Amyotrophic lateral sclerosis (ALS)-associated VAPB-P56S inclusions represent an ER quality control compartment

Abstract

BackgroundProtein aggregation and the formation of intracellular inclusions are a central feature of many neurodegenerative disorders, but precise knowledge about their pathogenic role is lacking in most instances. Here we have characterized inclusions formed in transgenic mice carrying the P56S mutant form of VAPB that causes various motor neuron syndromes including ALS8.ResultsInclusions in motor neurons of VAPB-P56S transgenic mice are characterized by the presence of smooth ER-like tubular profiles, and are immunoreactive for factors that operate in the ER associated degradation (ERAD) pathway, including p97/VCP, Derlin-1, and the ER membrane chaperone BAP31. The presence of these inclusions does not correlate with signs of axonal and neuronal degeneration, and axotomy leads to their gradual disappearance, indicating that they represent reversible structures. Inhibition of the proteasome and knockdown of the ER membrane chaperone BAP31 increased the size of mutant VAPB inclusions in primary neuron cultures, while knockdown of TEB4, an ERAD ubiquitin-protein ligase, reduced their size. Mutant VAPB did not codistribute with mutant forms of seipin that are associated with an autosomal dominant motor neuron disease, and accumulate in a protective ER derived compartment termed ERPO (ER protective organelle) in neurons.ConclusionsThe data indicate that the VAPB-P56S inclusions represent a novel reversible ER quality control compartment that is formed when the amount of mutant VAPB exceeds the capacity of the ERAD pathway and that isolates misfolded and aggregated VAPB from the rest of the ER. The presence of this quality control compartment reveals an additional level of flexibility of neurons to cope with misfolded protein stress in the ER.

VAP‐SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function

“…Subsequently, neurons were fixed and the fluorescence intensity of internalized Syt at individual presynaptic boutons was measured. VAP was depleted from neurons by expressing shRNAs targeting VAPA and VAPB, which we have validated in the previous studies (Teuling et al, 2007;Kuijpers et al, 2013 internalization compared to control cells ( Fig 1C and D). In addition, VAPA and VAPB knockdown resulted in a slight decrease in bouton size and bouton density (Figs 1C,and EV1B and C).…”

“…VAP was depleted from neurons by expressing shRNAs targeting VAPA and VAPB, which we have validated in the previous studies (Teuling et al, 2007;Kuijpers et al, 2013). VAP was depleted from neurons by expressing shRNAs targeting VAPA and VAPB, which we have validated in the previous studies (Teuling et al, 2007;Kuijpers et al, 2013).…”

The basal interstitial nucleus (BIN) of the cerebellum provides diffuse ascending inhibitory input to the floccular granule cell layer

“…Sections were serially collected in 8 (mouse, rat), 20 (ferret, rabbit, macaque) or 40 (human) vials with phosphate buffered saline (PBS, pH 7.2) such that each vial contained a complete series of sections. Sections were processed free‐floating for diaminobenzidine (DAB, 0.05%)‐immunoperoxidase histochemistry or immunofluorescence (Kuijpers et al, ). Following preincubation in PBS containing 0.4% Triton X‐100 (PBST) and normal horse serum (NHS) and for 1 hr at room temperature, sections were incubated 48–60 hr at 4 °C with the primary antibodies in PBST with 2% NHS.…”

VAMP-associated Proteins (VAP) as Receptors That Couple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Proteostasis with Lipid Homeostasis

“…However, the significance of such regulation is exemplified by the role of VAPB mutants in the development of familial amyotrophic lateral sclerosis (ALS) (31,(33)(34)(35)(36). A P56S mutation in the MSP domain of VAPB destabilizes the domain and supports the aggregation of VAPB with VAPA leading to a general loss of function.…”