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

Abstract

In neurons, the continuous and dynamic endoplasmic reticulum (ER) network extends throughout the axon, and its dysfunction causes various axonopathies. However, it remains largely unknown how ER integrity and remodeling modulate presynaptic function in mammalian neurons. Here, we demonstrated that ER membrane receptors VAPA and VAPB are involved in modulating the synaptic vesicle (SV) cycle. VAP interacts with secernin‐1 (SCRN1) at the ER membrane via a single FFAT‐like motif. Similar to VAP, loss of SCRN1 or SCRN1‐VAP interactions resulted in impaired SV cycling. Consistently, SCRN1 or VAP depletion was accompanied by decreased action potential‐evoked Ca2+ responses. Additionally, we found that VAP‐SCRN1 interactions play an important role in maintaining ER continuity and dynamics, as well as presynaptic Ca2+ homeostasis. Based on these findings, we propose a model where the ER‐localized VAP‐SCRN1 interactions provide a novel control mechanism to tune ER remodeling and thereby modulate Ca2+ dynamics and SV cycling at presynaptic sites. These data provide new insights into the molecular mechanisms controlling ER structure and dynamics, and highlight the relevance of ER function for SV cycling.

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

“…Dual-color gSTED imaging (as described above) was performed on PSD95-GFP, GFP-β-actin #1, GFP-GluN1 #1, GFP-CaMKIIα, GSG1L-GFP, and FRRS1L-GFP knock-in neurons stained with anti-GFP and anti-PSD95. pORANGE FRRS1L-GFP was cotransfected with pSyn tagRFP-ER [82]. (Dual-color) gSTED imaging was additionally performed on extracted cytoskeleton of the GFP-β-actin and β3-tubulin-GFP knock-in neurons.…”

Axonal Endoplasmic Reticulum Dynamics and Its Roles in Neurodegeneration

“…A specific role of ER Ca 2+ has been recently shown in mammalian neurons, where at presynaptic terminals, ER luminal Ca 2+ can promote PM Ca 2+ uptake via the ER Ca 2+ sensor STIM1, a process required for presynaptic Ca 2+ flux and exocytosis (de Juan-Sanz et al, 2017). Depletion of either the vesicle-associated membrane protein-associated proteins A (VAPA) and B (VAPB), which are ER membrane receptors, or the cytoplasmic VAP-associated protein Secernin-1 (SCRN1), reduces presynaptic Ca 2+ influx and synaptic vesicle cycling (Lindhout et al, 2019). Also, in Drosophila neurons, the ER-resident Ca 2+ sensor MCTP (multiple C2 domain and transmembrane region protein) promotes release of synaptic vesicles (Genç et al, 2017).…”

“…A role for ER MFN2 in ER tubulation has been proposed, since it is required to keep luminal continuity at the peripheral ER in mammalian cells (de Brito and Scorrano, 2008). VAPA/B are also found at ER membrane and required for axonal ER continuity (Lindhout et al, 2019). Finally, one characteristic feature of axonal ER is its very small diameter -ER tubules in most cells frequently has a diameter of ∼60 nm (varying between 25 and 90 nm), but in axons, ER tubules have a diameter on average around 40 nm, and often becoming small enough for the lumen not to be even visible (Wu et al, 2017;Yalçın et al, 2017;Terasaki, 2018).…”

ER – lysosome contacts at a pre-axonal region regulate axonal lysosome availability

“…The following sequences for rat-shRNAs, inserted in pSuper vector, were used in this study: RTN4-shRNA (5'-gtccagatttctctaatta-3'), DP1-shRNA (5'-gacatataaagttccagaa-3'), P180-shRNAs (5'tcagtgcaattgtctgtat-3' and 5'-taaaccaaccaacacagcg-3'), KTN1-shRNA (5'-ggaccttctcaagaggtta-3'), and CLIMP63-shRNA (5'-tcaaccgtattagtgaagttctaca-3') (Farías et al, 2019); VAPA-shRNA (5'gcatgcagagtgctgtttc-3') and VAPB-shRNA (5'-ggtgatggaagagtgc-3') (Teuling et al, 2007;Lindhout et al, 2019). A previously described sequence for Protrudin-shRNA (5'-aagcttcttgatccgactggaag-3'; Shirane and Nakayama, 2006) was cloned into pSuper vector after oligo annealing.…”

“…ER tubules and LEs/ lysosomes are translocated from the soma into the axon by the kinesin-1 motor (Farías et al, 2017;Farías et al, 2019). Local availability of ER tubules instructs axon formation and regulates axonal synaptic vesicle cycling (Farías et al, 2019;Lindhout et al, 2019) and active transport of LEs/ lysosomes into the axon is required for proper clearance of faulty proteins and organelles located far away from the cell soma (Farías et al, 2017; Farfel-Becker et al, 2019). Interestingly, mutations in genes encoding ER-shaping proteins cause the neurodegenerative disease hereditary spastic paraplegia, in which aberrant lysosomes have been observed (Westrate et al, 2015; Allison et al, 2017;Lee and Blackstone, 2020).…”