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.

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously-encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Accordingly, our findings establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis, but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

Charcot-Marie-Tooth Type 4B3 (CMT4B3) is a genetic disorder leading to peripheral axon degeneration and clinical manifestations of distal weakness and gait impairment. CMT4B3 is caused by mutations in SBF1 /MTMR5, a negative regulator of phosphoinositide signaling and autophagy. Although SBF1 mutations are ubiquitously expressed, how and why loss of SBF1 /MTMR5 exerts deleterious effects predominantly in neurons of the peripheral nervous system (PNS) is unknown. To investigate the effects of mutant SBF1 /MTMR5 on PNS neurons compared to non-neurons, we engineered a novel and unique model system of CMT4B3 using human induced pluripotent stem cells (iPSCs) differentiated into key components of the PNS: motor neurons (iMNs), sensory neurons (iSNs), or skeletal muscle (iMuscle). To model CMT4B3, we used iPSCs derived from a CMT43B patient, or genetically knocked down SBF1 in WT cells. Strikingly, CMT4B3 iMNs showed the highest degree of cell degeneration among all cell types, concordant with the clinical phenotype of patients. We also found that CMT4B3 iMNs and iSNs showed attenuated expression of MTMR5 and related paralogs MTMR2 and MTMR13. Knockdown of SBF1 most significantly augmented autophagy in iMNs than other cell types. Finally, we tested treatment with VPS34-IN1, a pharmacologic inhibitor of the Class III PI3-Kinase functioning in opposition to MTMR5 in regulating phosphoinositides, and found that VPS34-IN1 rescued cell death in CMT4B3 iMNs. Together, our results for the first time confirm PNS cell type-specific differences in myotubularin expression, autophagy, and vulnerabilities to SBF1 mutations, and identify a novel therapeutic strategy of high disease-modifying potential for CMT4B3.