrial dysfunction displayed in neurons from transgenic AD models [170]. Defective anterograde movement impairs the fusion of old mitochondria with newly formed organelle in soma, thus inhibiting their fusion-mediated repair. Additionally, a defective retrograde movement described in AD [129] reduces the removal of defective mitochondria in soma by autophagy. 

The reduced levels of SIRT1 described in AD also compromise the activation of autophagy proteins, stabilization of PINK1 in mitochondria, and the regulation of mitophagy receptors Nix/BNIP3L and LC3 [171]. 

Moreover, mitochondrial SIRT3 levels are decreased in AD, resulting in lower FOXO3-mediated activation of p62 [172]. Reduced intracellular levels of NAD+ were reported in AD [173], and depletion of NAD+ levels could also diminish sirtuins activity. 

Thus, reduced levels and activity of SIRTs can compromise mitophagy, leading to the accumulation of damaged mitochondria in AD.

3.2. Parkinson's Disease

PD is a neurodegenerative disease that results from the degenerative loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), leading to dopamine deficiency [174]. 

Postural instability, bradykinesia, and gaiting are the typical traits of the disease, often accompanied by hyposmia and marked gastrointestinal complications in the form of gastroparesis and constipation [175]. 

The histopathological hallmark of PD is the presence of fibrillar aggregates referred to as Lewy bodies (LBs), in which α-synuclein (aSyn) is a major constituent [176]. aSyn is a natively unfolded protein with many attributed functions in neuronal development and synaptic signaling [177]. 

However, some studies indicate that the lack of wild-type (WT) aSyn is not relevant for basal neuronal function [178], suggesting that a loss of function of aSyn does not cause PD. Despite intense debate, aSyn oligomers are toxic to neuronal cells [179].

3.2.1. Autophagy in PD

Autophagy was first associated with PD when Anglade and colleagues found that the death of dopaminergic neurons in SNpc in post-mortem brains of PD patients was associated with autophagic deregulation [180]. 

Monomeric aSyn is a short-lived protein and thus its physiological levels are maintained mainly by the ubiquitin-proteasome system (UPS) [181]. However, in the case of aSyn intracellular overload, the clearance of native aSyn by the UPS becomes deficient, thus shifting the elimination of aSyn monomers to the autophagosome–lysosomal pathway (ALP) [182]. 

Interestingly, a study using mice injected with human aSyn adenovirus in the substantia nigra demonstrated that many autophagic proteins are upregulated in the initial phase of the disease, suggesting an increment in autophagy in this stage [183]. However, post-mortem studies indicate an increase in the levels of LC3 II and a decrease in cathepsin D, pointing to an impaired function in the ALP pathway [121]. 

Moreover, histological findings demonstrate that LC3 II is often localized within aSyn inclusions [184], which implies not only a defective autophagic process but also that macroautophagy is responsible for the clearance of aSyn aggregates [185]. 

CMA is strongly impaired in PD, as reduced expression of LAMP2a and Hsc70 is observed in the post-mortem brains of PD patients [121,186]. Interestingly, the remaining LAMP2a-positive vesicles co-localized with an. These observations are consistent with the fact that aSyn bears a KFERQ-like pentapeptide, 95VKKDQ99, with a strong specificity to lysosomal vesicles [187,188]. 

In aSyn oligomers, this pentapeptide is still available and allows aggregates to bind to the lysosomal receptors, blocking the import of aSyn to the lysosome, which partially explains the defects observed in the CMA of neuronal cells in PD [185]. Further studies have demonstrated that post-translational modifications in aSyn also hinder the CMA pathway and contribute to overall cellular toxicity [189]. 

Indeed, specific inhibition of CMA achieved by the downregulation of LAMP2a appears to be responsible for increased levels of native aSyn in rat cortical neurons [190]. Additionally, the reduction in LAMP2a expression in the nigrostriatal circuit of rats leads to dopaminergic cell death, accompanied by an increase in aSyn protein levels and an increase in LC3 expression [191]. 

Hence, CMA appears to be an extremely relevant pathway for the degradation of WT aSyn. Glucocerebrosidase (GBA) is a lysosomal protein responsible for the cleavage of glucocerebroside and glucosyl sphingosine in the lumen of these vesicles. 

GBA deficiency leads to substrate accumulation in the lysosome, with prejudicial effects in the endolysosomal pathway. Interestingly, heterozygous mutations in the GBA locus are considered a major risk factor for the development of PD [192]. Homozygous GBA mutant individuals develop Gaucher disease, as a result of which a great part develops parkinsonism [193]. 

A link between GBA and aSyn pathologies was found in patients with Lewy Body disorders, a spectrum of diseases with alternative forms of Parkinsonism. In these patients, the presence of aSyn inclusions was highly correlated with the presence of mutant GBA in the SNpc of post-mortem brains [194]. 

Remarkably, in dopaminergic neurons derived from iPSC of heterozygous GBA PD patients, showing decreased activity of GBA, an increase in the number and enlargement of lysosomes were found [195], accompanied by increased levels of aSyn oligomers in PD-derived neurons, when compared to control cells. 

Moreover, GBA knockdown in rat striatum led to an accumulation of oligomeric aSyn, preceded by a disruption in the autophagic pathway. Beclin-1 might mediate the effects of GBA since its activity is downregulated upon a decrease in GBA activity, with a concomitant reduction in LC3 II [196]. 

Leucine-rich repeat kinase 2 (LRRK2) is linked to a form of autosomal dominant form of PD, being also a major risk factor for idiopathic PD. Apart from many cellular functions, LRRK2 has a prominent role in autophagy, being involved in several distinct phases of the process [197]. 

A study using an age-dependent LRRK2 knock-in mouse model showed that midbrain neurons had a higher number of LAMP2a-positive vesicles when compared to WT mice [197], suggesting a build-up of lysosomal vesicles. This increase was associated with poorer CMA efficiency since the clearance of lysosomal-specific substrates with a KFERQ motif was lower on LRRK2 knock-in midbrain cells. 

This is consistent with evidence that demonstrates that LRRK2 regulates the activity of a subset of Rab GTPases, which are responsible for membrane mobilization, vesicle assembly, and transportation [198,199]. G2019S is the most common mutation in LRRK2-associated PD, and mutated LRRK2 is thought to halt the autophagic process. 

For example, differentiated SH-SY5Y neuronal cells expressing this mutant form of LRRK2 presented much smaller neurites and an aberrant accumulation of LC3 vesicles [200]. Moreover, in mice carrying mutant G2019S LRRK2, there was an increment of early and late autophagic vesicles, with detrimental effects on the number of neurons bearing tyrosine hydroxylase (TH) and on cortex neuronal morphology [201]. 

These observations can be corroborated by the fact that LRRK2 activity is believed to affect macroautophagy via Beclin-1 in an mTOR-independent pathway [202]. Since higher expression of native aSyn is considered a risk factor for the development of idiopathic PD [203], macroautophagy assumes a crucial role in the maintenance of any cellular abundance. 

Interestingly, findings demonstrate that the expression of mutant forms of aSyn or overexpression of WT aSyn block autophagy [204], conferring a pivotal role in this process in disease progression. Corroborating these observations, Vogiatzi and coworkers found that WT aSyn increased 1.5- to 3.8-fold in PC12 cells after the administration of 3-methyladenine (3-MA), a macroautophagy inhibitor [190]. 

Additionally, the seeding of aSyn fibrils gives rise to degradation-resistant aSyn inclusions, with a negative outcome in macroautophagy. Cells harboring aSyn aggregates display an autophagosome build-up [205], which can be explained by the fact that aSyn inclusions promote the mislocalization of mATG9, a protein that facilitates membrane trafficking for phagophore formation [204]. 

Recently, it was observed that A30P aSyn expression in midbrain dopaminergic neuronal primary cultures arrests the autophagic flux, observed by a decrease in autophagosome-associated LC3 protein with a concomitant increase in SQSTM1/p62 levels [206]. 

Furthermore, in peripheral blood mononuclear cells derived from PD patients, autophagic proteins such as ULK1 and Beclin1 were found to be dysregulated, correlating with an accumulation [207]. The self-renewing nature of autophagy is essential for PD pathophysiology, and the inability to recycle misfolded or aggregated proteins has a profound effect on neuronal cells [61]. 

Hence, studies suggest that impaired autophagy could be the etiologic factor in PD pathogenesis. For example, specific ablation of ATG7, essential for autophagosome elongation in dopaminergic neurons from aged mice, induces characteristic PD features, such as motor frailty, loss of TH-positive neurons, and aSyn deposition [208]. 

Moreover, ATG7 depletion leads to the presence of p62-containing aSyn inclusions in SNpc dopaminergic neurons and a robust increase in polyubiquitinated substrates [209]. Although the defects in autophagy are transversal to PD models, the relation of causality needs to be further investigated.

3.2.2. Mitophagy in PD

Mitochondria play a central role in PD pathogenesis [210]. Mitochondrial failure, including decreased complex I activity, well-described in post-mortem brains of idiopathic PD patients [211,212], together with increased oxidative stress in nigrostriatal neurons [213] and lower mitochondrial capacity of buffering Ca2+ [214], are well-known features of PD neurodegenerative process, which highlights the paramount importance of clearing damaged mitochondria in diseased neurons. aSyn oligomers have been described to induce mitochondrial damage. 

Indeed, both WT and mutant aSyn are imported to the mitochondria in cell lines [215], cultured dopaminergic neurons, and PD patients' brains [216]. However, A53T aSyn has faster accumulation rates in the mitochondria, impairing the activity of complex I and causing exacerbated ROS production [217]. Furthermore, a study using dopaminergic neuronal cultures showed that aSyn aggregates retaining pSer129 aSyn bind preferentially to mitochondria rather than WT aSyn, leading to impaired oxidative phosphorylation [218]. 

Indeed, mutant aSyn stimulates mitochondrial fragmentation and promotes the presence of cardiolipin at the surface of mitochondria [219]. Cardiolipin is a danger signal elicited by mitochondria that link to LC3 to initiate mitophagy [220]. 

Remarkably, aSyn can bind to cardiolipin, thus competing with LC3 and halting mitophagy [219]. The dynamic interplay between Parkin and PINK1 allows for the correct identification of damaged mitochondria. 

Strikingly, mutations in Parkin and PINK1 genes are responsible for early-onset autosomal recessive forms of PD [221]. Pathological evidence was found in the brains of these PD patients, such as a reduced population of neurons in the SNpc and fibrillary gliosis [222]. In PD, Parkin activity upregulates the Drp1 function to promote mitochondrial fission and downregulates the function of Mfn1/2 to prevent fusion, resulting in smaller mitochondria that are easier to engulf in phagophores [223]. 

Interestingly, mutant forms of Parkin fail to localize within the mitochondria after a depolarizing insult, leading to the inhibition of mitophagy [224]. Moreover, proteins localized at the OMM that regulate apoptotic events, such as Bcl-XL and Mcl-1, inhibit Parkin recruitment to mitochondria, blocking mitophagy [225]. 

Additionally, p62-positive mitochondria prone to selective degradation are reduced in cells expressing mutant Parkin [226], which might happen due to Parkin sequestration into insoluble aggregates [227]. Transgenic mice with absent Parkin display an increase in pSer129 aSyn but not in aSyn total levels [228]. Such ablation can produce mild nigrostriatal defects [229], associated with altered mitochondrial morphology [230]. 

These findings can be explained by Parkin's ability to modulate the phosphorylation state of aSyn at Ser129 [231]. It was demonstrated that PINK1 mutant forms associated with PD fail to accurately recruit Parkin to the mitochondria, resulting in dysfunctional mitophagy [232]. Additionally, PINK1 ablation leads to an increase in the number of fragmented mitochondria [233], a common feature in PD patient's dopaminergic neurons [234]. 

Although the number of dopaminergic neurons remains unaffected by PINK1 depletion, striatal neurons of PINK1−/− mice display a reduced capacity for releasing dopamine [235] and are more susceptible to exogenous stressors such as oxidative stress [236]. 

These observations are accompanied by lower respiration rates in mutant animals and decreased activity of the mitochondrial complex I [237], which can explain the contribution of this mutation to PD. Surprisingly, Parkin or PINK1 depletion alone does not induce overt neuronal degeneration in rodents, which suggests that other Parkin/PINK1-independent mitophagic processes may be taking place [228]. 

The relevance of mitochondrial dysfunction and hence mitophagy is underlined by the fact that several mitochondrial toxins, including 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rotenone, often mimic the molecular dysfunction in PD and are used as models for idiopathic PD [238]. These toxins target the mitochondria by inhibiting complex I, a feature usually observed in post-mortem brains of PD patients [212]. 

MPTP induces parkinsonism by promoting dopaminergic neurodegeneration and aSyn accumulation [239]. MPTP is a lipophilic compound that can cross the blood-brain barrier (BBB), and in astrocytes, it is converted to its ionic form 1-methyl4-phenyl pyridinium (MPP+) [240]. This active form (MPP+) serves as a high-affinity substrate for dopaminergic transporters to get into neuronal cells [241], inhibiting mitochondrial complex I and consequently reducing respiration rates [239], among other cytotoxic effects. 

Indeed, MPP+ treatment in primary rat dopaminergic neurons and SH-SY5Y cells promotes Drp1-dependent mitochondrial fragmentation [242]. Additionally, in PC12 dopaminergic-differentiated cells, MPP+-induced mitochondrial damage is accompanied by a dramatic decrease in axonal transport rates, followed by a marked neurodegenerative process [243]. 

Likewise, rotenone, another strong complex I inhibitor, can mimic the phenotypic hallmarks of PD. When injected in mice, rotenone stimulates the selective death of dopaminergic neurons along with the appearance of aSyn aggregates and motor dysfunction [244]. In addition, rotenone administration disrupts the autophagic flux, denounced by the increase in LC3 and p62 and decreased levels of LAMP2a, halting the selective degradation of mitochondria [244]. 

Of note, rotenone has a severe impact on the microtubule network [245], impairing the autophagic flux in rats [244] and altering the movement rates of mitochondria in neurites of differentiated SH-SY5Y cells [246]. Since mitophagy is highly dependent on microtubule-dependent transport [247], mitophagy is expected to be impaired after rotenone exposure.

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