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.

Objective: DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling.Methods: Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy‐related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression.Results: Here we report a zebrafish model of Depdc5 loss‐of‐function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy‐associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein.Interpretation: This study demonstrates that Depdc5 knockdown leads to early‐onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy‐associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1‐dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5‐inherited forms of focal epilepsy.

The GGGGCC hexanucleotide repeat expansion (HRE) of the C9orf72 gene is the most frequent cause of amyotrophic lateral sclerosis (ALS), a devastative neurodegenerative disease characterized by motor neuron degeneration. C9orf72 HRE is associated with lowered levels of C9orf72 expression and its translation results in the production of dipeptide-repeats (DPRs). To recapitulate C9orf72-related ALS disease in vivo, we developed a zebrafish model where we expressed glycine-proline (GP) DPR in a c9orf72 knockdown context. We report that C9orf72 gain- and loss-of-function properties act synergistically to induce motor neuron degeneration and paralysis with poly(GP) accumulating preferentially within motor neurons along with Sqstm1/p62 aggregation indicating macroautophagy/autophagy deficits. Poly(GP) levels were shown to accumulate upon c9orf72 downregulation and were comparable to levels assessed in autopsy samples of patients carrying C9orf72 HRE. Chemical boosting of autophagy using rapamycin or apilimod, is able to rescue motor deficits. Proteomics analysis of zebrafish-purified motor neurons unravels mitochondria dysfunction confirmed through a comparative analysis of previously published C9orf72 iPSC-derived motor neurons. Consistently, 3D-reconstructions of motor neuron demonstrate that poly(GP) aggregates colocalize to mitochondria, thus inducing their elongation and swelling and the failure of their processing by mitophagy, with mitophagy activation through urolithin A preventing locomotor deficits. Finally, we report apoptotic-related increased amounts of cleaved Casp3 (caspase 3, apoptosis-related cysteine peptidase) and rescue of motor neuron degeneration by constitutive inhibition of Casp9 or treatment with decylubiquinone. Here we provide evidence of key pathogenic steps in C9ALS-FTD that can be targeted through pharmacological avenues, thus raising new therapeutic perspectives for ALS patients.

Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis episodes precipitated by fasting. Autophagy functioning was analyzed in vitro, in primary skeletal myoblasts from TANGO2 patients, in basal and fasting conditions, and TANGO2 mutations were associated with reduced LC3-II levels upon starvation. In zebrafish larvae, tango2 inhibition induced locomotor defects which were exacerbated by exposure to atorvastatin, a compound known to cause rhabdomyolysis. Importantly, rhabdomyolysis features of tango2 knockdown were associated with autophagy and mitophagy defects in zebrafish. Calpeptin treatment was sufficient to rescue the locomotor properties thanks to its beneficial effect on autophagy functioning in zebrafish and to improve LC3-II levels in starved primary muscle cells of TANGO2 patients. Overall, we demonstrated that TANGO2 plays an important role in autophagy thus giving rise to new therapeutic perspectives in the prevention of RM life-threatening episodes.

Pathogenic mutations within the TBK1 gene leading to haploinsufficiency are causative of amyotrophic lateral sclerosis (ALS). This gene is linked to autophagy and inflammation, two cellular mechanisms reported to be dysregulated in ALS patients, although its functional role in the pathogenesis could involve other players. We targeted the TBK1 ortholog in zebrafish, an optimal vertebrate model for investigating genetic defects in neurological disorders. We generated zebrafish models with invalidating tbk1 mutations using CRISPR-Cas9 or tbk1 knockdown models using antisense morpholino oligonucleotide (AMO). The early motor phenotype of zebrafish injected with tbk1 AMO beginning at 2 days post fertilization (dpf) is associated with the degeneration of motor neurons. In parallel, CRISPR-induced tbk1 mutants exhibit impaired motor function beginning at 5 dpf and increased lethality beginning at 9 dpf. A metabolomic analysis showed an association between tbk1 loss and severe dysregulation of nicotinamide metabolism, and incubation with nicotinamide riboside rescued the motor behavior of tbk1 mutant zebrafish. Furthermore, a proteomic analysis revealed increased levels of inflammatory markers and dysregulation of programmed cell death pathways. Necroptosis appeared to be strongly activated in TBK1 fish, and larvae treated with the necroptosis inhibitor necrosulfonamide exhibited improved survival. Finally, a combined analysis of mutant zebrafish and TBK1-mutant human motor neurons revealed dysregulation of the KEGG pathway “ALS”, with disrupted nuclear-cytoplasmic transport and increased expression of STAT1. These findings point toward a major role for necroptosis in the degenerative features and premature lethality observed in tbk1 mutant zebrafish. Overall, the novel tbk1-deficient zebrafish models offer a great opportunity to better understand the cascade of events leading from the loss of tbk1 expression to the onset of motor deficits, with involvement of a metabolic defect and increased cell death, and for the development of novel therapeutic avenues for ALS and related neuromuscular diseases.

Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis (RM) episodes precipitated by fasting. Since starvation promotes autophagy induction, we wondered whether TANGO2-related muscle symptoms result from autophagy insufficiency to meet cellular demands in stress conditions. Autophagy functioning was analyzed in vitro , in primary skeletal muscle cells from TANGO2 patients in basal and fasting conditions. In addition, we developed a tango2 morphant zebrafish model to assess the effect of tango2 knockdown (KD) on locomotor function and autophagy efficiency in vivo . We report that TANGO2 mutations are associated with decreased LC3-II levels upon starvation in primary muscle cells, but not in fibroblasts. In zebrafish larvae, tango2 knockdown induces locomotor defects characterized by reduced evoked movements which are exacerbated by exposure to atorvastatin, a compound known to cause RM. Importantly, RM features of tango2 KD are also associated with autophagy and mitophagy defects in zebrafish. Calpeptin treatment, a known activator of autophagy, is sufficient to rescue the locomotor properties, thanks to its beneficial effect on autophagy functioning in zebrafish and independently to its effect on calpain activity. LC3-II levels of primary muscle cells of TANGO2 patients are also improved by calpeptin treatment. Overall, we demonstrate that TANGO2 plays an important role in autophagy, and that autophagy efficiency is critical to prevent RM, thus giving rise to new therapeutic perspectives in the prevention of these life-threatening episodes in TANGO2 pathology.