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Background: Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. Disease pathophysiology is complex and not yet fully understood, but is proposed to include the accumulation of misfolded proteins, as aggregates are present in spinal cords from ALS patients and in ALS model organisms. Increasing autophagy is hypothesized to be protective in ALS as it removes these aggregates. Rapamycin is frequently used to increase autophagy, but is also a potent immune suppressor. To properly assess the role of rapamycin-induced autophagy, the immune suppressive role of rapamycin should be negated., Findings: Autophagy is increased in the spinal cord of ALS mice. Dietary supplementation of rapamycin increases autophagy, but does not increase the survival of mutant SOD1 mice. To measure the effect of rapamycin in ALS independent of immunosuppression, we tested the effect of rapamycin in ALS mice deficient of mature lymphocytes. Our results show that rapamycin moderately increases the survival of these ALS mice deficient of mature lymphocytes., Conclusions: Rapamycin could suppress protective immune responses while enhancing protective autophagy reactions during the ALS disease process. While these opposing effects can cancel each other out, the use of immunodeficient mice allows segregation of effects. Our results indicate that maximal therapeutic benefit may be achieved through the use of compounds that enhance autophagy without causing immune suppression.

Dysfunction of lysosomes, the primary hydrolytic organelles in animal cells, is frequently associated with aging and age‐related diseases. At the cellular level, lysosomal dysfunction is strongly linked to cellular senescence or the induction of cell death pathways. However, the precise mechanisms by which lysosomal dysfunction participates in these various cellular or organismal phenotypes have remained elusive. The ability of lysosomes to degrade diverse macromolecules including damaged proteins and organelles puts lysosomes at the center of multiple cellular stress responses. Lysosomal activity is tightly regulated by many coordinated cellular processes including pathways that function inside and outside of the organelle. Here, we collectively classify these coordinated pathways as the lysosomal processing and adaptation system (LYPAS). We review evidence that the LYPAS is upregulated by diverse cellular stresses, its adaptability regulates senescence and cell death decisions, and it can form the basis for therapeutic manipulation for a wide range of age‐related diseases and potentially for aging itself. [ABSTRACT FROM AUTHOR]

Background: Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72-ALS. Methods: In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72-ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO). Results: Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72-ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72-ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients. Conclusions: Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72-ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72-ALS. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motor neurons (MNs). Since the identification of the first ALS mutation in 1993, more than 40 genes have been associated with the disorder. The most frequent genetic causes of ALS are represented by mutated genes whose products challenge proteostasis, becoming unable to properly fold and consequently aggregating into inclusions that impose proteotoxic stress on affected cells. In this context, increasing evidence supports the central role played by autophagy dysfunctions in the pathogenesis of ALS. Indeed, in early stages of disease, high levels of proteins involved in autophagy are present in ALS MNs; but at the same time, with neurodegeneration progression, autophagy-mediated degradation decreases, often as a result of the accumulation of toxic protein aggregates in affected cells. Autophagy is a complex multistep pathway that has a central role in maintaining cellular homeostasis. Several proteins are involved in its tight regulation, and importantly a relevant fraction of ALS-related genes encodes products that directly take part in autophagy, further underlining the relevance of this key protein degradation system in disease onset and progression. In this review, we report the most relevant findings concerning ALS genes whose products are involved in the several steps of the autophagic pathway, from phagophore formation to autophagosome maturation and transport and finally to substrate degradation. [ABSTRACT FROM AUTHOR]

Autophagy regulates cellular homeostasis by degrading and recycling cytosolic components and damaged organelles. Disruption of autophagic flux has been shown to induce or facilitate neurodegeneration and accumulation of autophagic vesicles is overt in neurodegenerative diseases. The fruit fly Drosophila has been used as a model system to identify new factors that regulate physiology and disease. Here we provide a historical perspective of how the fly models have offered mechanistic evidence to understand the role of autophagy in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Charcot–Marie–Tooth neuropathy, and polyglutamine disorders. Autophagy also plays a pivotal role in maintaining tissue homeostasis and protecting organism health. The gastrointestinal tract regulates organism health by modulating food intake, energy balance, and immunity. Growing evidence is strengthening the link between autophagy and digestive tract health in recent years. Here, we also discuss how the fly models have advanced the understanding of digestive physiology regulated by autophagy. [ABSTRACT FROM AUTHOR]

Mechanisms of protein homeostasis are crucial for overseeing the clearance of misfolded and toxic proteins over the lifetime of an organism, thereby ensuring the health of neurons and other cells of the central nervous system. The highly conserved pathway of autophagy is particularly necessary for preventing and counteracting pathogenic insults that may lead to neurodegeneration. In line with this, mutations in genes that encode essential autophagy factors result in impaired autophagy and lead to neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). However, the mechanistic details underlying the neuroprotective role of autophagy, neuronal resistance to autophagy induction, and the neuron-specific effects of autophagy-impairing mutations remain incompletely defined. Further, the manner and extent to which non-cell autonomous effects of autophagy dysfunction contribute to ALS pathogenesis are not fully understood. Here, we review the current understanding of the interplay between autophagy and ALS pathogenesis by providing an overview of critical steps in the autophagy pathway, with special focus on pivotal factors impaired by ALS-causing mutations, their physiologic effects on autophagy in disease models, and the cell type-specific mechanisms regulating autophagy in non-neuronal cells which, when impaired, can contribute to neurodegeneration. This review thereby provides a framework not only to guide further investigations of neuronal autophagy but also to refine therapeutic strategies for ALS and related neurodegenerative diseases. Abbreviations: ALS: amyotrophic lateral sclerosis; Atg: autophagy-related; CHMP2B: charged multivesicular body protein 2B; DPR: dipeptide repeat; FTD: frontotemporal dementia; iPSC: induced pluripotent stem cell; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PINK1: PTEN induced kinase 1; RNP: ribonuclear protein; sALS: sporadic ALS; SPHK1: sphingosine kinase 1; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK-binding kinase 1; TFEB: transcription factor EB; ULK: unc-51 like autophagy activating kinase; UPR: unfolded protein response; UPS: ubiquitin-proteasome system; VCP: valosin containing protein. [ABSTRACT FROM AUTHOR]

The hexanucleotide repeat (GGGGCC) expansion in C9orf72 is accounted for a large proportion of the genetic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The hypotheses of how the massive G4C2 repeats in C9orf72 destroy the neurons and lead to ALS/FTD are raised and improving. As a multirole player, C9orf72 exerts critical roles in many cellular processes, including autophagy, membrane trafficking, immune response, and so on. Notably, the partners of C9orf72, through which C9orf72 participates in the cell activities, have been identified. Notably, the structures of the C9orf72-SMCR8-WDR41 complex shed light on its activity as GTPase activating proteins (GAP). In this manuscript, we reviewed the latest research progress in the C9orf72-mediated ALS/FTD, the physiological functions of C9orf72, and the putative function models of C9orf72/C9orf72-containing complex. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two clinically distinct classes of neurodegenerative disorders. Yet, they share a range of genetic, cellular, and molecular features. Hexanucleotide repeat expansions (HREs) in the C9orf72 gene and the accumulation of toxic protein aggregates in the nervous systems of the affected individuals are among such common features. Though the mechanisms by which HREs cause toxicity is not clear, the toxic gain of function due to transcribed HRE RNA or dipeptide repeat proteins (DPRs) produced by repeat-associated non-AUG translation together with a reduction in C9orf72 expression are proposed as the contributing factors for disease pathogenesis in ALS and FTD. In addition, several recent studies point toward alterations in protein homeostasis as one of the root causes of the disease pathogenesis. In this review, we discuss the effects of the C9orf72 HRE in the autophagy-lysosome pathway based on various recent findings. We suggest that dysfunction of the autophagy-lysosome pathway synergizes with toxicity from C9orf72 repeat RNA and DPRs to drive disease pathogenesis. Abbreviation: ALP: autophagy-lysosome pathway; ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ASO: antisense oligonucleotide; C9orf72: C9orf72-SMCR8 complex subunit; DENN: differentially expressed in normal and neoplastic cells; DPR: dipeptide repeat protein; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; ER: endoplasmic reticulum; FTD: frontotemporal dementia; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HRE: hexanucleotide repeat expansion; iPSC: induced pluripotent stem cell; ISR: integrated stress response; M6PR: mannose-6-phosphate receptor, cation dependent; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: neurodegenerative disorder; RAN: repeat-associated non-ATG; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SLC66A1/PQLC2: solute carrier family 66 member 1; SMCR8: SMCR8-C9orf72 complex subunit; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; WDR41: WD repeat domain 41. [ABSTRACT FROM AUTHOR]

Progressive neuronal demise is a key contributor to the key pathogenic event implicated in many different neurodegenerative disorders (NDDs). There are several therapeutic strategies available; however, none of them are particularly effective. Targeted neuroprotective therapy is one such therapy, which seems a compelling option, yet remains challenging due to the internal heterogeneity of the mechanisms underlying various NDDs. An alternative method to treat NDDs is to exploit common modalities involving molecularly distinct subtypes and thus develop specialized drugs with broad-spectrum characteristics. There is mounting evidence which supports for the theory that dysfunctional ryanodine receptors (RyRs) disrupt intracellular Ca2+ homeostasis, contributing to NDDs significantly. This review aims to provide direct and indirect evidence on the intersection of NDDs and RyRs malfunction, and to shed light on novel strategies to treat RyRs-mediated disease, modifying pharmacological therapies such as the potential therapeutic role of dantrolene, a RyRs antagonist. [ABSTRACT FROM AUTHOR]

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2. Abbreviations: 3ʹREADS: 3ʹ region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A1; BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5ʹ-3ʹ exoribonuclease 2. [ABSTRACT FROM AUTHOR]

A pathogenic GGGCCC hexanucleotide expansion in the first intron/promoter region of the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS). The C9orf72 gene product forms a complex with SMCR8 (Smith-Magenis Syndrome Chromosome Region, Candidate 8) and WDR41 (WD Repeat domain 41) proteins. Recent studies have indicated roles for the complex in autophagy regulation, vesicle trafficking, and immune response in transgenic mice, however a direct connection with ALS etiology remains unclear. With the aim of increasing understanding of the multi-functional C9orf72-SMCR8-WDR41 complex, we determined by mass spectrometry analysis the proteins that directly associate with SMCR8. SMCR8 protein binds many components of the ubiquitin-proteasome system, and we demonstrate its poly-ubiquitination without obvious degradation. Evidence is also presented for localization of endogenous SMCR8 protein to cytoplasmic stress granules. However, in several cell lines we failed to reproduce previous observations that C9orf72 protein enters these granules. SMCR8 protein associates with many products of genes associated with various Mendelian neurological disorders in addition to ALS, implicating SMCR8-containing complexes in a range of neuropathologies. We reinforce previous observations that SMCR8 and C9orf72 protein levels are positively linked, and now show in vivo that SMCR8 protein levels are greatly reduced in brain tissues of C9orf72 gene expansion carrier individuals. While further study is required, these data suggest that SMCR8 protein level might prove a useful biomarker for the C9orf72 expansion in ALS. [ABSTRACT FROM AUTHOR]

Nutrient utilization and energy metabolism are critical for the maintenance of cellular homeostasis. A mutation in the C9orf72 gene has been linked to the most common forms of neurodegenerative diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we have identified an evolutionarily conserved function of C9orf72 in the regulation of the transcription factor EB (TFEB), a master regulator of autophagic and lysosomal genes that is negatively modulated by mTORC1. Loss of the C. elegans orthologue of C9orf72, ALFA-1, causes the nuclear translocation of HLH-30/TFEB, leading to activation of lipolysis and premature lethality during starvation-induced developmental arrest in C. elegans. A similar conserved pathway exists in human cells, in which C9orf72 regulates mTOR and TFEB signaling. C9orf72 interacts with and dynamically regulates the level of Rag GTPases, which are responsible for the recruitment of mTOR and TFEB on the lysosome upon amino acid signals. These results have revealed previously unknown functions of C9orf72 in nutrient sensing and metabolic pathways and suggest that dysregulation of C9orf72 functions could compromise cellular fitness under conditions of nutrient stress. Author summary: An expansion of repeated nucleotides in the non-coding region of the C9orf72 gene has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion leads to a reduced expression of the C9orf72 gene and loss of function of the C9orf72 protein may contribute to the pathogenesis. In this study, we identified a new mechanism through which C9orf72 influences nutrient sensing, autophagy, and metabolism. In the multi-cellular organism Caenorhabditis elegans, the C9orf72 orthologue regulates the activity of TFEB, a crucial transcriptional regulator of autophagic and lysosomal genes, through which the lipid metabolism and survival are influenced especially under nutrient stress conditions. The regulatory effect of C9orf72 on TFEB is conserved in mammals, and this is mediated by the dynamic regulation of the Rag GTPases by C9orf72. Given the critical role of the Rag GTPases in nutrient sensing and autophagy, we propose that the C9orf72 function is important for metabolic homeostasis in the cell and its deficiency can lead to compromised fitness under stress conditions. [ABSTRACT FROM AUTHOR]

Expansion of G4C2 repeats within the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Such repeats lead to decreased expression of the autophagy regulator C9ORF72 protein. Furthermore, sense and antisense repeats are translated into toxic dipeptide repeat (DPR) proteins. It is unclear how these repeats are translated, and in which way their translation and the reduced expression of C9ORF72 modulate repeat toxicity. Here, we found that sense and antisense repeats are translated upon initiation at canonical AUG or near‐cognate start codons, resulting in polyGA‐, polyPG‐, and to a lesser degree polyGR‐DPR proteins. However, accumulation of these proteins is prevented by autophagy. Importantly, reduced C9ORF72 levels lead to suboptimal autophagy, thereby impairing clearance of DPR proteins and causing their toxic accumulation, ultimately resulting in neuronal cell death. Of clinical importance, pharmacological compounds activating autophagy can prevent neuronal cell death caused by DPR proteins accumulation. These results suggest the existence of a double‐hit pathogenic mechanism in ALS/FTD, whereby reduced expression of C9ORF72 synergizes with DPR protein accumulation and toxicity. Synopsis: An expansion of G4C2 repeats in the C9ORF72 gene causes amyotrophic lateral sclerosis (ALS). Here, cell transfection and mass spectrometry indicate that G4C2 repeats are translated through classical initiation to cognate ATG or near‐cognate start codons. Furthermore, impaired autophagy due to reduced expression of the C9ORF72 protein synergizes toxicity of G4C2 translation. Sense G4C2 repeats are translated into polyGA and polyGR‐containing proteins, which initiate at a CUG and an AGG near‐cognate codon, respectively.Antisense C4G2 repeats are translated into a polyPG‐containing protein that initiates at an ATG start codon.Loss of C9ORF72 protein impairs autophagy, which promotes the accumulation and thus the toxicity of polyGA, polyPG and polyGR proteins. [ABSTRACT FROM AUTHOR]

Microsatellite repeat expansion disease loci can exhibit pleiotropic clinical and biological effects depending on repeat length. Large expansions in C9orf72 (100s–1000s of units) are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). However, whether intermediate expansions also contribute to neurodegenerative disease is not well understood. Several studies have identified intermediate repeats in Parkinson's disease patients, but the association was not found in autopsy-confirmed cases. We hypothesized that intermediate C9orf72 repeats are a genetic risk factor for corticobasal degeneration (CBD), a neurodegenerative disease that can be clinically similar to Parkinson's but has distinct tau protein pathology. Indeed, intermediate C9orf72 repeats were significantly enriched in autopsy-proven CBD (n = 354 cases, odds ratio = 3.59, p = 0.00024). While large C9orf72 repeat expansions are known to decrease C9orf72 expression, intermediate C9orf72 repeats result in increased C9orf72 expression in human brain tissue and CRISPR/cas9 knockin iPSC-derived neural progenitor cells. In contrast to cases of FTD/ALS with large C9orf72 expansions, CBD with intermediate C9orf72 repeats was not associated with pathologic RNA foci or dipeptide repeat protein aggregates. Knock-in cells with intermediate repeats exhibit numerous changes in gene expression pathways relating to vesicle trafficking and autophagy. Additionally, overexpression of C9orf72 without the repeat expansion leads to defects in autophagy under nutrient starvation conditions. These results raise the possibility that therapeutic strategies to reduce C9orf72 expression may be beneficial for the treatment of CBD. [ABSTRACT FROM AUTHOR]

The intronic hexanucleotide expansion in the C9orf72 gene is one of the leading causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), two devastating neurodegenerative diseases. C9orf72 forms a heterodimer with SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8) protein. However, the physiological function of SMCR8 remains to be characterized. Here we report that ablation of SMCR8 in mice results in splenomegaly with autoimmune phenotypes similar to that of C9orf72 deficiency. Furthermore, SMCR8 loss leads to a drastic decrease of C9orf72 protein levels. Many proteins involved in the macroautophagy-lysosome pathways are downregulated upon SMCR8 loss due to elevated activation of MTORC1 and AKT, which also leads to increased spine density in the Smcr8 knockout neurons. In summary, our studies demonstrate a key role of SMCR8 in regulating MTORC1 and AKT signaling and tissue homeostasis. Abbreviations: ALS: amyotrophic lateral sclerosis; C9orf72: chromosome 9 open reading frame 72; FTLD: frontotemporal lobar degeneration; GEF: guanosine nucleotide exchange factor; GTPase: guanosine tri-phosphatase; KO: knockout; MTOR: mechanistic target of rapamycin kinase; SMCR8: Smith-Magenis chromosome region, candidate 8; WDR41: WD repeat domain 41; WT: wild type [ABSTRACT FROM AUTHOR]

Axon degeneration is a pathophysiological process of axonal dying and breakdown, which is characterized by several morphological features including the accumulation of axoplasmic organelles, disassembly of microtubules, and fragmentation of the axonal cytoskeleton. Autophagy, a highly conserved lysosomal-degradation machinery responsible for the control of cellular protein quality, is widely believed to be essential for the maintenance of axonal homeostasis in neurons. In recent years, more and more evidence suggests that dysfunctional autophagy is associated with axonal degeneration in many neurodegenerative diseases. Here, we review the core machinery of autophagy in neuronal cells, and provide several major steps that interfere with autophagy flux in neurodegenerative conditions. Furthermore, this review highlights the potential role of neuronal autophagy in axon degeneration, and presents some possible molecular mechanisms by which dysfunctional autophagy leads to axon degeneration in pathological conditions. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS. [ABSTRACT FROM AUTHOR]

The objective of this study is to investigate the autophagy activity of cells by the intracellular release of rapamycin (Rapa) of an autophagy inducer. Rapa was incorporated into nanospheres of poly (lactic-co-glycolic acid) (PLGA) for the controlled release of Rapa. Rapa was released from the PLGA nanospheres incorporating rapamycin (Rapa-PLGA-NS) with time while the Rapa-PLGA-NS were hydrolytically degraded. When human hepatocellular carcinoma (HepG2) cells were incubated with the Rapa-PLGA-NS, the Rapa-PLGA-NS were internalized, and the intracellular concentration was maintained over four days, indicating the intracellular Rapa release. The microtubule-associated protein 1 light chain (LC3) of an autophagy marker was significantly high for the Rapa-PLGA-NS group compared with the free Rapa group even after four days incubation. In addition, intracellular harmful ubiquitinated proteins were degraded by the intracellular release of Rapa even after four days incubation in contrast to free Rapa. It is concluded that the intracellular Rapa release is effective in modulating the autophagy activity over a longer time period. [ABSTRACT FROM PUBLISHER]