Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation

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.

Sequestosome 1 Is Part of the Interaction Network of VAPB

“…This recruitment to cytoplasmic aggregates was also observed for endogenous VAPA, as described previously [38]. However, we did not detect the recruitment of FUS or TDP-43 to the cytoplasmic aggregates, in contrast to a previous publication [36] and no colocalization with autophagosome or lysosome membrane proteins, LC3 and LAMP1 was observed in our study (Figure S1). Furthermore, two established interaction partners of VAPB, ACBD5 and OSBPL9 were not affected by the P56S mutation (Figure 2).…”

“…Evidently, biotinylation of SQSTM1 depended on the presence of P56S-VAPB in the transfected cells. A coaccumulation of SQSTM1 with P56S-VAPB aggregates has been shown very recently in atrophic muscle fibres as well as in skin fibroblasts from an ALS8-patient [36]. Furthermore, SQSTM1 colocalized with cytoplasmic P56S-VAPB inclusions in motor neurons derived from transgenic mice [58].…”

“…The mutant P56S-VAPB has previously been shown to sequester several RNA binding proteins including FUS and TDP-43 in transfected cells [36]. It also causes nuclear envelope defects with effects on proteins of the inner nuclear membrane and the NPC (nuclear pore complex) [37].…”

“…A gain-of-function effect, on the other hand, could result from aberrant interactions of P56S-VAPB that are normally not observed for the wild-type protein. Several proteins have been reported to recruit to P56S-VAPB-aggregates, including ACBD5 [33], the HCN2 channel [34], the Yip1-interacting factor homologue A (YIF1A; [35]), stress granule markers such as TIAR1 or G3BP [36] and the ubiquitin-binding protein sequestosome 1 [36].…”

Current Concepts on Genetic Aspects of Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis

“…VAPB protein is involved in lipid and calcium ion transfers between endoplasmic reticulum and mitochondria and mutant VAPB is compromising mitochondrial functioning through reducing OXPHOS due to insufficient Ca 2+ import in mitochondria [ 66 ]. VAPB mutations are described in autosomal dominant ALS as well as in late-onset spinal muscular atrophy [ 67 ] and recent study pointed out that dysfunctional autophagy may be one of the pathomechanisms provoked by common mutation in gene VAPB [ 68 ].…”