CAPS-1 requires its C2, PH, MHD1 and DCV domains for dense core vesicle exocytosis in mammalian CNS neurons

Linda van Keimpema; Robbelien Kooistra; Ruud F Toonen; Matthijs Verhage

doi:10.1038/s41598-017-10936-4

CAPS-1 requires its C2, PH, MHD1 and DCV domains for dense core vesicle exocytosis in mammalian CNS neurons

Linda van Keimpema, Robbelien Kooistra, Ruud F Toonen, Matthijs Verhage

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Abstract

CAPS (calcium-dependent activator protein for secretion) are multi-domain proteins involved in regulated exocytosis of synaptic vesicles (SVs) and dense core vesicles (DCVs). Here, we assessed the contribution of different CAPS-1 domains to its subcellular localization and DCV exocytosis by expressing CAPS-1 mutations in four functional domains in CAPS-1/-2 null mutant (CAPS DKO) mouse hippocampal neurons, which are severely impaired in DCV exocytosis. CAPS DKO neurons showed normal development and no defects in DCV biogenesis and their subcellular distribution. Truncation of the CAPS-1 C-terminus (CAPS Δ654-1355) impaired CAPS-1 synaptic enrichment. Mutations in the C2 (K428E or G476E) or pleckstrin homology (PH; R558D/K560E/K561E) domain did not. However, all mutants rescued DCV exocytosis in CAPS DKO neurons to only 20% of wild type CAPS-1 exocytosis capacity. To assess the relative importance of CAPS for both secretory pathways, we compared effect sizes of CAPS-1/-2 deficiency on SV and DCV exocytosis. Using the same (intense) stimulation, DCV exocytosis was impaired relatively strong (96% inhibition) compared to SV exocytosis (39%). Together, these data show that the CAPS-1 C-terminus regulates synaptic enrichment of CAPS-1. All CAPS-1 functional domains are required, and the C2 and PH domain together are not sufficient, for DCV exocytosis in mammalian CNS neurons.

Original language	English
Article number	10817
Pages (from-to)	10817
Journal	Scientific Reports
Volume	7
Issue number	1
DOIs	https://doi.org/10.1038/s41598-017-10936-4
Publication status	Published - 7 Sept 2017

Funding

This work is funded by the Agentschap NL (NeuroBasic PharmaPhenomics, FES0908 to MV) and the EU (ERC-ADG-322966-DCVexocytosis to MV). The authors thank Ingrid Saarloos and Robbert Zalm for producing constructs and lentiviral particles, Joost Hoetjes and Frank den Oudsten for genotyping, Frank den Oudsten and Desiree Schut for producing glia islands and support during culturing, Joke Wortel for housing and breeding mice, and Margherita Farina for valuable discussion and input.

Funders	Funder number
Seventh Framework Programme	322966
European Commission
Agentschap NL	FES0908

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abstract = "CAPS (calcium-dependent activator protein for secretion) are multi-domain proteins involved in regulated exocytosis of synaptic vesicles (SVs) and dense core vesicles (DCVs). Here, we assessed the contribution of different CAPS-1 domains to its subcellular localization and DCV exocytosis by expressing CAPS-1 mutations in four functional domains in CAPS-1/-2 null mutant (CAPS DKO) mouse hippocampal neurons, which are severely impaired in DCV exocytosis. CAPS DKO neurons showed normal development and no defects in DCV biogenesis and their subcellular distribution. Truncation of the CAPS-1 C-terminus (CAPS Δ654-1355) impaired CAPS-1 synaptic enrichment. Mutations in the C2 (K428E or G476E) or pleckstrin homology (PH; R558D/K560E/K561E) domain did not. However, all mutants rescued DCV exocytosis in CAPS DKO neurons to only 20% of wild type CAPS-1 exocytosis capacity. To assess the relative importance of CAPS for both secretory pathways, we compared effect sizes of CAPS-1/-2 deficiency on SV and DCV exocytosis. Using the same (intense) stimulation, DCV exocytosis was impaired relatively strong (96% inhibition) compared to SV exocytosis (39%). Together, these data show that the CAPS-1 C-terminus regulates synaptic enrichment of CAPS-1. All CAPS-1 functional domains are required, and the C2 and PH domain together are not sufficient, for DCV exocytosis in mammalian CNS neurons.",

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CAPS-1 requires its C2, PH, MHD1 and DCV domains for dense core vesicle exocytosis in mammalian CNS neurons

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