Macroautophagy And Mitophagy in Neurodegenerative Disorders: Focus On Therapeutic Interventions Part 3

3.3. Huntington's Disease

Huntington's Disease (HD) is an autosomal-dominant inherited neurodegenerative disease, characterized by cognitive dysfunction, psychiatric and behavioral disturbances, involuntary motor movement, and later dementia [248,249]. 

Huntington's disease is a genetic disease that often causes symptoms such as changes in body shape, movement disorders, and cognitive decline. Because of the disease's effects on the brain, memory problems are often a concern for patients and their families. Although this is a challenging issue, proactive coping and support can help alleviate symptoms and improve quality of life.

First, it is important to note that Huntington's disease itself is not the only source of memory problems. As with any disease, there are many ways for patients to fight the disease, such as controlling their diet, maintaining a healthy lifestyle, and getting enough rest. Although these measures cannot cure the disease, they can reduce the memory problems caused by the disease.

Second, the support of family members, friends, community, and medical teams plays a vital role in the memory recovery of Huntington's patients. Family members can help patients remember time and make daily arrangements, and they should also give patients enough understanding and encouragement to make patients feel that they are not alone. The medical team can provide professional help and adopt some neuroprotective treatments, including medication, physical therapy, etc.

Finally, the patient's attitude and psychological quality also have a direct impact on the recovery of memory. Patients should learn to actively cope with the disease. Although it may seem difficult, memory can be restored through continuous efforts and communication.

In short, Huntington's disease does have a certain impact on the patient's memory, but we should not give up help and treatment. With the company of relatives and friends and the support of the medical team, patients can reverse the impact of the disease on memory and regain the bright side of life through active coping and various means. It can be seen that we need to improve memory. Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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HD selectively causes neurodegeneration of medium spiny neurons (MSNs) in the striatum, more specifically, in the caudate and putamen, with a dorsomedial to the ventrolateral direction [250]. 

Later in the disease, the parietal cortex is also largely affected, while neurodegeneration in other brain regions, namely the cerebellum, thalamus, and white matter, has also been described [251]. 

The genetic cause of HD is a repeat expansion mutation of the trinucleotide cytosine, adenine, and guanine (CAG) in the coding region of the ubiquitously expressed HTT gene (with 36 CAG repeats being the pathological threshold), encoding an abnormally long N-terminal polyglutamine (polyQ) tract [252]. 

The polyQ expansion leads to mutant HTT (mHTT) aggregation, resulting in mitochondrial and synaptic dysfunction, altered mitochondrial Ca2+ handling, endoplasmic reticulum (ER) stress, impaired gene transcription, and protein translation, inhibited protein clearance pathways, and neuronal loss [253]. Abnormal deposition of misfolded mHTT in the nucleus (forming inclusion bodies) and perinuclear/cytoplasmic subcellular regions is another HD feature. 

These aggregates mostly comprise N-terminal fragments of polyQ-expanded HTT, as shown in neuronal nuclei and dystrophic neurites throughout the cortex and striatum of HD patients [254,255]. 

These inclusions are enriched in ubiquitin and ubiquitinated HTT, components of the UPS, and chaperones such as heat-shock proteins [254], reflecting a generalized deficiency in UPS-mediated degradation of mHTT. 

Indeed, UPS function is diminished in different HD models, which can be accounted for by proteasome sequestration by mHTT aggregates [256].

3.3.1. Autophagy in HD

Two putative KFERQ-like CMA-targeting motifs were identified in the HTT protein: one at amino acids 99–103 (KDRVN), and one at amino acids 248–252 (NEIKV). Upon phosphorylation at Ser16, the very N-terminus (14-LKSFQ-18) is considered a KFERQ-like third motif [257]. 

While normal HTT is degraded by the UPS, polyQ-HTT is a target for CMA-degradation, since purified intact lysosomes showed mHTT fragments in a LAMP2Adependent manner [257]. 

However, CMA-dependent mHTT degradation is less efficient. This may be explained by polyQ expansion-mediated delay of mHTT CMA transport across the lysosomal membrane, leading to accumulation in the cytosol. 

Furthermore, LAMP2A and Hsc70 can strongly interact with polyQ-HTT, which results in CMA pathway traffic blockage [257]. However, LAMP2 mRNA expression is increased in the caudate nucleus of HD patients, which is consistent with increased levels of LAMP2 protein and concomitant induction of CMA in HD patient cells and mouse models [258], suggesting an attempt to increase the activity of this system. 

Indeed, full-length polyQ-HTT is primarily targeted to macroautophagy, whereas N-terminal fragments of HTT are selectively targeted to CMA [37]. Interestingly, R6/2 HD mice showed diminished inclusion formation and improved HD phenotypes after the N-terminal mHTT fragment was targeted to the CMA pathway. 

Furthermore, clearance of phosphorylated N-terminal fragments of polyQ-HTT depends on LAMP2A and Hsc70 proteins [259]. Fibroblasts from HD patients, striatal Hdh knock-in cells, and pre-symptomatic HTT Q111 knock-in mice showed augmented CMA activity [258]. 

This increase in CMA activity was not sustained when aged mice became symptomatic. Moreover, a significant decrease in LAMP-2A staining was detected in older HTT Q111 knock-in mice [258]. 

Since LAMP-2A localization and CMA activity are regulated by lipid composition at the lysosomal membrane, and altered lipid metabolism occurs in HD [260], modifications in lipid homeostasis might contribute to HD dysfunction in CMA. Thus, to compensate for a malfunctioning macroautophagy pathway, HD neurons might upregulate CMA. 

However, CMA-competent lysosomes are less efficient at degrading substrates over time, while polyQ-HTT interferes with LAMP-2A and Hsc70 cargo uptake and lipids accumulation. As an attempt to maintain normal proteostasis, since UPS-mediated protein degradation and CMA fails, macroautophagy may be upregulated in HD [261]. 

PolyQ-HTT is targeted for autophagic clearance, suggesting selectivity of macroautophagy towards pathogenic HTT [262]. However, UPS and autophagy degradation pathways are significantly compromised in HD, which results in polyQ-HTT aggregate accumulation, ultimately leading to neuronal dysfunction and death [263]. 

Furthermore, progressive failure of degradation pathways and proteostasis lead to the accumulation of damaged organelles such as mitochondria and ER. Interestingly, in addition to polyQ-HTT aggregates being a substrate for autophagy-mediated degradation, HTT protein is further involved in autophagy pathway regulation [264], as described below in this section. 

Moreover, HTT has a role in the axonal transport of autophagosomes, as suggested by live-cell imaging studies in striatal neurons. After both the loss of HTT and the expression of mHTT, decreased autophagosome transport and subsequent inhibition of substrate degradation were observed [265]. 

Brain biopsies from HD patients showed significant abnormalities in compartments of the vesicular–endocytic pathway, with abnormal proliferation of multivesicular bodies, endosomes, and lysosomes, accompanied by disruption of the Golgi apparatus and disorganization of the ER [266]. 

Concordantly, striatal neurons in brains from HD patients stained for HTT showed significant increases in endosome–lysosome-like organelles and HTT-positive tubulovesicular structures [255]. Normal HTT acts as a scaffold protein to selective autophagy, since HTT can physically interact with p62 to facilitate its association with LC3-II and ubiquitinated substrates. 

HTT can also bind to ULK1, releasing ULK1 from negative regulation by mTOR [267]. Indeed, ULK1 was shown to have reduced activity in HD cellular and animal models (Q175) [268,269]. 

Moreover, HTT can contribute to the microtubular transport of autophagosomes which is essential for autophagosome-lysosome fusion [265]. Indeed, different cell types, such as primary neurons, striatal cell lines, fibroblasts, and hepatocytes from two HD mouse models and lymphoblasts derived from HD patients showed an increased number of autophagic vacuoles without an augmented autophagy-mediated degradation [260]. 

Additionally, two HD mice models, R6/2 (which expresses exon 1 of the human HTT gene with ~150 CAG repeats) and YAC128 (transgenic yeast artificial chromosome (YAC) mice expressing full-length human HTT gene with 128 CAG repeats) mice showed specifically increased levels of the autophagy markers p62 and LC3-II in the striatum at a later age (18 months) [270], suggesting deficit(s) in autophagy-mediated protein degradation. 

Conversely, polyQ-HTT aggregates can sequester mTOR, leading to autophagy induction [271]. One possible explanation for this contradiction is a substantial alteration in macroautophagy cargo recognition in HD cells, which results in 'empty' autophagosome formation [260]. 

Autophagosomes have a reduced amount of cytosolic cargo inside, possibly due to abnormal p62–polyQ-HTT interaction, leading to reduced protein degradation rate in HD samples [260], which can result in serious deleterious effects on cell homeostasis and consequent neuronal loss. 

Compensatory activation of macroautophagy in response to polyQ-HTT aggregates may be an important approach to HD neuron survival. Organelles are turned over by macroautophagy, requiring the recognition of targeted membranes by the autophagy machinery. 

Because polyQ-HTT can interact with different organelle membranes [264,271], polyQ-HTT may interfere with organelle recognition using autophagic vacuoles. Concordantly with this hypothesis, HD cells show an accumulation of defective mitochondria, which suggests that compromised mitochondria are not efficiently recognized or marked up to autophagy. 

Furthermore, polyubiquitination is another way for protein aggregates to be degraded by macroautophagy, and polyQ-HTT can bind to polyubiquitinated protein aggregates, which can restrict their recognition by the autophagy system [262]. 

Several studies revealed that HTT interacts with autophagy-associated proteins indirectly influencing the autophagy pathway. Clearance of polyubiquitinated inclusions is mediated by the selective autophagy receptor, p62, which can exist in polyubiquitinated mHTT aggregates [272]. 

A shell of p62 and LC3 proteins can surround mHTT aggregates to promote recruitment to autophagosomes, whereas p62 knock-down leads to augmented cell death in the presence of mHTT [273]. p62 expression levels are diminished in the HD R6/1 mouse model brain, as a result of decreased protein synthesis, at the early stage of the pathology. 

However, at late stages, p62 accumulates in the striatum and hippocampus [274]. STHdhQ111/Q111 striatal cells, derived from HD knock-in mice, also showed increased p62 levels, in response to proteotoxic stress [275]. Thus, p62 can bind mHTT aggregates and progressively accumulate in HD mice and patient's cell nuclei, contributing to the onset of HD symptoms. 

Previously, Kurosawa and colleagues revealed that p62 depletion in three HD mouse models (R6/2, HD190QG, and HD120QG mice) improved HD phenotypes and life expectancy, showing reduced nuclear inclusions and increased cytoplasmic inclusions of mHTT [276].

The selective autophagy receptor NBR1, which sequestrates protein aggregates, shares a similar function and structure to p62 and has been shown to interact with p62 [277]. As explained before, mHTT forms nuclear inclusions with p62, leading to neurotoxicity. 

In contrast to what happens with p62, NBR1 does not accumulate within neuronal nuclei or at a late stage of HD in mice or patients [274], suggesting that NBR1 may take over p62 nuclear accumulation to maintain some basic rate of selective macroautophagy. 

The adaptor protein autophagy-linked FYVE protein (ALFY) acts as a scaffold for aggregates, p62, and the autophagosome and has a central role in the autophagic degradation of mHTT aggregates [278]. 

Concordantly, ALFY mRNA expression is decreased in the caudate nucleus of HD patients [279]. The autophagy adaptor OPTN can also act as an autophagy receptor, which recognizes and promotes autophagic clearance of protein aggregates [280]. OPTN enables intranuclear mHTT inclusion clearance via selective autophagy [281]. 

Intriguingly, OPTN shows a cell-specific expression pattern in the striatum, which is related to the neuronal loss pattern in this HD brain region [282]. Moreover, OPTN overexpression reduced mHTT aggregation via increased selective autophagy [283]. Toll-interacting protein (Tollip) is another selective autophagy cargo receptor protein with protein aggregate clearance function [284]. 

A study using the stable Neuro2a cell line expressing mHTT (HD60Q and HD150Q) showed that Tollip associates with mHTT-derived N-terminal peptides, stimulating their aggregation and enhancing their clearance via autophagy, protecting the cells from proteotoxicity [285]. 

Additionally, the striatum and cortex from the HD R6/2 mice showed Tollip protein in nuclear inclusions [286]. As estimated, Tollip depletion led to cell death, while Tollip overexpression enhanced aggregate clearance [284]. 

Thus, by acting as a mediator of selective autophagy associated with HD, Tollip might be a good candidate for an HD therapeutic strategy. Beclin 1 is a major autophagy regulator, the levels of which are reduced with aging. 

Beclin 1 can be recruited to mHTT aggregates, reducing its activity, and further contributing to autophagy dysfunction in HD [287]. A study from Ashkenazi and colleagues, using the HD-N171-82Q mice (expressing the first 171 amino acids of human HTT) and primary fibroblasts from HD patients, showed that mHTT outcompetes ataxin 3 in binding Beclin 1, avoiding its activation [288]. 

This is a striatal-specific protein and a Beclin 1 activator that recruits Beclin 1 away from Bcl-2, preventing its inhibitory action. However, mHTT can inhibit Rhes interaction with Beclin 1, reducing its role in promoting autophagy [289]. 

Conversely, Beclin 1 overexpression increased mHTT aggregate clearance and reduced neuronal damage [287]. Expression of Beclin-1 in the human brain gradually decreases with age [287], as well as age-related decreased trafficking of the lysosomal protein LAMP2 [8]. Interestingly, HD patients fibroblasts follow the same pattern [288].

However, Beclin-1 seems to accumulate into polyQ-HTT inclusions in R6/2 HD transgenic mice and HD patients' brains [287], demonstrating a region-specific impairment. Embryonic fibroblasts from Q111-Htt knock-in HD mice showed an increased number of lipid droplets and markedly reduced the association of lipid droplets with HD autophagosomes [260], which may contribute to impaired intracellular lipid storage observed in HD. 

PolyQ-HTT aggregates also sequestered mTOR, as observed in COS-7 cells expressing mutant (Q74) huntingtin exon 1, brain tissue of mice expressing mHTT (Q82), and brain tissue from HD patient's caudate and putamen [271]. 

Additionally, ULK1 activity is also decreased in the brains of zQ175 HD mice, since ULK1 substrates such as Beclin-1 and ATG14 are less phosphorylated, together with the redistribution of ULK1 to an insoluble fraction where aggregated mHTT was found [269]. Moreover, Metzger and coworkers showed that the V471A polymorphism in ATG7 is related to an earlier onset form of HD [290].

As described before in this review, TFEB is a master regulatory transcription factor of the autophagy-lysosome pathway (lysosomal biogenesis and autophagy), which was shown to be upregulated by peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha [291], a member of transcription coactivators that plays a central role in the regulation of cellular energy metabolism, mitochondrial biogenesis, and adaptive thermogenesis. N171-82Q HD transgenic mice showed abnormal TFEB expression and activity, suggesting that TFEB signaling is impaired in HD [291]. 

Concordantly, TFEB can promote polyQ-HTT clearance [291], which emphasizes TFEB as a possible therapeutic target for HD or other diseases with protein aggregate accumulation. 

Therefore, it is necessary to further understand autophagy dysfunction in HD, since preventing mHTT accumulation and/or improving neuronal proteostasis may constitute relevant therapeutic opportunities in HD.

3.3.2. Mitophagy in HD

Previous studies reported ultrastructural defects in mitochondria isolated from post-mortem HD cortical tissue and compromised oxidative function and ATP synthesis in pre-symptomatic HD carriers [292], suggesting that mitochondrial dysfunction is an early relevant pathogenic mechanism. 

HD mutant cells and striatal and cortical neurons isolated from YAC128 transgenic mice presented altered mitochondrial bioenergetics, including pyruvate dehydrogenase (PDH) dysfunction [293], as well as ∆ψm deregulation [294], augmented mitochondrial ROS production [295], and increased Ca2+ uptake in isolated mitochondria from pre-symptomatic R6/2 and YAC128 brain mice [296]. 

We further showed similar mitochondrial results in HD human lymphoblasts [297]. Profound changes in ∆ψm associated with apoptotic events and reduced ATP levels were also observed in symptomatic HD cybrids (an ex-vivo peripheral model obtained from the fusion of HD human platelets with mtDNA-depleted rho0 cells) and in HD human B-lymphocytes [298–300]. 

Concordantly, when compared with WT cells, striatal STHdhQ111/Q111 cells showed significant ∆ψm reduction after increasing Ca2+ concentrations [301]; in the case of primary striatal neurons derived from YAC128 mice, reduced ∆ψm and reduced mitochondrial Ca2+ handling occurred due to cytosolic Ca2+ overload after selective activation of N-methylD-aspartate (NMDA) receptors [302]. 

Furthermore, abnormal mitochondrial morphology and trafficking were observed in post-mortem HD patients' brain specimens and HD human lymphoblasts [303]. We previously showed that HD patients' mitochondrial platelets presented reduced activity of citrate synthase in pre-symptomatic and Cx-I in pre-symptomatic and symptomatic HD carriers [304]. 

By this, isolated brain mitochondria from the caudate nucleus of HD patients [305] and from different HD cells (human neuroblastoma cells; STHdhQ111/Q111 striatal cells), animal models (Hdh(CAG)150 knock-in mouse) showed HTT fragments in close contact with mitochondria [304,306], suggesting a direct effect of mHTT on mitochondrial function. 

Indeed, mHTT may cause direct effects by physically interacting with the organelle and associated proteins and further indirect effects on mitochondria and energy metabolism via transcriptional deregulation (e.g., by interfering with PGC-1alpha transcriptional activity). 

Thus, abnormal mitochondrial membrane potential (∆ψm), oxidative stress, impaired oxidative phosphorylation (OXPHOS), and loss of mitochondrial dynamics, accompanied by mitochondrial morphology modifications and/or decreased biogenesis, favor the accumulation of damaged mitochondria in HD [307]. Abnormal elimination of dysfunctional mitochondria observed in HD suggests an impairment in mitophagy. Levels of basal mitophagy were seen to be reduced in the DG of HD mice crossed with the mito-Keima mouse line [308]. 

Moreover, a recent study showed that mitophagy is affected in STHdhQ111/Q111 striatal cells [309]. These authors showed that mHTT affects the initiation of the mitophagy process, as well as the recruitment of mitophagy receptors and its interaction with LC3-II during mitophagy [309]. 

As explained earlier, PINK1-Parkin-mediated mitophagy starts with PINK1 stabilization at the OMM of damaged mitochondria to recruit Parkin. In the caudate nucleus from HD patients, mRNA expression of PINK1 is significantly decreased [310]. 

Augmented mitochondrial fragmentation [311] and inefficient incorporation of mitochondria into autophagosomes [265] are HD features; thus, decreased PINK1 may exacerbate the dysfunctional autophagosome recruitment in HD cells. Concordantly, PINK1 overexpression in HD flies and STHdhQ111/Q111 cells proved to be protective in these models [312]. 

Moreover, juvenile HD fibroblasts showed increased Parkin levels [313]. Although there is some lack of information regarding the alterations in PINK1/Parkin-dependent mitophagy in HD, its activation may constitute an interesting therapeutic approach. 

Moreover, mHTT can interact with Drp1, resulting in increased Drp1 GTPase activity and consequently increased mitochondrial fission, causing reduced mitochondrial function [303]. Recently, Aladdin and co-workers showed that skin fibroblasts from juvenile HD patients had significantly lower levels of mitochondrial fusion and fission proteins and reduced branching in the mitochondrial network. 

Furthermore, juvenile HD fibroblasts revealed higher proteasome activity, which was associated with elevated gene and protein expression of Parkin, as well as increased proteasomal degradation of the mitochondrial fusion protein Mfn1 in diseased cells [313]. 

These data suggest that expansion of mHTT is linked to increased proteasomal activity and faster turnover of specific substrates of the ubiquitin-proteasome system to protect cells, which could contribute to altered mitochondrial dynamics in the early phases of the disease.

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