Abstract
Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.
Highlights
Amyotrophic lateral sclerosis (ALS) is a devastating disease characterised by progressive loss of upper and lower motor neurons (MNs), eventually leading to paralysis and death
P56S-vesicle-associated membrane proteinassociated protein B (VAPB) leads to defective autophagy in ALS8 Over expressed P56S-VAPB forms ER-associated aggregates in cells, hiPSC MNs (Supplementary Fig. 1a, b) as well as in the P56S-VAPB transgenic mouse models (Supplementary Fig. 1c); in contrast, P56S-VAPB inclusions were absent in induced pluripotent stem cellderived MNs of ALS8 patients[43]
Skin fibroblasts obtained from this ALS8 patient revealed smaller prominent punctate VAPB aggregates (Fig. 1c), which formed larger globular aggregates when challenged with proteasome or autophagy inhibitors (Fig. 1c; arrowheads right panel)
Summary
Amyotrophic lateral sclerosis (ALS) is a devastating disease characterised by progressive loss of upper and lower motor neurons (MNs), eventually leading to paralysis and death. Majority of the fALS- linked genes either regulate protein quality control and autophagy and/ or RNA binding protein (RBP) homeostasis[1,2]. Pathogenic mutations in these genes induce neuronal toxicity coupled. Tripathi et al Cell Death and Disease (2021)12:466 with protein aggregation, defective proteostasis/autophagy and aberrant RBP homeostasis. A dominantly inherited mutation (P56S) in the gene encoding for the vesicle-associated membrane proteinassociated protein B (VAPB) has been associated with typical ALS (ALS8), atypical ALS and late-onset spinal muscular atrophy (SMA)[3,4,5]. VAPB is a ubiquitously expressed membrane-anchored protein of ER and ERGolgi intermediate vesicles[6,7].
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