Autophagy and its neuroprotection in neurodegenerative diseases***☆○

Ping Gu , Avaneesh Jakkoju, Mingwei Wang Weidong Le,

1Department of Neurology, First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China

2Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA

3Institute of Neurology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

INTRODUCTION

Normal cell growth and development requires a dynamic equilibrium between protein synthesis and degradation.

Presence of either aggregates or soluble toxic forms of the proteins is particularly harmful in neurons which often results in neuronal death. The ubiquitin-proteasomal system (UPS) is mainly involved in the degradation of short-lived misfolded proteins that are restricted to the cytosol and nucleus or endoplasmic reticulum. In contrast, the autophagy-lysosome pathway (ALP)comprises vesicular organelles with various hydrolases, which contribute to degradate extracellular proteins (endocytosis or pinocytosis), long-lived intracellular protein and damaged organelles (such as mitochondria)[1]. ALP usually acts in parallel with UPS to control the homeostasis of various cell functions, and ALP is the most important degradation mechinary when UPS is impaired[2].

Autophagy has been implicated in various physiological processes including stress response, cellular differentiation, and programmed cell death and in a few pathological conditions serving as a quality control mechanism for misfolded proteins.

ALP has important roles in development,immune defense, programmed cell death,tumor suppression, pathogen infection, and many diseases[3]. Recently, there has been a growing interest in identifying the role of ALP in numerous neurodegenerative diseases such as Parkinson’s disease (PD),Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and polyglutamine diseases that includes Huntington’s disease(HD). These diseases have a common pathological characteristic deposits of protein aggregates in association with neurodegeneration in central nervous system (CNS). These aggregated proteins can be cytoplasmic, nuclear or extracellular.

Preventing aggregation or disaggregating misfolded or conformationally altered proteins may provide potential therapeutic benefit for these neurodegenerative diseases[4]. In this review, we will discuss the various aspects of ALP and its dual roles in neuronal cell death and survival.

Then, we will focus on the neuroprotection and putative therapeutic potential of autophagy in several neurodegenerative diseases.

THE PATHWAYS UNDERLYING AUTOPHAGY AND GENERAL FUNCTIONS

Autophagy is a cellular process of “self-eating or self-digest”. There are three forms of autophagy named macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) based on the mechanism and function of phagocytic activity in cell[4].

Of these three forms, macroautophagy is often referred to as autophagy. Autophagy is designated to process cytoplasm, senescent intracellular organelles, long-lived stable proteins, large membrane proteins and protein complexes (including oligomers and aggregates that are too big to pass through the proteasome) to form double-membrane vesicles (autophagic vacuoles, AVs or autophagosomes) and subsequently fused with lysosome as a mature autophagolysosome, where their contents are then degraded by acidic lysosomal hydrolases. The degradated products are recycled for the synthesis of new molecules and reused in the cytosol[3]. The core of the molecular machinery of autophagy consists of a specific family of genes called the autophagy related gene (Atg)[3]. The microtubule-associated protein 1 light chain 3 (LC3, a mammalian homolog of Atg8 in yeast) is commonly used as a specific marker of an autophagosome. LC3 is localized to the autophagosomal membrane during autophagy formation[1]. Microautophagy is responsible for the gradual, continuous turnover of cytosolic proteins.

Lysosomes can directly engulf cytoplasm by invagination,protrusion, and separation of lysosomal membrane even under resting conditions. In contrast to micro and macroautophagy, where whole regions of the cytosol are sequestered and delivered to lysosome, CMA targets single cytosolic proteins to the lysosomal surface and mediates their one-by-one translocation across the lysosomal membrane for degradation. The unfolded and soluble cytosolic proteins containing a specific penta peptide motif are selectively targeted to the lysosomal lumen for degradation via the lysosomal chaperon in heat shock cognate 70 (hsc70) and lysosome-associated membrane protein type 2A (LAMP-2A) complex binding[5].

Although basal macroautophagy and CMA activities can be detectable in different cell types and tissues, they can be activated by oxidative stress or nutrient stress;alteration in their activities has been implicated in CNS injury and diseases[4-5]. Blockage of macroautophagy can lead to up-regulation of CMA in both normal condition and stress[6]. CMA works in a coordinated manner with other autophagic pathways to compensate each other.

CMA is the only pathway for the selective degradation of soluble protein in ALP and can clear damaged proteins without interfering nearby functional ones[7].

ALP breaks down the unwanted and damaged organelles and proteins as housekeeping process to prevent the accumulation of protein aggregates which affect the function of neurons[5]. The dysfunction of the ALP is an important process in a variety of neurodegenerative diseases that are believed to be mediated by toxic, aggregate-prone, intracytosolic proteins which become inaccessible to the UPS when they form oligomers[4].

AUTOPHAGY AND PROTEIN DEGRADATION HOMEOSTASIS IN NEURONS

A steady state of basal and induced autophagy is important for the physiological control of the number and quality of organelles by eliminating superfluous, aged and damaged organelles and proteins, particularly in the nervous system. Neurons, as highly metabolically active,post-mitotic cells, are especially vulnerable to the accumulation of defective proteins and degeneration[8].

Extreme polar morphology of a neuron makes its autophagic function vulnerable for disruption as autophagosomes generated at the distal end of axons and dendrites will have to travel long distances to fuse with lysosomes concentrating in or near perikaryon[9]. By virtue of a neuron’s non division nature and inability to re distribute defective proteins and organelles, basal and induced autophagy are essential for its survival[5].

Dysfunction of the UPS has already been strongly implicated in the pathogenesis of many neurodegenerative diseases; autophagy can serve to degrade ubiquitinated proteins[10]. Proteasome inhibitor lactacystin induces autophagy enhancement and increases levels of p53. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) or beclin 1 siRNA further activates p53 as transcription regulator and causes neuron apoptosis. While the autophagy inducer rapamycin protects dopaminergic (DA) neurons from lactacystin-mediated cell death by down regulating p53 and its related apoptotic pathways and by inducing autophagy to degrade aggregated proteins[11].

Suppression of neuronal constitutive autophagy by targeted deletion of Atg5, Atg7 genes in mice models[12-13]and Atg8a gene in adult drosophila[14]resulted in severe neuronal degeneration. Autophagy-deficient neurons accumulate ubiquitinated proteins as inclusion bodies and degenerate progressively.

Neurons are predominantly vulnerable to develop autophagic stress with aging, because of an increased requirement for autophagic clearance of worn-out components. Both macroautophagy and CMA have been shown to decline with aging in rodents and probably in humans[4,15]. The decreased autophagic activity occurs concomitantly with the accumulation of insoluble ubiquitinated and oxidized proteins with age. Enhanced autophagy-related gene 8a (Atg8a) expression in older fly brains extends the average lifespan by 56% and promotes resistance to oxidative stress and accumulation of ubiquitinated proteins[14]. This data supports the critical role of autophagy as an anti-aging mechanism. Maintenance of proper autophagy prevents the age-dependent accumulation of damage in neurons and promotes longevity[14].

If autophagy is impaired, misfolded and aggregate-prone proteins are often seen accumulated in the brain of neurodegenerative diseases[4,8-9]. As we know, proteins found to be mutated including amyloid precursor protein(APP) for AD, α-synuclein for PD and huntingtin for HD[16-19]. Mutant copper-zinc superoxide dismutase(SOD1) for familial ALS (fALS) can be recognized by p62 linked with LC3-II, targeted for ALP[19]. If autophagy is up-regulated, the clearance of such proteins is efficient and the neurotoxicity is attenuated in the models of these neurodegenerative diseases. Conversely, inhibition of autophagy by ablating autophagy genes or pharmacological inhibitors such as 3-MA, bafilomycin A1 can ultimately lead to cell death, further confirming a critical role for autophagy in various pathological states of the nervous system[4,12-13].

Mitochondria play a critical role in neuron homeostasis and survival/death by regulating energy production and apoptosis. Dysfunction of mitochondria and alterations of dynamics are also strongly implicated in neurodegenerative diseases[4,8]. Mitophagy is the major pathway for autophagic clearance of damaged mitochondria in neurons, defending the cells against apoptosis. Damaged mitochondria induced by neurotoxins 1-methyl-4-phenylpyridinium (MPP+) or 6-hydroxydopamine (6-OHDA) are selectively degraded by autophagy in neurons[20-21]. It has been shown that the neuroprotective effect of autophagy-inducers such as rapamycin, lithium and trehalose are at least partially mediated through upregulating macroautophagy and enhancing degradation of misfolded proteins[11,20,22-24].

Considering all of the available data, there is no doubt that autophagy has a beneficial effect of protecting against neurodegeneration. While autophagy dysfunction may induce or participate in accumulation of toxic aggregates there by leading to neuronal cell death causing neurodegenerative diseases. Thus regulation of autophagy can have favorable outcomes in neurodegenerative diseases depending on the stage of neurodegeneration.

DUAL ROLES OF AUTOPHAGY IN NEURONAL CELL DEATH AND SURVIVAL

As above, under most circumstances, autophagy constitutes a stress adaptation pathway that promotes neuron survival. During the last few years, studies have indicated that enhanced autophagy may be either an alternative pathway of neural cell death[15]or an ultimate attempt for cells to survive[4,12]. Increasing attention has been focused on the role of autophagy in neuronal death associated with chronic neurodegenerative diseases, in which either impaired autophagic degradation or excessively activated autophagy may lead to the disruption of the intracellular organelles and accumulation of AVs in neurons[25]. Increased autophagic markers (such as AVs or LC3) have been associated with characteristic of degenerative or dying neurons described in neurodegenerative diseases, which has led to the hypothesis that autophagy may also have a causative role in stress-induced cell death[26]. A recent study done by Zhang et al[27]showed paradoxical increase in disability in presymptomatic ALS mouse model treated with rapamycin which is known to enhance autophagy in nerve cells.

Generally, there are at least three possible mechanisms of neuron death: apoptosis, necrosis and autophagic cell death. Programmed cell death in neurons involves apoptosis (termed type I cell death). However, autophagy has been observed as a primary death mediator (called type II cell death) in several models ranging from unicellular organisms to Drosophila melanogaster. It is characterized by the massive accumulation of cytoplasmic AVs[28]. Autophagy may not only be a cause of cell death, it may also precede apoptosis as a defense mechanism. At low levels of stress, autophagy could represent a primary attempt to reestablish neuron homeostasis. When the autophagic capacity is overwhelmed, apoptosis is triggered. Blocking autophagy can significantly decrease caspase-3 activity;whereas blocking apoptosis can increase the formation of autophagosomes[29]. Autophagy caused by Atg1 over expression in Drosophila is sufficient to induce cell death.

The affected cells are caspase-dependent and display apoptotic features[30].

Apoptosis and autophagy may be co-regulated in the same directions. Autophagy can lead to apoptosis or necrosis, presumably via common regulators such as the anti-apoptotic Bcl-2 family[31-33]and the transcription factor p53[11,32]. Apoptotic cell death is mediated by caspase activation as the anti-apoptotic Bcl-2 and Bcl-xL proteins[31,33]. The anti-apoptotic proteins Bcl-2 and Bcl-XL bind Beclin 1 (mammalian Atg6, an autophagic effector) and inhibit autophagic activity[31,33]. Disruption of these interactions by the pro-apoptotic BH3-only proteins or BH3 mimetic induces autophagy.

Apoptosis can also suppress autophagy. Pro-apoptotic protein Bax reduces autophagy by enhancing caspase-mediated cleavage of Beclin 1 at D149. The cleavage of Beclin 1 is a critical event whereby caspases inhibit autophagy, as a non-cleavable Beclin 1 mutant restored autophagy in cells overexpressing Bax[34]. The activation of autophagy beyond a certain threshold may promote autophagic cell death directly by causing the cellular dysfunction as a result of cellular atrophy, while inefficient activation of autophagy increases cell vulnerability and often results in cell death[35]. Therefore,it is all depending on the actual cellular environment that both over activation and deregulation of autophagy seems to induce neuronal degeneration. Indeed, the consequences of autophagy activation may well depend on a particular physiological condition[4]. How the precise balance between autophagy and apoptosis is maintained in neurons requires further investigation.

AUTOPHAGY AND NEURODEGENERATIVE DISEASES

Formation of intracellular aggregates is characteristic of many neurodegenerative diseases and alterations in the autophagic pathway leads to the accumulation of neurotoxic proteins and neuronal cell death. The accumulation of AVs has been observed in affected brain regions in many neurodegenerative diseases including AD, PD, HD and ALS. Gene mutations, defects in protein translation, and post-translational modifications can lead to molecular conformation changes and these proteins become aggregate-prone[3-4]. Increasing line of evidence indicates that neurodegeneration is frequently associated with UPS dysfunction and failure in the clearance of proteins composed of ubiquitinated conjugates. It has been suggested that autophagy is induced in the early stage of disease and impaired at the later period of the neurodegenerative process.

Alzheimer’s disease

AD, as a major cause of disability and death in the elderly population, is the most prevalent human degenerative disease. Extracellular aggregated β-amyloid (Aβ) in neuritic plaques and abnormally phosphorylated tau in intraneuronal fibrillary tangles are generally thought to be the major culprits in the neurodegenerative process of AD. Progressively striking accumulation of autophagosomes is a hallmark of AD neuropathology. Autophagy is found induced in the brain of early stage of sporadic AD and in PS1/APP transgenic mouse, independent of Aβ deposition. In the PS1/APP model, autophagosomes proliferate in vulnerable neuronal dendrites at young ages before the extracellular deposition of Aβ, implying autophagy as an initial protective response to the pathopoiesis factors at the early stage of disease[16]. PS1 gene mutation is the main cause of early onset familial AD. PS1 is not only a component of the γ-secretase complex but may be required for AVs maturation and specifically fusion with lysosomes. PS1 mutation leads to altered trafficking of APP[36]. With the progression of AD, AVs are accumulated abnormally in damaged or dystrophic neurites due to a progressive impairment of autophagy[37].

Indeed, the accumulation of AVs are a major site for producing intracellular pathogenic Aβ in AD brain. The abnormally accumulated autophagosomes contain APP,Aβ, and exceptional enrichment of the β-secretase and γ-secretase complex as well as APP-rich organelle membranes. Studies have shown that autophagy pathway directly participates in the metabolism of APP and intracellular Aβ generation. Within AVs, APP is cleaved by the β and γ-secretase and eventually generates cytotoxic Aβ[16]. Either stimulating AVs production or impairment of maturation of AVs to lysosomes might be expected to increase the number of AVs and raise intracellular Aβ levels. With the deterioration of AD, increased autophagy in the neuropil could be a significant source of Aβ overproduction[37]. It is widely believed that Aβ is a major intracellular toxic peptide and plays a key role in the pathogenesis of AD.

The dysfunctional autophagy promotes β-amyloidogenesis and neuronal cell loss[16]. Regulating autophagy in a neuron can change the levels of intracellular Aβ.

The incidence of AD is greatly increased by hypoxic injury. It is believed that cerebral ischemia or stroke plays an important role in the onset and development of AD.

Increasing evidence suggests that hypoxia facilitates the pathogenesis of AD through accelerating the accumulation of Aβ and increasing the hyperphosphorylation of tau. Hypoxia may accelerate Aβ production by modulating the APP processing through activating autophagy. Long-term and repeated hypoxia treatment can increase Aβ by elevating β-cleavage of APP and lead to more and larger senile plaque formations in APPSwe+PS1A246Edouble transgenic mice.

Hypoxia enhanced the expression of APH-1a, a component of γ-secretase complex, which in turn may lead to increase in γ-cleavage activity[38]. Therefore, the ALP is considerably more complex in AD because it is simultaneously both induced and impaired. A strong link seems to exist between AD and autophagic dysfunction and amyloidogenesis.

In contrast to UPS, autophagy could efficiently degrade both soluble mutant tau and its aggregates. Activation of autophagy can reduce tau pathology[39]. Wild-type (WT)tau protein has been proposed to be necessary for proper autophagy function. Inhibition of ALP can not only increase tau levels but also enhanced aggregation and cytotoxicity of the mutant tau. Hyperphosphorylation of tau, causing neurofibrillary tangles in AD, may influence its accessibility to autophagy[40]. A recent study has also revealed the connection between CMA and AD by tau protein. CMA is involved in the delivery of cytosolic tau to lysosomes for cleavage. However, the pathogenic fragment does not fully enter the lysosome but remains associated with the lysosomal membrane. This inefficient translocation seems to promote formation of tau oligomers at the surface of these organelles which may act as precursors of aggregation and interfere with lysosomal function[40]. Induction of autophagy by rapamycin has been shown to promote degradation of mutant htau40/P301L. This would indicate enhanced autophagy as a potential therapeutic route for AD.

Parkinson’s disease

PD is a neurodegenerative disease characterized by a massive loss of nigrostriatal DA neurons. A classic pathological hallmark is the occurrence of filamentous intracellular inclusions named Lewy bodies (LB) that consist of mainly α-synuclein. The DA neurons in the nigrostriatal system when injured are prone to developing autophagic stress. Increased AVs are described not only in DA neurons of midbrain in patients with PD[41], but also in genetic and toxic PD models[20-21,42]. The increased AVs in the early stages and progressive stages of PD is considered as a defensive or a protective response by the auto-regulative mechanism of autophagy, in order to accelerate the degradation of cytoplasimic misfolded proteins through upregulated autophagy. However, with the progression of the disease, this compensatory auto-regulative mechanism is ultimately unable to maintain the cellular homeostasis and eventually results in neurodegeneration[37,43]. All three forms of autophagy have been reported to be affected in PD, including macroautophagy, mitophagy and CMA[24]. Atg5 knockout mice models accumulate ubiquitin-positive inclusion bodies in neurons and develop parkinsonism[12].

Both mutation of α-synuclein and increased levels of WT α-synuclein are thought to be crucial in PD pathogenesis[17]. Over expression, misfolding, or decreased degradation are all pathways that may lead to the accumulation of α-synuclein in neurons. Excessive WT α-synuclein can lead to PD in rare familial cases and α-synuclein protein accumulation also is detected in sporadic PD[36]. Both ALP and UPS might be involved in α-synuclein turnover[44]. CMA and macroautophagy are important pathways for WT α-synuclein degradation in neurons[45]. CMA has been identified as the major degradation pathway used for WT α-synuclein in neurons[18]. The mutants A53T and A30P could be degradated by macroautophagy[18,45]. Interestingly, while soluble forms of α-synuclein can be cleared by CMA,α-synuclein mutants (A53T and A30P, the cause of autosomal dominant early-onset PD) actually inhibits CMA by tightly binding to LAMP-2A and blocks mutated α-synuclein and other CMA substrates entering lysosomes, hereby inhibiting themselves from degradation[18]. Thus, accumulated mutant α-synucleins and other substrates further disturb cellular homoeostasis and contribute to neuronal toxicity. PC12 cells over expressing the A53T mutant form of α-synuclein exhibit autophagic cell death[46]. Impairment of CMA and/or macroautophagy may be major factors in the initiation of the pathological cascade in sporadic PD[43]. All the above evidence supports the hypothesis that impaired autophagic degradation of α-synuclein is an important mechanism of neurodegeneration in PD.

Lysosomal dysfunction could help explain α-synuclein accumulation in PD. Lysosomal malfunction also accompanies α-synuclein aggregation in a progressive mouse model of PD treated chronically with MPTP and probenecid[47]. Mutation in the gene encoding for lysosomal protein glucocerebrosidase increases the risk for PD at least 5 fold, which also provides a further link between the lysosomal pathway, CMA and PD[48].

Moreover, loss of function mutations in the lysosomal ATPase ATP13A2 in patients with autosomal recessive early-onset parkinsonism lead to a failure of autophagy execution and aggregation of α-synuclein[49]. All these data support the association of PD with lysosomal dysfunction.

Several genes associated with PD have been linked to ALP. Recent study shows that mutant ubiquitin carboxylterminal hydrolase L1 (UCH-L1) also inhibits CMA by binding to LAMP-2a and blocking the interaction of normal substrates with LAMP-2a. Impairment of CMA by mutant UCH-L1 and α-synuclein activates macroautophagy in order to compensate for the impaired cellular degradation[50]. LRRK2 mutations are identified to cause autosomal-dominant PD, indistinguishable from the sporadic PD. LRRK2 is a negative regulator of autophagy and interacts with parkin. Mutant LRRK2 increases autophagic activity and causes neuronal degeneration[51]. Modulation of LRRK2 function may be a promising therapeutic target to help restore the balance of autophagy in PD.

Recent report indicates that the neuroprotective effects of ubiquitin ligase Parkin and the protein kinase PINK1 links ubiquitylation with selective autophagy of impaired mitochondria[52]. Mitophagy employs the ubiquitin ligase Parkin. Parkin has been reported to facilitate not only the ubiquitin mediated sequestration but also the engulfment of dysfunctional mitochondria by selectively recruiting mitochondria and contributes to their degradation by autophagy[53-54]. Parkin over expression is protective in PINK1-deficient cells by enhancement of autophagy[55].

Mitochondria are important regulators of autophagy-induced cell death. It has been demonstrated that exposure to pesticide rotenone and herbicide paraquat can be risk factors in the incidence of PD.

Parkinsonism neurotoxin MPP+, 6-OHDA as well as mitochondria inhibitor paraquat, rotenone elicit mitochondrial injury and autophagic stress[20-21,56-57].

MPP+ induces autophagy and mitochondrial degradation that is inhibited by siRNA knockdown of autophagy proteins Atg5, Atg7 and Atg8. Interestingly, all treatments that inhibit autophagy also confer protection from MPP+induced cell death[56]. Paraquat induces the accumulation of AVs and increases the degradation of long-lived proteins in SH-SY5Y cells[57]. Rotenone-induced α-synuclein aggregates are cleared following stimulation of autophagy by rapamycin[24]. All of these evidences indicate that enhanced oxidative stress possibly activates autophagy during the early stages of mitochondrial dysfunction and may help to resist the later oxidative stress-induced injury. Enhancement of autophagy through a mTOR-dependent and independent pathway by autophagy-inducers has been reported to have the protective effect in PD models[11,22,24].

Huntington’s disease

HD is a progressive, autosomal dominant,neurodegenerative disease caused by a CAG trinucleotide repeat expansion encoding an abnormally long polyglutamine (polyQ) tract in the huntingtin protein. The intracellular inclusions of mutant huntingtin in neurons are pathological hallmarks of HD and they cause cytotoxicity critical for the pathogenesis of HD[58].

Autophagy is crucial in HD, both full-length mutant huntingtin and PolyQ-expanded huntingtin aggregates could be cleared by autophagy. Many animal and cell HD models were detected to have increased autophagic activity and AVs[17,58-59]. Increased number of AVs has been also reported in the brains of HD patients. The highly ubiquitinated huntingtin is accumulated in the AVs and affects the function of neuron in HD[60]. Inhibition of autophagy at the levels of autophagosome formation by 3-MA or at the levels of autophagosome -lysosome fusion using bafilomycin A1 results in increased aggregate formation and cell death in HD cell models[61]. A recent study has shown that expanded polyQ72 aggregates induce endoplasmic reticulum stress-mediated cell death with caspase-12 activation and autophagy activation[62]. These results suggest that inhibiting ALP might interfere with the degradation of mutant huntingtin. Up regulation of autophagy by pharmacological strategy via mTOR-dependent and independent pathway has been reported to be a potential therapeutic avenue for HD.

Autophagic gene beclin 1 regulates the accumulation of mutant huntingtin. Beclin 1 reduces the levels of mutant huntingtin by promoting autophagy. The expression of beclin 1 decreases with aging in human brains. Beclin 1-mediated long-lived protein degradation pathway plays a key role to the catabolism of mutant huntingtin.

Mutant huntingtin has a negative effect on beclin 1-mediated protein degradation[63]. This suggested that autophagy plays a potentially important role in both initiation and progression of HD.

Recent studies have identified novel small-molecule enhancers (SMERs)[10,19,28], which enhance the clearance of mutant huntingtins by inducing autophagy.

SMERs mediated autophagy induction occurs at the stage of autophagosome formation and protected against polyglutamine toxicity in neuron[59].

Small-molecule ‘chemical chaperones’ such as trehalose decreases polyglutamine aggregates and improves motor dysfunction and extends lifespan in a transgenic mouse model of HD[22,64]. In addition, a microtubule-associated deacetylase-histone deacetylase (HDAC) is reported to sufficiently rescue neurodegeneration caused by polyQ toxicity by turnover of aberrant proteins by autophagy[10]. These results suggest that autophagy is a major degradation route for mutant huntingtin and protects neurons from cytotoxicity.

Amyotrophic lateral sclerosis

ALS is a progressive neurodegenerative disease, which selectively affects motor neurons (MNs). Although most cases are sporadic ALS (sALS), 5-10% of ALS cases run in families. Mutations in the gene that encodes SOD1 are responsible for 20-25% of fALS cases[65]. In both sALS and fALS patients, the pathological hallmark is the presence of ubiquinated inclusions and altered mitochondria. Mice overexpressing mutant SOD1 develop an ALS-like phenotype comparable with human ALS. The activation status of autophagy is detected in the spinal cord of G93A SOD1 mutant mice. The autophagic marker, LC3-II protein is increased at the presymptomatic stage of the mice[66]. Expression of cathepsins and LC3-II is elevated, while the levels of phosphorylated mTOR/Ser2448 (an activated form) are decreased in SOD1G93A mutant mice at an early symptomatic stage. Therefore, it is possible that autophagy is partially regulated by the mTOR intracellular signaling pathway in the processing of degradation of mutant SOD1[67-68]. Expression of mutant SOD1 alone does not induce toxicity, whereas inhibition of autophagy induced mutant SOD1-mediated cell death and increased SOD1 levels in detergent-soluble and insoluble fractions, suggesting WT SOD1and the A4V, G85R, and G93A SOD1 mutants are degraded by autophagy and reduces neurotoxicity[69]. Defective autophagy can lead to accumulation of AVs in diseased MNs[70]. A recent study done in ALS mouse model treated with rapamycin, a known autophagy enhancer showed paradoxical worsening of motor function. This study also showed the accumulation of AVs and increased levels of autophagy marker proteins in MNs.

This finding is apparently related to increased level of apoptotic activity in MNs of animal model. It also indicates that autophagic alteration in MNs of spinal cord of mouse model occurs at an early stage of disease[27].

ALS with frontotemporal dementia due to mutation of the CHMP2B, subunit of the endosomal sorting complexes required for the transport of (ESCRT)-III may directly affect autophagy[71].Functional ESCRTs are required for efficient fusion of AVs with the endocytic pathway and for degradation of ALP. The proteins LC3-II, p62 and LAMP-2 are accumulated in neurons and astrocytes of the cortical sections in Fig4 and Vac14 mutant mice[72]. Fig4 and Vac14 are two components of the PI (3, 5) P2 regulatory complex.

Cytoplasmic inclusion bodies containing insoluble p62 and ubiquinated protein aggregates are present in regions of the mutant mouse brain. Co-localization of p62 and lamp-2 in the affected cells indicates that autophagic flux is impaired[72].

Above all, the autophagy pathway is altered at different levels in ALS. Inhibition in the autophagy machinery produces the accumulation of autophagy substrates including mitochondria and misfolded proteins, which represent a constant pathological finding in most fALS and sALS. The increased clearance of the mutated SOD1 by enhanced autophagy activity may thus be a common therapeutic strategy for the treatment of ALS[73].

THERAPEUTIC POTENTIAL OF AUTOPHAGY IN NEURODEGENERATIVE DISEASES

Currently, there are no effective therapies to slow or prevent neurodegeneration. Several neurodegenerative diseases are associated with the misfolded and aggregated proteins. The disease severity frequently correlates with the expression levels of the protein. Clearance of these aggregate-prone proteins via the modulation of autophagy may represent a potential strategy to ameliorate the progression of disease.

Data obtained so far suggest that upregulation of autophagy in an mTOR-dependent and independent pathway enhances the clearance of different forms of mutant and aggregate-prone toxic proteins including α-synuclein, huntingtin, tau[17,22-23,59,61,74-75]or damaged mitochondria[21,32,53]. mTOR is a pivotal regulator of autophagy induction, rapamycin induce autophagy by inhibiting mTOR. Studies show that rapamycin can protect neurons from degeneration in several neurodegenerative models[22,59,61,74-75]. Additionally,improvement of behavior tasks by rapamycin has been seen in both a Drosophila HD model and transgenic HD mice[17].

Several pharmacological agents have been shown to activate autophagy in an mTOR-independent pathway by inhibiting IMPase and depleting free inositol and myoinositol-1, 4, 5-triphosphate (IP3) levels including lithium, carbemazepine and valproate[62,71]. Like rapamycin, lithium increases the clearance of mutant huntingtin and delays onset of disease progression and augment the life span in HD fly model[23,75]. Treatment with lithium delays neurodegeneration and disease onset via enhancing autophagy and decreasing the ubiquitin and α-synuclein accumulation in G93A ALS mice and slow down the disease progression in patients suffering from ALS[73].

Researchers explore that combined treatment with different autophagy inducers has additive effect on autophagy and enhance neuroprotection. Combination mTOR inhibitor rapamycin with mTOR-independent autophagy inducer lithium has more beneficial effects and counteracts the autophagy inhibitory effects of mTOR activation by lithium. This rational combination treatment approach shows higher protection against neurodegeneration in a fly model of HD, compared with inhibition of either pathway alone[23]. Combination of lithium and valproate produces a greater consistent effect in delaying disease onset, prolonging survival and decreasing the neurological deficit scores in ALS model,as compared to mono-treatment with lithium or VPA[71].

SMERs in combination with rapamycin resulted in greater rates of autophagy substrate clearance,compared to either the SMERs or rapamycin alone in cell and fly models of HD[59].

Trehalose has also been shown to reduce α-synuclein and huntingtin aggregation and induce autophagy in an mTOR-independent manner[23,64]. Recent studies have shown several autophagy inducers including verapamil(an L-type alcium channel antagonist) and clonidine (an imidazoline receptor agonist that reduces cAMP levels)in an mTOR-independent pathway is regulated by intracellular calcium levels and cAMP[76].

All data have shown that autophagy induction may protect against neurodegenerative diseases caused by aggregate-prone proteins. It should be noted that combination therapies provide more effective treatment for neurodegeneration; meanwhile the low dose of each compound is safer for long-term treatment. Hence, the ability to maintain proper autophagic activity, rather than massive upregulation of autophagy, should be the therapeutic approach in the treatment of neurodegerative diseases.

CONCLUSION

Increasing lines of evidence suggest that autophagy can be induced and activated in neurodegeneration processing, especially at the early stage of the diseases. Autophagy mainly functions to remove impaired organelles and misfolded protein aggregates,and promote cell survival during stress. Autophagy activity usually declines as a result of aging and disease-related factors, which become inadequate to eliminate damaged organelles and intracellular toxic protein aggregates. Progressive dysfunction of autophagy caused by genetic and environmental risk factors may further contribute to neuronal cell demise in neurodegenerative diseases, whereas autophagy enhancement through mTOR-dependent and independent pathways can attenuate neuronal loss in animal models of neurodegenerative diseases.

Several drugs that modulate autophagy have highlighted the potential therapeutic target of autophagy for the protein misfolding related neurodegenerative diseases. However, the modulation of autophagy is a very delicate process because autophagy is a double edge sword, which may have both protective and deleterious effect on neurons in AD and PD models depending on their pathological conditions. Also worsening of motor function in ALS mouse model treated with rapamycin, which increases the autophagy in MNs further proves this point. Further research is necessary to elucidate the molecular mechanisms of regulation for autophagy and the role of autophagy in CNS functions and the neurodegenerative diseases. A better understanding of factors that influence the balance of formation and degradation of proteins in different neurodegenerative conditions is critical. Thus, modulation of autophagy should be carefully applied to treat these diseases and a successful therapy will rely specifically on targeting on autophagy pathways to restore dysfunction of the ALP.

Author contribution:Weidong Le participated in the study conception and design. Ping Gu, Mingwei Wang, and Avaneesh Jakkoju were in charge of study implementation.

Conflicts of Interest:None declared.

Funding:the National Nature Science Foundation of China,No. 30970925, 30730096, and Shanghai Pujiang Project, No.09PJD014.

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