RIM-Binding Protein 2 Organizes Ca2+ Channel Topography and Regulates Release Probability and Vesicle Replenishment at a Fast Central Synapse.

Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivoSIGNIFICANCE STATEMENT Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (CaV)2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing.

Lipopolysaccharide-responsive beige-like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain-containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein-truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.

Modeling and Correction of Protein Conformational Disease in iPSC-derived Neurons through Personalized Base Editing.

Altered protein conformation can cause incurable neurodegenerative disorders. Mutations in SERPINI1 , the gene encoding neuroserpin, alter protein conformation resulting in cytotoxic aggregation in neuronal endoplasmic reticulum. Aggregates cause oxidative stress impairing function, leading to neuronal death. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant progressive myoclonic epilepsy. Patients present with seizures and cognitive impairments that progress to dementia and premature death. We developed HEK293T and induced pluripotent stem cell (iPSC) models of FENIB, harboring the patient's pathogenic SERPINI1 variant or stably overexpressing mutant neuroserpin fused to GFP (MUT NS-GFP). FENIB cells form neuroserpin inclusions which increase in size and number. Here, we utilized a personalized adenine base editor (ABE)-mediated approach to efficiently correct the pathogenic variant and to restore neuronal dendritic morphology. ABE-treated MUT NS-GFP cells demonstrated reduced inclusion size and number. Using an inducible MUT NS-GFP neuron system, we identified early prevention of toxic protein expression allowed aggregate clearance, while late prevention halted neuronal impairments. To address several challenges for clinical applications of gene correction, we developed a neuron-specific engineered virus-like particle to optimize neuronal ABE delivery. Preventing mutant protein with altered conformation production improved toxic protein clearance. Our findings provide a targeted strategy and may treat FENIB and potentially other neurodegenerative diseases due to altered protein conformation such as Alzheimer's and Huntington's diseases.

Cortical and thalamic inputs drive distinct hippocampal microcircuits to modulate synchronized activity during development.

Synchronized activity, a hallmark of hippocampal network dynamics, appears early during development. Whether extrinsic inputs drive such activity remains unknown. In this issue of Neuron, Leprince et al.1 show that synchronized activity, while modulated by both cortical and thalamic inputs ex vivo, depends solely on cortical inputs in vivo.

Piccolo Promotes Vesicle Replenishment at a Fast Central Auditory Synapse.

Piccolo and Bassoon are the two largest cytomatrix of the active zone (CAZ) proteins involved in scaffolding and regulating neurotransmitter release at presynaptic active zones (AZs), but have long been discussed as being functionally redundant. We employed genetic manipulation to bring forth and segregate the role of Piccolo from that of Bassoon at central auditory synapses of the cochlear nucleus-the endbulbs of Held. These synapses specialize in high frequency synaptic transmission, ideally poised to reveal even subtle deficits in the regulation of neurotransmitter release upon molecular perturbation. Combining semi-quantitative immunohistochemistry, electron microscopy, and in vitro and in vivo electrophysiology we first studied signal transmission in Piccolo-deficient mice. Our analysis was not confounded by a cochlear deficit, as a short isoform of Piccolo ("Piccolino") present at the upstream ribbon synapses of cochlear inner hair cells (IHC), is unaffected by the mutation. Disruption of Piccolo increased the abundance of Bassoon at the AZs of endbulbs, while that of RIM1 was reduced and other CAZ proteins remained unaltered. Presynaptic fiber stimulation revealed smaller amplitude of the evoked excitatory postsynaptic currents (eEPSC), while eEPSC kinetics as well as miniature EPSCs (mEPSCs) remained unchanged. Cumulative analysis of eEPSC trains indicated that the reduced eEPSC amplitude of Piccolo-deficient endbulb synapses is primarily due to a reduced readily releasable pool (RRP) of synaptic vesicles (SV), as was corroborated by a reduction of vesicles at the AZ found on an ultrastructural level. Release probability seemed largely unaltered. Recovery from short-term depression was slowed. We then performed a physiological analysis of endbulb synapses from mice which, in addition to Piccolo deficiency, lacked one functional allele of the Bassoon gene. Analysis of the double-mutant endbulbs revealed an increase in release probability, while the synapses still exhibited the reduced RRP, and the impairment in SV replenishment was exacerbated. We propose additive roles of Piccolo and Bassoon in SV replenishment which in turn influences the organization and size of the RRP, and an additional role of Bassoon in regulation of release probability.

RIM-Binding Protein 2 Promotes a Large Number of CaV1.3 Ca2+-Channels and Contributes to Fast Synaptic Vesicle Replenishment at Hair Cell Active Zones.

Ribbon synapses of inner hair cells (IHCs) mediate high rates of synchronous exocytosis to indefatigably track the stimulating sound with sub-millisecond precision. The sophisticated molecular machinery of the inner hair cell active zone realizes this impressive performance by enabling a large number of synaptic voltage-gated CaV1.3 Ca2+-channels, their tight coupling to synaptic vesicles (SVs) and fast replenishment of fusion competent SVs. Here we studied the role of RIM-binding protein 2 (RIM-BP2)-a multidomain cytomatrix protein known to directly interact with Rab3 interacting molecules (RIMs), bassoon and CaV1.3-that is present at the inner hair cell active zones. We combined confocal and stimulated emission depletion (STED) immunofluorescence microscopy, electron tomography, patch-clamp and confocal Ca2+-imaging, as well as auditory systems physiology to explore the morphological and functional effects of genetic RIM-BP2 disruption in constitutive RIM-BP2 knockout mice. We found that RIM-BP2 (1) positively regulates the number of synaptic CaV1.3 channels and thereby facilitates synaptic vesicle release and (2) supports fast synaptic vesicle recruitment after readily releasable pool (RRP) depletion. However, Ca2+-influx-exocytosis coupling seemed unaltered for readily releasable SVs. Recordings of auditory brainstem responses (ABR) and of single auditory nerve fiber firing showed that RIM-BP2 disruption results in a mild deficit of synaptic sound encoding.