Brain-derived neurotrophic factor scales presynaptic calcium transients to modulate excitatory neurotransmission.

Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic physiology, as well as mechanisms underlying various neuropsychiatric diseases and their treatment. Despite its clear physiological role and disease relevance, BDNF's function at the presynaptic terminal, a fundamental unit of neurotransmission, remains poorly understood. In this study, we evaluated single synapse dynamics using optical imaging techniques in hippocampal cell cultures. We find that exogenous BDNF selectively increases evoked excitatory neurotransmission without affecting spontaneous neurotransmission. However, acutely blocking endogenous BDNF has no effect on evoked or spontaneous release, demonstrating that different approaches to studying BDNF may yield different results. When we suppressed BDNF-Tropomyosin receptor kinase B (TrkB) activity chronically over a period of days to weeks using a mouse line enabling conditional knockout of TrkB, we found that evoked glutamate release was significantly decreased while spontaneous release remained unchanged. Moreover, chronic blockade of BDNF-TrkB activity selectively downscales evoked calcium transients without affecting spontaneous calcium events. Via pharmacological blockade by voltage-gated calcium channel (VGCC) selective blockers, we found that the changes in evoked calcium transients are mediated by the P/Q subtype of VGCCs. These results suggest that BDNF-TrkB activity increases presynaptic VGCC activity to selectively increase evoked glutamate release.

Spatially non-overlapping Ca2+ signals drive distinct forms of neurotransmission.

Calcium (Ca2+) signaling is tightly regulated within a presynaptic bouton. Here, we visualize Ca2+ signals within hippocampal presynaptic boutons using GCaMP8s tagged to synaptobrevin, a synaptic vesicle protein. We identify evoked presynaptic Ca2+ transients (ePreCTs) that derive from synchronized voltage-gated Ca2+ channel openings, spontaneous presynaptic Ca2+ transients (sPreCTs) that originate from ryanodine sensitive Ca2+ stores, and a baseline Ca2+ signal that arises from stochastic voltage-gated Ca2+ channel openings. We find that baseline Ca2+, but not sPreCTs, contributes to spontaneous glutamate release. We employ photobleaching as a use-dependent tool to probe nano-organization of Ca2+ signals and observe that all three occur in non-overlapping domains within the synapse at near-resting conditions. However, increased depolarization induces intermixing of these Ca2+ domains via both local and non-local synaptic vesicle turnover. Our findings reveal nanosegregation of Ca2+ signals within a presynaptic terminal that derive from multiple sources and in turn drive specific modes of neurotransmission.

Bi-allelic ATG4D variants are associated with a neurodevelopmental disorder characterized by speech and motor impairment.

Autophagy regulates the degradation of damaged organelles and protein aggregates, and is critical for neuronal development, homeostasis, and maintenance, yet few neurodevelopmental disorders have been associated with pathogenic variants in genes encoding autophagy-related proteins. We report three individuals from two unrelated families with a neurodevelopmental disorder characterized by speech and motor impairment, and similar facial characteristics. Rare, conserved, bi-allelic variants were identified in ATG4D, encoding one of four ATG4 cysteine proteases important for autophagosome biogenesis, a hallmark of autophagy. Autophagosome biogenesis and induction of autophagy were intact in cells from affected individuals. However, studies evaluating the predominant substrate of ATG4D, GABARAPL1, demonstrated that three of the four ATG4D patient variants functionally impair ATG4D activity. GABARAPL1 is cleaved or "primed" by ATG4D and an in vitro GABARAPL1 priming assay revealed decreased priming activity for three of the four ATG4D variants. Furthermore, a rescue experiment performed in an ATG4 tetra knockout cell line, in which all four ATG4 isoforms were knocked out by gene editing, showed decreased GABARAPL1 priming activity for the two ATG4D missense variants located in the cysteine protease domain required for priming, suggesting that these variants impair the function of ATG4D. The clinical, bioinformatic, and functional data suggest that bi-allelic loss-of-function variants in ATG4D contribute to the pathogenesis of this syndromic neurodevelopmental disorder.

Probing the segregation of evoked and spontaneous neurotransmission via photobleaching and recovery of a fluorescent glutamate sensor.

Synapses maintain both action potential-evoked and spontaneous neurotransmitter release; however, organization of these two forms of release within an individual synapse remains unclear. Here, we used photobleaching properties of iGluSnFR, a fluorescent probe that detects glutamate, to investigate the subsynaptic organization of evoked and spontaneous release in primary hippocampal cultures. In nonneuronal cells and neuronal dendrites, iGluSnFR fluorescence is intensely photobleached and recovers via diffusion of nonphotobleached probes with a time constant of ~10 s. After photobleaching, while evoked iGluSnFR events could be rapidly suppressed, their recovery required several hours. In contrast, iGluSnFR responses to spontaneous release were comparatively resilient to photobleaching, unless the complete pool of iGluSnFR was activated by glutamate perfusion. This differential effect of photobleaching on different modes of neurotransmission is consistent with a subsynaptic organization where sites of evoked glutamate release are clustered and corresponding iGluSnFR probes are diffusion restricted, while spontaneous release sites are broadly spread across a synapse with readily diffusible iGluSnFR probes.

BDNF signaling in context: From synaptic regulation to psychiatric disorders.

Brain-derived neurotrophic factor (BDNF) is a neuropeptide that plays numerous important roles in synaptic development and plasticity. While its importance in fundamental physiology is well established, studies of BDNF often produce conflicting and unclear results, and the scope of existing research makes the prospect of setting future directions daunting. In this review, we examine the importance of spatial and temporal factors on BDNF activity, particularly in processes such as synaptogenesis, Hebbian plasticity, homeostatic plasticity, and the treatment of psychiatric disorders. Understanding the fundamental physiology of when, where, and how BDNF acts and new approaches to control BDNF signaling in time and space can contribute to improved therapeutics and patient outcomes.

Novel mutations in CLN6 cause late-infantile neuronal ceroid lipofuscinosis without visual impairment in two unrelated patients.

CLN6 is a transmembrane protein located in the endoplasmic reticulum that is involved in lysosomal acidification. Mutations in CLN6 cause late-infantile neuronal ceroid lipofuscinosis (LINCL), and teenage and adult onset NCL without visual impairment. Here we describe two pediatric patients with LINCL from unrelated families who were evaluated at the National Institutes of Health. Both children exhibited typical phenotypes associated with LINCL except that they lacked the expected visual impairment. Whole exome sequencing identified novel biallelic mutations in CLN6, i.e., c.218-220dupGGT (p.Trp73dup) and c.296A > G (p.Lys99Arg) in Proband 1 and homozygous c.723G > T (p.Met241Ile) in Proband 2. Expression analysis in dermal fibroblasts showed a small increase in CLN6 protein levels. Electron micrographs of these fibroblasts demonstrated large numbers of small membrane-bound vesicles, in addition to lipofuscin deposits. LysoTracker™ Red intensity was increased in fibroblasts from both patients. This study supports a role for CLN6 in lysosomal homeostasis, and highlights the importance of considering CLN6 mutations in the diagnosis of Batten Disease even in patients with normal vision.