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

2. Mitophagy

Mitochondria harbor a double membrane, composed of an inner mitochondrial membrane (IMM) and an outer mitochondrial membrane (OMM), separated by the intramembrane space. 

The inner mitochondrial membrane is an important component of the cell. Its main function is to generate the energy required by the cell and control the metabolism of the entire cell. Modern biological research shows that the inner mitochondrial membrane is also closely related to human cognitive ability, especially memory.

The molecules of the inner mitochondrial membrane also contain some components related to cognition and the nervous system, such as membrane proteins and oxidative phosphorylation systems. These components are directly involved in the metabolic process of brain cells, and their activity status will affect the normal function of neurons, thereby affecting human cognition and memory.

In addition, a molecule called mitochondrial transporter in the inner mitochondrial membrane also plays an important role in memory storage and recall. This molecule can help neurons transmit chemical signals between synapses and strengthen the connectivity of synapses, thereby achieving long-term storage and accurate recall of memory.

In summary, the connection between the inner mitochondrial membrane and cognitive ability and memory has long been scientifically verified. Optimizing the metabolic state of the inner mitochondrial membrane helps to improve the working efficiency of the brain and improves people's cognitive and memory abilities. Therefore, we can promote the health of the inner mitochondrial membrane by properly adjusting our diet, exercising, and maintaining a good attitude, thereby enhancing our memory and cognitive abilities. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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This organelle configuration is essential for maintaining an electrochemical gradient, allowing mitochondria to generate the necessary amount of ATP to be used by cells [77]. The elimination of defective mitochondria is an essential quality control mechanism to sustain a healthy pool of mitochondria and maintain a viable network in neuronal cells [78]. 

These cells are more sensitive to mitochondrial defects due to their high metabolic demands; thus, the selective elimination of dysfunctional organelles is vital for reliable neurotransmission. 

Mitochondria that are irreversibly damaged or become no longer efficient are eliminated by mitophagy, a selective mechanism where specialized receptors recognize flawed mitochondria and mediate cargo targeting autophagic vesicles [79]. Cells make use of different methods to recycle defective mitochondria, namely ubiquitin-dependent and independent pathways. 

Majorly, neuronal cells resort to the ubiquitin-dependent mechanism system controlled by PTEN-induced kinase 1 (PINK1)/Parkin to preserve an active and dynamic pool of mitochondria. 

PINK1 is a mitochondrial protein whose accumulation in the OMM acts as a sensor for maintaining mitochondrial function since it flags damaged mitochondria for degradation [80]. 

PINK1 exhibits a mitochondrial-targeting sequence, thus being systematically imported to the IMM by complexing with translocases of the outer membrane 20 and 40 (TOM20 and TOM40) [81], where it is cleaved by the intramembrane serine protease presenilin associated rhomboid-like (PARL) [82] and therefore maintained at low levels under physiological conditions. 

Upon a given damage, the molecular interactions between TOM20/40 and PINK1 decrease, and PINK1 clusters in the OMM of damaged mitochondria, promoting Parkin phosphorylation and its subsequent recruitment [83]. 

Parkin, a cytosolic E3 ubiquitin ligase, ubiquitinates a myriad of proteins of the OMM, which are recognized by receptors such as p62, also denominated sequestosome 1 (SQSTM1). p62/SQSTM1 is the ubiquitin-binding receptor responsible for driving ubiquitinated mitochondria to the autophagosome assembly site by interacting with the ATG8 family LC3/GABARAP receptors [84]. 

These receptors also recognize the LIR motifs of many OMM proteins for specific targeting of damaged mitochondria at the autophagosome assembly site [85]. In neuronal cells, ubiquitin-independent mitophagy orchestrated by two bi-functional proteins, BCL2 and adenovirus E1B 19 kDa-interacting protein 3 (BNIP3L) and BNIP3- like protein X (NIX), is also responsible for the differentiation of retinal ganglion cells (RGCs). BNIP3 and NIX contain a BH3 domain and are mainly located at the OMM. 

These proteins have a WXXL-like motif with an affinity for LC3 and its homolog GABARAP, driving mitochondria to autophagic vesicles. During retinal neurogenesis, a metabolic shift takes place, accompanied by an increase in the expression of both proteins [86]. 

NIX activity can be mediated by increasing reactive oxygen species (ROS) levels in several cell lines [87], adding a new layer of complexity apart from developmental functions. Indeed, ROS production increases as a consequence of damaged mitochondria [88] and can induce mitophagy [88]. NIX can also regulate the translocation of Parkin to mitochondria upon a depolarizing stimulus, such as CCCP [87]. 

In addition, BNIP3L and NIX-regulated mitophagy have neuroprotective functions. In a model of brain ischemia, the selective degradation of mitochondria-mediated by these proteins was crucial for neuronal survival and function [89]. Additionally, NIX overexpression alone is sufficient to restore mitophagy defects observed in cell lines derived from PD patients [90]. 

Fun14 domain-containing protein 1 (FUNDC1) is an OMM protein that interacts with LC3 and GABARAP and mediates mitophagy in hypoxic conditions. At homeostatic levels, FUNDC1-dependent mitophagy is repressed by Src kinase-mediated phosphorylation in the moiety of the LIR motif at Tyr18. 

In a hypoxic state, reduced Src activity results in FDUNC1 Tyr18 dephosphorylation, facilitating its interaction with LC3 and subsequent mitophagy [91]. Moreover, hypoxia-driven phosphorylation of ULK1 by AMPK at Ser555 favors ULK1 translocation to the mitochondria, enhancing FUNDC1 activity [92]. Additionally, Optineurin (OPTN) is a cytosolic receptor located at the OMM that promotes mitophagy through its interaction with LC3 [93]. 

Interestingly, OPTN dysfunction is associated with neurodegenerative diseases, as well as other pathologies [94]. Overall, mitophagy constitutes an essential quality control mechanism for maintaining neuronal metabolism and homeostasis. 

The transport of mitochondria within neurons relies on anterograde and retrograde movements and is essential to fulfill the energetic and metabolic requirements along the neuronal structure. These movements are also essential for mitochondrial repair and degradation and are assisted by transport adaptor proteins (Figure 3).

3. Autophagy and Neurodegenerative Diseases

3.1. Alzheimer's Disease

AD is a neurodegenerative disorder and the most prevalent form of dementia in the elderly population, affecting almost 50 million people worldwide [95]. Early-onset familial AD (EOAD, fAD) accounts for less than 5% of the cases and develops before the age of 65 years. 

Autosomal dominant mutations in three genes have been associated with EOAD: the amyloid precursor protein (APP) gene and the presenilin-1 (PSEN1) and presenilin-2 (PSEN2) genes, subunits of the γ-secretase complex [95]. However, the most common form is the late-onset sporadic form of AD (LOAD, sAD) with a prevalence higher than 95% and with unknown etiology. 

The neuropathology of AD is characterized by progressive cognitive impairment and memory decline, which has been correlated with the presence of extracellular deposition of amyloid-β plaques (Aβ, aggregation of β-amyloid peptides) and intraneuronal neurofibrillary tangles (NFTs, aggregation of hyperphosphorylated tau protein) that compromise synaptic integrity and homeostasis, leading to irreversible damage in specific brain regions such as the hippocampus and the cortex [96]. 

The Aβ peptides are generated by the sequential proteolytic processing of the amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1/β-secretase (BACE1) and γ-secretase through the amyloidogenic pathway. Cleavage of APP by BACE1, the activity of which is elevated in the brain of sporadic AD patients [97], occurs mainly in endosomes, providing an acidic environment for optimal enzyme activity [98]. 

Intracellular trafficking of APP occurs by secretory and endocytic pathways. APP is secreted to the plasma membrane being then internalized by clathrin-mediated or caveolin-mediated endocytosis [99]. 

In endosomes, APP can be cleaved by BACE1 giving rise to the N-terminal APP β fragment. (sAPPβ) and the C-terminal fragment; the latter can undergo further cleavage by γ-secretase, resulting in the formation of Aβ and the APP intracellular domain. In the non-amyloidogenic pathway, APP is cleaved by α-secretase in the middle of the Aβ domain, preventing the formation of the Aβ peptides. 

The cleavage by γ-secretase occurs at different positions of the C-terminal fragment, resulting in the formation of Aβ peptides with different lengths. The most common soluble peptides formed are Aβ1-38 and Aβ1-40, followed by Aβ1-42, the latter being more hydrophobic and more prone to form trimeric and tetrameric oligomers, thus representing the most toxic isoform [100]. 

Aβ oligomers tend to form Aβ fibrils with β-pleated sheets that assemble extracellularly into amyloid plaques [101]. Aβ has been also associated with the hyperphosphorylation and aggregation of tau, a microtubule-associated protein that normally assists in the polymerization and assembly of axonal microtubules and vesicle transport. 

The phosphorylation status of tau is regulated by distinct kinases and phosphatases that are modulated in the presence of Aβ oligomers; for instance, glycogen synthase kinase-3β (GSK3β) and protein phosphatase 2A (PP2A) directly regulate tau phosphorylation status [102]. The hyperphosphorylated state of tau inhibits its association with microtubules, affecting its stabilization and leading to microtubule network disassembly [103,104]. 

The proteolytic cleavage of hyperphosphorylated tau triggers the formation of tau oligomers [105] that are more toxic than aggregates, leading to progressive neuronal death [106].

3.1.1. Autophagy Impairment in AD

In AD, distinct molecular pathogenic mechanisms resulting from the accumulation of Aβ oligomers and tau hyperphosphorylation have been reported, namely mitochondrial dysfunction [107,108] and disruption of proteostasis systems, such as unfolded protein response and autophagy [109]. 

Although Aβ peptides exhibit a KFERQ-related motif, which is important for the normal processing and degradation of APP, these fragments are not substrates for CMA degradation [110], and macroautophagy assists in their clearance [111]. 

Indeed, macroautophagy plays an important role in Aβ metabolism because it is involved in the degradation and clearance of APP [112] and its cleavage products, including Aβ [113] and APP-CTFs (amyloid precursor protein cleaved C-terminal fragment) [114].

Autophagy dysfunction has been demonstrated in both AD patients and animal models. In AD patient's brains, an accumulation of immature autophagic vesicles containing filamentous tau was observed in dystrophic neurites [115]. 

These autophagic vesicles are often found in proximal regions of amyloid plaques in the brains of patients and rodent models [116]. Curiously, accumulation of autophagosomes was also observed in dendrites and soma of APP/PS1 transgenic mouse neurons even before the appearance of Aβ plaques [117]. Additionally, it was observed an accumulation of immature autophagic vesicles before neuronal loss in the hippocampus of the PS1M146L/APP751SL mouse model [118]. 

The abnormal accumulation of autophagosomes in neurons suggests that the defective autophagic–lysosomal pathway contributes to the accumulation of Aβ and tau oligomers in AD [117]. APP and PSEN1 localize in autophagosomes; thus, an accumulation of these organelles is associated with an increase in Aβ generation, which in turn disturbs autophagosome membranes [115]. 

Consequently, the immature autophagosomes described in AD brains and APP/PS1 transgenic mice may constitute another source for Aβ generation. Brains from PSEN1-EOAD patients display lysosomal impairment associated with amyloid pathology and neurodegeneration [119]. 

It has been suggested that autophagosome accumulation might result from an impairment of autophagosome-lysosome fusion or defective lysosome digestion [120]. Studies showed an alteration of autophagy initiation, specifically a decrease in Beclin 1 expression in the cortex of AD patients and mouse models [121]. The increased activity of caspase 3 in AD patients might explain the excessive cleavage of Beclin 1 [122]. 

The genetic reduction of Beclin 1 in a transgenic mouse model of AD expressing human APP increased Aβ accumulation, whereas overexpression of Beclin 1 significantly reduced Aβ levels [113]. In addition to Beclin-1, other ATG proteins were downregulated in an age-dependent manner, resulting in an accumulation of Aβ [123]. ATG7, a key protein in the regulation of autophagy, was implicated in AD pathology. 

The levels of ATG7 were found to be reduced in the cerebral cortex and hippocampus of an AD mouse model, but no changes were detected in temporal cortices on AD patients [124]; ATG7 knockdown mice exhibited a reduction of Aβ secretion accompanied by an increase of intracellular Aβ [125], which further supports a role for ATG7 in the transport of Aβ peptides to multivesicular bodies to be secreted. 

The expression of p62 was shown to be decreased in a triple transgenic mouse model, compromising the initial steps of selective autophagy in AD [126]. However, a genome-wide analysis revealed transcriptional up-regulation of positive regulators of autophagy in AD. 

Consistent with activation of autophagy, an upregulation of ATG protein levels and a higher rate of autophagosome formation and lysosomal biogenesis were observed at the early stages of AD [127]. This controversy suggests differential autophagy regulation in the early and late stages of AD, which should be considered when evaluating the mechanism in different models and when searching for therapeutic strategies. 

The presence of immature autophagosomes in dystrophic neurites suggests that retrograde transport of autophagy-related vesicles and their maturation are impaired in AD [128]. In support of this, inhibition of autophagosome delivery to lysosomes leads to their accumulation in neurites with similar morphology, as described in AD [64]. 

Aβ oligomers can disrupt the dynein–snapin complex, impairing the axonal vesicle transport, which could contribute to defective delivery of autophagosomes to lysosomes in AD [129]. Degradation of tau, which is primarily located in axons and less found in neurites and soma, occurs through proteasome or by macroautophagy, depending on its oligomerization state. 

AD-associated tau modifications drive its degradation through macroautophagy when the proteasome-mediated proteolysis becomes impaired by disease progression [130]. Of relevance, the degradation of tau can produce different fragments that bind to Hsc70 being targeted to LAMP-2A by the CMA [131,132]. 

Still, the accumulation of these fragments in the lysosomal membrane causes the aggregation of tau, which disrupts lysosomal integrity and blocks CMA [132]. The impairment of the autophagic–lysosomal pathway results in the aggregation of tau oligomers, which, in turn, are degraded when macroautophagy is induced with rapamycin [132,133]. 

Tau is essential for the retrograde movement of autophagosomes towards lysosomes through the stabilization of microtubules, a role that is compromised in AD [134]. Hyperphosphorylation of tau and its oligomerization results in the displacement of microtubule machinery and consequently in the impairment of autophagosome movement and maturation, which also contributes to autophagy dysfunction. 

Apart from defects in autophagosome transport, an impairment in lysosomal function was also described in AD. PSEN1 is a regulator of lysosomal function acting as a chaperone for the vacuolar-ATPase that acidifies the lysosomal lumen. Mutations in PSEN1 impaired lysosomal acidification and autophagosome-lysosome fusion, resulting in the accumulation of Aβ-loaded autophagosomes [135]. 

Additionally, it was suggested that lysosomal proteolysis is affected in AD; high levels of Aβ, LC3-II, and ubiquitinated proteins were present both in lysosome and autophagosome fractions isolated from the AD transgenic mouse model [136]. Consistent with this, the disruption of lysosomal proteolysis by inhibiting lysosomal cathepsins in healthy neurons results in the accumulation of autophagic vesicles with similar morphology to those present in AD patients and transgenic mouse models [64]. 

Aβ also compromises lysosomal membrane integrity, resulting in the release of cathepsins into the cytoplasm [137,138]. The presence of cathepsin D in exosomes isolated from the blood of preclinical AD patients [139] supports the hypothesis of an ineffective lysosomal function. Curiously, genetic variations in the Cathepsin D codifying gene were also described as risk factors for AD [140]. 

The analysis of the autophagy process in neurons from early to late stages of AD showed an increased expression of autophagy genes at early stages but the lysosomal clearance defect resulted in the accumulation of autolysosomal structures with the progression of disease [141]. Sirtuins (SIRTs) encompass a group of nicotinamide adenine dinucleotide (NAD+ )- dependent proteins that are regulators of multiple cellular pathways. 

Among them, Sirt1 has been associated with autophagy regulation. Lower levels of SIRT1 compromise the deacetylation-mediated activation of downstream autophagy proteins (e.g., ATG5, ATG7, and LC3) [142], which may also contribute to autophagy impairment in AD. The levels of SIRT1 are decreased in the parietal cortex of AD brain patients and correlate with the accumulation of Aβ and tau tangles [143]. 

Activation of mTOR, the central coordinator of autophagy, was described in AD in response to Aβ accumulation [144]. Aβ increases mTOR activity, which also results in higher tau expression and GSK3β-mediated phosphorylation [145], supporting a role for mTOR in tau-mediated pathogenesis. Additionally, secretion of phospho-tau through exocytotic vesicles by mTOR signaling was observed in the brains of AD patients [146]. 

Dysregulation of the PI3K/Akt/mTOR pathway has been also implicated in AD pathogenesis [147]. Activation of PI3K by growth factors (e.g., insulin-like growth factor-1, IGF1) promotes the phosphorylation of Akt and indirect activation of mTORC1, promoting autophagy. In AD, Aβ interacts with and inhibits the PI3K/Akt/mTOR pathway [148], leading to activation of GSK3β and increased tau hyperphosphorylation. 

Moreover, the transcription factor EB (TFEB), the master regulator of lysosome biogenesis, is a downstream target of mTORC1. A decrease in mTORC1 activity induces dephosphorylation of TFEB and its translocation to the nucleus to activate the expression of autophagy and lysosomal biogenesis-related genes [149]. 

The levels of TFEB were found to be decreased in the brain samples (hippocampus) of AD patients, particularly decreasing nuclear levels of TFEB in the advanced stages of the disease [150]. 

Additionally, TFEB translocation to the nucleus was attenuated in presenilin-deficiency fibroblasts and induced pluripotent stem cell-derived human AD neurons, resulting in reduced CLEAR network activation [151]. 

However, in a double-transgenic APP/PS1 animal model, increased levels of total and nuclear TFEB were detected in the hippocampus of 8-month-old AD mice with a concomitant increase of its downstream targets, suggesting that TFEB is involved in lysosomal-mediated AD progression [152]. The upregulation of TFEB target genes was also reported in PS1 conditional knockout mice and 5xFAD mice [153]. 

The activation of the TFEB network might reflect an attempt to compensate for the impaired autophagy in AD animal models. The exact role of TFEB as a master regulator of lysosomal function in AD pathogenesis is far from being fully understood and requires further investigation.

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