Neuron-specific translational control shift ensures proteostatic resilience during ER stress.

Proteostasis is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R-like endoplasmic reticulum (ER) kinase (PERK)-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK-deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK-deficient neurons. Haem-regulated inhibitor (HRI) mediates p-eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back-up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.

Munc18-1 is essential for neuropeptide secretion in neurons.

Neuropeptide secretion from dense-core vesicles (DCVs) controls many brain functions. Several components of the DCV exocytosis machinery have recently been identified, but the participation of a SEC1/MUNC18 (SM) protein has remained elusive. Here, we tested the ability of the three exocytic SM proteins expressed in the mammalian brain, MUNC18-1/2/3, to support neuropeptide secretion. We quantified DCV exocytosis at a single vesicle resolution upon action potential train-stimulation in mouse CNS neurons (of unknown sex) using pHluorin- and/or mCherry-tagged Neuropeptide-Y (NPY) or Brain-Derived Neurotrophic Factor (BDNF). Conditional inactivation of Munc18-1 abolished all DCV exocytosis. Expression of MUNC18-1, but not MUNC18-2 or MUNC18-3, supported DCV exocytosis in Munc18-1 null neurons. Heterozygous (HZ) inactivation of Munc18-1, as a model for reduced MUNC18-1 expression, impaired DCV exocytosis, especially during the initial phase of train-stimulation, when the release was maximal. These data show that neurons critically and selectively depend on MUNC18-1 for neuropeptide secretion. Impaired neuropeptide secretion may explain aspects of the behavioral and neurodevelopmental phenotypes that were observed in Munc18-1 HZ mice.SIGNIFICANCE STATEMENT:Neuropeptide secretion from dense-core vesicles (DCVs) modulates synaptic transmission, sleep, appetite, cognition and mood. However, the mechanisms of DCV exocytosis are poorly characterized. Here, we identify MUNC18-1 as an essential component for neuropeptide secretion from DCVs. Paralogs MUNC18-2 or -3 cannot compensate for MUNC18-1. MUNC18-1 is the first protein identified to be essential for both neuropeptide secretion and synaptic transmission. In heterozygous Munc18-1 neurons, that have a 50% reduced MUNC18-1 expression and model the human STXBP1 syndrome, DCV exocytosis is impaired, especially during the initial phase of train-stimulation, when the release is maximal. These data show that MUNC18-1 is essential for neuropeptide secretion and that impaired neuropeptide secretion upon reduced MUNC18-1 expression may contribute to the symptoms of STXBP1 syndrome.

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.

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.

Munc13 controls the location and efficiency of dense-core vesicle release in neurons.

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.