Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 125 4 basal Ca 2+ levels were obtained by determining the ratio (F 0 /F max ) of fluorescent GCaMP6f or R-GECO1 signals before (F 0 ) and after (F max ) ionomycin treatment. Ionomycin is an ionophore which induces Ca 2+ permeability at membranes, thereby enables effectively saturating Ca 2+ indicators to determine F max. In VAP knockdown neurons, relative basal Ca 2+ levels at single boutons were markedly elevated (~2.5-fold) compared to control (Fig 5G–I). This is in line with the observed decreased evoked Ca 2+ influx, as higher basal Ca 2+ levels will result in a lower extracellular-cytoplasmic Ca 2+ concentration gradient. Consistently, presynaptic Ca 2+ levels were significantly increased (~2-fold) with dominant- negative SCRN1-F402A expression, but not with SCRN1 wild-type expression, indicating that the observed effect is mediated by VAP-SCRN1 interactions (Fig 5J–L). Together, these data imply that ER-localized VAP-SCRN1 interactions are engaged in modulating basal Ca 2+ levels at presynaptic sites. DISCUSSION The dynamic and continuous ER network extends throughout the axon, and evidence for its role in controlling presynaptic neurotransmitter release begins to emerge (Summerville et al, 2016; De Gregorio et al, 2017; de Juan-Sanz et al, 2017). In this study, we identified novel control mechanisms for ER remodeling, presynaptic Ca 2+ homeostasis, and Ca 2+ - induced SV cycling, which are mediated by ER receptor VAP together with VAP-interacting protein SCRN1. Together, these data point toward a model where VAP-SCRN1 interactions tune ER integrity and dynamics, and thereby could modulate basal Ca 2+ levels and subsequently SV cycling at presynaptic sites. SCRN1 is a VAP-interacting protein Here, we demonstrated that loss of ER membrane receptor VAP results in discontinuous ER structures, impaired ER dynamics, and decreased SV cycling. This is in line with previous studies which already hinted for a role of VAP in maintaining ER morphology and regulating synaptic function (Skehel et al, 1995; Kaiser et al, 2005; Gomez-Suaga et al, 2019). To gain further mechanistic insights into the VAP-mediated phenotypes, we sought to identify a VAP-associated protein involved in this function. Hence, we selected SCRN1 as a potential candidate for several reasons. First, SCRN1 was abundantly expressed in brain tissue, as indicated by various expression databases (Protein Atlas, Expression Atlas, Alan Brain Atlas) and confirmed by our Western blot analysis of different rat tissues. Second, in non-neuronal cells, SCRN1 was found to play a role in regulating Ca 2+ -controlled exocytosis, which is also a key process of the SV cycle (Way et al, 2002; Lin et al, 2015). We confirmed the interaction between VAP and SCRN1, and identified a single FFAT-like motif responsible for the interaction. Consistent with our hypothesis, we found that either SCRN1 depletion or dominant-negative SCRN1-F402A expression phenocopied the effect of VAP depletion on both ER remodeling and SV cycling. The dominant-negative effect was consistently more potent than the shRNA knockdown effect, similar to what is often observed when comparing dominant-negative and shRNA silencing expression constructs