Macroautophagy And Mitophagy in Neurodegenerative Disorders: Focus On Therapeutic Interventions Part 4

Additionally, besides mitophagy, mHTT disturbs mitochondrial trafficking, since mHTT aggregates may physically obstruct intracellular organellar dynamics, whereas diffuse mHTT can also interfere with trafficking adaptor proteins, affecting mitochondrial and autophagosomal dynamics [314]. 

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An expected consequence of mHTT-induced mitochondrial dysfunction is a decrease in ATP production, restricting ATP-dependent activities of the proteostasis network [315]. 

Besides ATP limitation, mitochondrial dysfunction can damage the autophagic component of proteostasis by other mechanisms. For example, mitochondrial dysfunction might disrupt ER-mitochondrial contact sites, which compromises autophagosome formation [316]. 

Likewise, mitochondrial dysfunction is related to abnormal mitochondria–lysosome reciprocal regulation [316] and impaired lysosomal function [317], which may compromise the completion of autophagy.

4. Therapeutic Strategies

4.1. Targeting Macroautophagy and Mitophagy in AD

Current AD therapies largely delay the symptoms without targeting the pathogenic mechanisms. The presence of misfolded proteins and impairment of autophagy are two relevant causative factors in AD; thus, autophagy constitutes a possible therapeutic approach for AD. 

Although it is speculative that induction of autophagy might be a promising therapeutic strategy, the disruption of several steps of autophagic flux described in AD complicates this assumption. Moreover, caution must be taken when studying the effect of autophagy-targeting drugs in the different phases of disease progression. 

A possible mechanism to activate autophagy is through the modulation of upstream regulatory signaling pathways towards autophagy induction. The mTORC1 pathway is a well-established signaling player in autophagy regulation and is dysregulated in AD, establishing a possible therapeutic target. Rapamycin is an FDA-approved drug that inhibits mTORC1 activity, resulting in autophagy activation. 

Long-term treatment with rapamycin rescued cognitive deficits in several mouse models of AD [318]. The beneficial effects mediated by rapamycin include decreasing Aβ deposition and reducing phosphorylation and accumulation of misfolded tau into NFTs [319,320]. 

SMER28, an enhancer of rapamycin, increased autophagy by an ATG5-dependent pathway and reduced Aβ peptide accumulation [321]. Notably, rapamycin-mediated improvements were observed when the treatment was initiated before symptoms onset [322]. Consistently, autophagy induction with rapamycin was not effective in ameliorating cognition if Aβ plaques and tangles were already present [322]. 

In the late stages of the disease, the lysosomal system is severely compromised, and rapamycin treatment might even exacerbate the pathology. Despite all the preclinical evidence showing that modulation of mTORC1 with rapamycin is a promising therapeutic strategy at early stages of AD, no clinical trial has been carried out so far [323]. 

It is noteworthy to highlight that mTOR is a central hub of intracellular signaling, including regulation of gene translation and cell homeostasis. It is reasonable to speculate that long-term inhibition of mTOR might have side toxic effects, so it is urgent to develop novel autophagy modulators. Another promising strategy is the genetic modulation of autophagy-related proteins that are disturbed in AD. 

For instance, a study showed that injection of a lentivirus encoding beclin-1 in the hippocampus and frontal cortex of APP-transgenic mice induced autophagy and reduced intracellular and extracellular Aβ deposition [113]. 

Moreover, increasing p62 expression in an APP/PS1 mouse model resulted in autophagy activation by a mTOR-independent pathway, leading to a reduction of Aβ pathology and amelioration of cognitive deficits [324]. Genetic-based strategies to modulate autophagy might constitute a more accurate therapy in the AD context, but a deeper characterization of all the autophagic steps and proteins affected in AD is required before therapeutic strategies' implementation. 

Carbamazepine (CBZ) is an anti-epileptic drug that showed promising results in the management of agitative behavior in AD patients [325]. The CBZ was found to inhibit mTORC1 activity, thus enhancing autophagy and decreasing amyloid burden and Aβ42 levels in transgenic AD mouse models [326]. Nevertheless, its secondary interactions with other drugs make it less popular among anticonvulsants used in the context of AD. 

Memantine is an antagonist of the N-methyl-D-aspartate (NMDA) receptor and one of the few FDA-approved drugs for the symptomatic treatment of moderate to severe AD. 

Some studies demonstrated its efficiency in ameliorating synaptic function, brain amyloidosis, and memory decline [327]. A study showed that memantine might exert its function by inhibiting autophagy through mTORC1 or Beclin-1 signaling in AD [328]. 

Contrariwise, memantine was identified as an autophagy enhancer in a large screening study to characterize clinically approved molecules for age-related disorders and neurodegeneration [329]. 

Combination therapy of memantine with cholinesterase inhibitors has been used in moderate to severe AD cases and shown improvement in cognition [330], but more studies are required to clarify the mechanism of action of memantine as an autophagy modulator in AD.

Metformin is an antidiabetic drug that activates AMPK by increasing AMP levels and has been used in diabetes type II patients to prevent dementia and Alzheimer-like phenotypes associated with hyperinsulinemia [331]. The use of metformin as monotherapy for AD is contradictory. 

AMPK activation suppresses mTOR activity and induces autophagy, thus exacerbating autophagosome accumulation and also Aβ generation by γ- and β-secretase [332,333]. Metformin was found to aggravate Aβ pathology through increased Aβ generation by transcriptional up-regulation of BACE1 and Aβ secretion in neuronal cell models [332]. 

A very promising work showed that metformin can increase microglial phagocytosis and lysosomal acidification, enhancing extracellular Aβ clearance and preventing plaque formation [334], which may compensate for Aβ generation and secretion in neuronal cells. 

Other studies showed that metformin decreases hyperphosphorylation of tau through an AMPK-independent mechanism [335]. Further research demonstrated that metformin increases the levels of insoluble tau species despite having a beneficial effect on the hyperphosphorylation state of tau [336]. The overall studies performed so far to characterize the effect of metformin in AD have generated conflicting results [337]. 

These discrepancies are probably an outcome of the effect of metformin on distinct biological pathways (reviewed in detail in [338]) and further research is required to address whether metformin should be used as an AD therapeutic drug targeting autophagy. 

The use of natural bioactive compounds extracted from food to modulate biological processes has been in the spotlight for the last few years. One of the best-characterized targets is AMPK, both in neurodegenerative and metabolic disorders. Resveratrol, a polyphenol found in red grapes and a recognized SIRT1 activator, was shown to stimulate AMPK in neurons [339]. 

Additionally, resveratrol lowers Aβ levels and secretion through AMPKmediated inhibition of mTOR activity and an increase of lysosomal degradation in mouse primary neurons [340]. Moreover, dietary supplementation with resveratrol lowered amyloid plaque formation and rescued memory loss in transgenic AD mouse models [341]. 

Resveratrol can indirectly activate SIRT1 and promote the clearance of abnormal proteins through mTOR-dependent and independent mechanisms [342]. In a 52-week phase II clinical trial, resveratrol was orally administered to AD patients with mild-to-moderate dementia and was detected in low nanomolar concentrations in the CSF of these patients, meaning this compound can cross the BBB but with poor bioavailability. 

Surprisingly, Aβ40 levels were present in CSF and plasma of placebo-control groups at lower levels compared to the resveratrol-treated group in the final phase of the study [343], supporting the crossing through the BBB and a central effect on the brain. 

However, a positive effect of resveratrol on AD biomarkers such as CSF and plasma Aβ42 or p-tau was not detected in this pilot study, possibly due to the small size sample. 

Nevertheless, resveratrol might have a beneficial effect on AD through the activation of autophagy by SIRT1 and mTOR-mediated signaling pathways. Quercetin is a flavonol mainly found in black and green tea that can also activate AMPK. Recent studies showed that quercetin administration reduced Aβ deposition and tau hyperphosphorylation and ameliorated cognitive function in a triple transgenic AD mouse model [344]. 

Moreover, long-term treatment with quercetin was shown to be preventive in neurodegeneration if administered before the appearance of AD histopathological alterations in the same model [344]. The possible modulation of autophagy by resveratrol or quercetin in AD supports further research as therapeutic strategies. Modulation of GSK3 activity may also constitute a new therapeutic strategy for AD [345]. 

GSK3β regulates phosphorylation levels of tau and PSEN and can also modulate lysosomal acidification and reduce Aβ production [346]. Curiously, it was proposed that a beneficial effect of GSK3β inhibition occurs by reactivation of mTOR and repression of autophagy, which, in turn, ameliorate cognitive functions in the 5xFAD mice model. 

Other studies contested the protective effect of mTOR modulation in AD; activation of mTOR improves cognitive deficits and was associated with a reduction in Aβ-associated events [347,348]. 

Lithium, a mood stabilizer used for bipolar disease, has been tested as a potential therapeutic drug for AD [349,350]. Lithium regulates several biochemical processes, among them the inhibition of GSK3β and modulation of the phosphatidylinositide cascade [351]. The intake of lithium by mutant tau transgenic mice delayed the formation of neurofibrillary tangles [352] and reduced Aβ pathology in the Drosophila AD model [353]. 

Moreover, lithium is a positive regulator of autophagy both by an mTOR-dependent pathway [354] and by depletion of intracellular free inositol and 1,4,5-inositol trisphosphate (IP3) [355], possibly through the inhibition of the IP3 receptor signaling cascade, a distinct autophagy-modulatory mechanism [356]. 

A clinical trial is currently ongoing to evaluate the effect of lithium on the onset of dementia in elderly people and its correlation with AD progression (Clinical trial: NCT03185208). 

Long-term treatment with nicotinamide reduced the accumulation of Aβ and hyperphosphorylated tau in the cortex and hippocampus of 3xTg-AD model mice with attenuation of cognitive decline [357]. 

This was accompanied by improvement of lysosomal acidification, autophagic–lysosomal function, and reduction of LC3-II, which indicates improvement of autolysosomal degradation. Consistently, another study with a 3xTg-AD mice model showed that nicotinamide administration was able to modulate SIRT1 activity and lysosomal proton gradient, thus promoting autolysosome acidification [358]. 

Despite its apparent beneficial effects, nicotinamide showed good tolerance but limited efficacy in cognitive improvement in phase II clinical trials with AD patients after 24 weeks of treatment (Clinical trial: NCT00580931) [359]. 

Nevertheless, other clinical trials were approved to evaluate nicotinamide's effect on tau levels in CSF of AD patients during 48 weeks (Clinical trials: NCT03061474), but no results have been published so far. Despite potential beneficial effects, nicotinamide that is released from nicotinamide adenine dinucleotide (NAD+, a nucleotide required for sirtuins activity) during the deacylation reaction of sirtuins was shown to act as a non-competitive inhibitor [360] and thus may abrogate neuroprotective effects exerted by these class III lysine deacetylases. 

Because the late steps of autophagic flux are also compromised in AD, it is possible that therapeutic targeting of early steps may not be sufficient to fully restore the mechanism and alleviate protein aggregate accumulation. 

Therefore, an interesting target for drug development is TFEB, which modulates autophagosome and lysosome biogenesis and is also a regulator of autophagy [149]. It was shown that overexpression of TFEB reduced Aβ accumulation and the presence of phospho-tau in tangles and ameliorated cognitive and synaptic deficits in both the rTg4510 and APP/PS1 mouse model of AD [361]. 

Another study showed that injection of TFEB into the hippocampus of APP/PS1 transgenic mice by an adeno-associated virus promoted its translocation into the nucleus and enhanced lysosomal function and autophagy, which lowered Aβ levels and amyloid plaques [155]. 

Moreover, activation of astroglial TFEB resulted in the clearance of extracellular tau through lysosomal degradation, contributing to the reduction of tau spreading [362], which supports a neuroprotective effect of TFEB modulation in AD tau pathology. Therapeutics targeting TFEB activation should consider avoidance of interfering with mTOR activity and related signaling pathways. 

The search for new TFEB-targeting molecules has increased in the last few years by attenuating Aβ generation and amyloid plaque deposition in AD. Since neuronal mitophagy is compromised in AD, the discovery of new mitophagy inducers constitutes a promising therapeutic approach to promote the clearance of damaged mitochondria and associated pathogenic hallmarks of the disease [363]. 

Modulation of autophagy, particularly the late steps, is expected to enhance mitophagy in AD. For instance, metformin stimulates mitophagy by restoring mTOR-dependent activation of autophagy and the Parkin-mediated mechanism [364]. 

Another study showed that modulation of autophagy with Nilotinib promotes the increase of intracellular levels of Parkin that interacts with Beclin1, reducing Aβ accumulation [365]. Nevertheless, is still unknown if the modulation of Parkin levels by Nilotinib also promotes mitophagy in AD.

Modulation of the NAD+/sirtuins pathway is a potential therapeutic target that is expected to improve mitophagy in AD. A recent drug-screening study showed that a combination of NAD+ precursors with mitophagy inducers, urolithin A and actinonin, restored mitophagy and ameliorated cognitive impairment by reducing phospho-tau and Aβ deposition in AD transgenic nematodes and mouse models [163]. 

Supplementation with NAD+ precursors attenuated Aβ and tau pathological hallmarks in AD models in part via the activation of mitophagy [163]. Overall, the conflicting data demonstrate that the induction of autophagy as a generalized therapy for AD is questionable. Stimulation of autophagy is not always beneficial and might even exacerbate Aβ pathology, resulting in elevated Aβ production and secretion. 

Autophagy is regulated by a complex crosstalk network of upstream signaling pathways that also regulate other cellular processes. The autophagy-inducing effect of each drug targeting one of these regulatory signaling pathways or proteins will most probably affect the other signaling branch with compensatory or opposite effects. Additionally, some autophagy-targeting therapies directly modulate Aβ generation. 

The differences among AD models might also complicate the conclusions about autophagy modulation and/or Aβ- and tau-associated pathologies and consequent improvement of cognitive deficits. 

Additionally, the lack of a standard method to measure autophagy in vivo also compromises the clinical assessment of the therapeutic efficiency of drugs targeting autophagy in patients. In conclusion, therapeutic development targeting autophagy should take into account the multifactorial causes of AD. 

Thus, the rational design of AD therapeutic drugs or the identification of combined therapeutic strategies should consider targeting both autophagy and APP processing or Aβ generation and tau pathology. 

The exact autophagy defect, when to use autophagy-modulating therapies during the disease progression, and the duration of the treatment should all be considered when developing novel therapeutic strategies for AD (Figure 4).

Figure 4. Overview of altered regulation of autophagy and mitophagy mechanisms in AD-role of protective compounds. Autophagy activity and related proteins are decreased in AD. mTORC1 was shown to be increased in AD, whereas its inhibitors (referred to in the purple dashed box) were shown to have positive effects in different AD models, most of them through diminished mTORC1 levels. 

Concordantly, AMPK inducer agents (referred to in the pink dashed box) were also shown to have positive effects in AD models. Several AD models showed increased autophagosome accumulation and reduced lysosomal function, which results in Aβ and Tau oligomer accumulation. 

Moreover, inducers of lysosomal function (referred to in the brown dashed box) were shown to significantly improve AD features in different AD models. Regarding augmented damaged mitochondria, AD models showed reduced mitophagy activity, whereas the inducer agents (referred to in the black dashed box) were also shown to increase mitophagy in AD models. 

Additionally, PINK1 levels were shown to be reduced in AD models, while the inducer agents (referred to in the yellow dashed box) were shown to increase those levels with positive effects.

4.2. Targeting Macroautophagy and Mitophagy in PD

Growing evidence provided by PD models reveals that the autophagic machinery is deeply hampered in PD, so macroautophagy emerges as a relevant therapeutic target to promote the clearance of toxic aggregates. 

For instance, the administration of rapamycin is sufficient to protect dopaminergic neurons from degenerating in mice subjected to MPTP-induced parkinsonism [366]. The protective effect of rapamycin is associated with increased autophagy, since mice exposed to MPTP with prior rapamycin treatment had no observable accumulation of late autophagosomes, unlike the group that was exposed solely to MPTP. 

In another model of PD, using 6-OHDA-treated mice, rapamycin was also effective in restoring motor impairment with decreased aSyn deposition [367]. Likewise, Crews and co-workers elegantly demonstrated that intra-cerebral infusions of rapamycin in the cortical layer of mice overexpressing WT aSyn mitigated the anomalies observed in the autophagic process, such as LC3 and cathepsin D expression, along with augmented clearance of aSyn deposits [368]. 

A study using metformin to enhance AMPK1-dependent autophagy demonstrated that this drug was able to rescue motor impairment and the proinflammatory profile of MPTP-treated mice, through an increased activity of autophagy and the rescue of the autophagic flux [369]. 

Although these studies highlight the importance of autophagy to clear aSyn deposits and to halt the neurodegenerative process, upstream regulation of autophagy produces many undesirable effects, which affect treatment efficacy in many disease models [370]. 

Beclin-1, for instance, is essential in the initial steps of autophagosome formation and thus plays a key role in controlling autophagy. Effectively, Beclin-1 lentiviral injection in mice overexpressing aSyn rescues the negative impact of aSyn pathology in the cortical layer of the brain by ameliorating the number of LC3-positive vesicles and consequently enhancing the clearance of aSyn aggregates present in these mice [371]. 

In consonance with this study, the activation of Beclin-1-dependent autophagy with isorhynchophylline induced the degradation of aSyn oligomers in dopaminergic neurons constitutively overexpressing the A53T aSyn mutant form [372]. Mitophagy is likely to have a significant contribution since Beclin-1 activity contributes to the translocation of Parkin to the OMM for the initiation of mitophagy [373]. 

As described before in this review, the removal of damaged mitochondria by mitophagy is highly significant for normal neuronal function. Indeed, an increasing body of evidence demonstrates the therapeutic value of removing damaged mitochondria in several PD models [374]. 

Pharmacological activation of PINK1 through the treatment of dopaminergic SH-SY5Y cells with a neo-substrate, kinetin triphosphate, increases the extent of phosphorylation of its substrates and promotes faster recruitment of Parkin to mitochondria, resulting in an amelioration of the negative impacts caused by oxidative stress [375]. 

Additionally, an interesting study using Drosophila melanogaster demonstrated that, by overexpressing PINK1, the removal of damaged mitochondria improves through higher facilitation of mitochondrial axonal motility [376]. 

Consistent with this approach, overexpression of Parkin is beneficial in an MPTP mouse model. Transgenic mice overexpressing Parkin are much less susceptible to MPTP-induced dopaminergic neurodegeneration and present lower levels of aSyn oligomers in striatal homogenates [377]. 

Likewise, flies who were administered with SR3677 a ROCK inhibitor that promotes mitophagy enhancement through Parkin activation demonstrated alleviated motor behavior after a paraquat insult. 

ROCK is consistently activated in several neurodegenerative models and is also considered to be an autophagy modulator [378]. Remarkably, damaged mitochondria are driven for lysosomal degradation upon SR3677 treatment [379].

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