Quantitative analysis of dense-core vesicle fusion in rodent CNS neurons.

Neuropeptides are essential signaling molecules secreted by dense-core vesicles (DCVs). They contribute to information processing in the brain, controlling a variety of physiological conditions. Defective neuropeptide signaling is implicated in several psychiatric disorders. Here, we provide a protocol for the quantitative analysis of DCV fusion events in rodent neurons using pH-sensitive DCV fusion probes and custom-written analysis algorithms. This method can be used to study DCV fusion mechanisms and is easily adapted to investigate fusion principles of other secretory organelles. For complete details on the use and execution of this protocol, please refer to Persoon et al. (2019).

The RAB3-RIM Pathway Is Essential for the Release of Neuromodulators.

Secretion principles are conserved from yeast to humans, and many yeast orthologs have established roles in synaptic vesicle exocytosis in the mammalian brain. Surprisingly, SEC4 orthologs and their effectors, the exocyst, are dispensable for synaptic vesicle exocytosis. Here, we identify the SEC4 ortholog RAB3 and its neuronal effector, RIM1, as essential molecules for neuropeptide and neurotrophin release from dense-core vesicles (DCVs) in mammalian neurons. Inactivation of all four RAB3 genes nearly ablated DCV exocytosis, and re-expression of RAB3A restored this deficit. In RIM1/2-deficient neurons, DCV exocytosis was undetectable. Full-length RIM1, but not mutants that lack RAB3 or MUNC13 binding, restored release. Strikingly, a short N-terminal RIM1 fragment only harboring RAB3- and MUNC13-interacting domains was sufficient to support DCV exocytosis. We propose that RIM and MUNC13 emerged as mammalian alternatives to the yeast exocyst complex as essential RAB3/SEC4 effectors and organizers of DCV fusion sites by recruiting DCVs via RAB3.

Pool size estimations for dense-core vesicles in mammalian CNS neurons.

Neuropeptides are essential signaling molecules transported and secreted by dense-core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo Super-resolution and electron microscopy reveal a total pool of 1,400-18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo-cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1-6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.

UFM1 founder mutation in the Roma population causes recessive variant of H-ABC.

OBJECTIVE: To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations. METHODS: We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression. RESULTS: Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1. Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%-25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines. CONCLUSIONS: UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks.

CAPS-1 promotes fusion competence of stationary dense-core vesicles in presynaptic terminals of mammalian neurons.

Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role.