Heterogeneity of glutamatergic synapses: cellular mechanisms and network consequences.

Chemical synapses are commonly known as a structurally and functionally highly diverse class of cell-cell contacts specialized to mediate communication between neurons. They represent the smallest "computational" unit of the brain and are typically divided into excitatory and inhibitory as well as modulatory categories. These categories are subdivided into diverse types, each representing a different structure-function repertoire that in turn are thought to endow neuronal networks with distinct computational properties. The diversity of structure and function found among a given category of synapses is referred to as heterogeneity. The main building blocks for this heterogeneity are synaptic vesicles, the active zone, the synaptic cleft, the postsynaptic density, and glial processes associated with the synapse. Each of these five structural modules entails a distinct repertoire of functions, and their combination specifies the range of functional heterogeneity at mammalian excitatory synapses, which are the focus of this review. We describe synapse heterogeneity that is manifested on different levels of complexity ranging from the cellular morphology of the pre- and postsynaptic cells toward the expression of different protein isoforms at individual release sites. We attempt to define the range of structural building blocks that are used to vary the basic functional repertoire of excitatory synaptic contacts and discuss sources and general mechanisms of synapse heterogeneity. Finally, we explore the possible impact of synapse heterogeneity on neuronal network function.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in cardiac failure if left untreated. We hypothesized that the tail-anchored protein dysferlin with multiple Ca2+-binding C2-domains is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. OBJECTIVE: To reveal the impact of the membrane fusion and repair protein dysferlin on TAT network stabilization and proliferation necessary for the hypertrophic growth of cardiomyocytes. METHODS AND RESULTS: Super-resolution light and electron microscopy of mouse cardiomyocytes identified a specific localization of dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Mass spectrometry was used to characterize the cardiac dysferlin interactome, thereby identifying a novel protein interaction with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. While the dysferlin knockout caused a mild progressive phenotype of dilated cardiomyopathy in the mouse heart, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging demonstrated a profound reorganization of the TAT network in wild-type left-ventricular myocytes post-transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Piccolino is required for ribbon architecture at cochlear inner hair cell synapses and for hearing.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene-trap mutant rats (Pclogt/gt ). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.

Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.

Tubular microdomains of Rab7-positive endosomes retrieve TrkA, a mechanism disrupted in Charcot-Marie-Tooth disease 2B.

Axonal survival and growth requires signalling from tropomyosin receptor kinases (Trks). To transmit their signals, receptor-ligand complexes are endocytosed and undergo retrograde trafficking to the soma, where downstream signalling occurs. Vesicles transporting neurotrophic receptors to the soma are reported to be Rab7-positive late endosomes and/or multivesicular bodies (MVBs), where receptors localize within so-called intraluminal vesicles (herein Rab7 corresponds to Rab7A unless specified otherwise). Therefore, one challenging question is how downstream signalling is possible given the insulating properties of intraluminal vesicles. In this study, we report that Rab7-positive endosomes and MVBs retrieve TrkA (also known as NTRK1) through tubular microdomains. Interestingly, this phenotype is absent for the EGF receptor. Furthermore, we found that endophilinA1, endophilinA2 and endophilinA3, together with WASH1 (also known as WASHC1), are involved in the tubulation process. In Charcot-Marie-Tooth disease 2B (CMT2B), a neuropathy of the peripheral nervous system, this tubulating mechanism is disrupted. In addition, the ability to tubulate correlates with the phosphorylation levels of TrkA as well as with neurite length in neuronal cultures from dorsal root ganglia. In all, we report a new retrieval mechanism of late Rab7-positive endosomes, which enables TrkA signalling and sheds new light onto how neurotrophic signalling is disrupted in CMT2B. This article has an associated First Person interview with the first author of the paper.

Methods for multiscale structural and functional analysis of the mammalian cochlea.

The mammalian cochlea is a snail-shaped structure deeply that is embedded in the temporal bone and harbors the auditory sensory epithelium - the organ of Corti. Since the discovery of this remarkable hearing organ in the middle of the 19th century, generations of anatomists and physiologists have been attracted to study the structural and functional details of this intricate and delicate structure and thereby contributed to establishing our current understanding of peripheral sound encoding. Since these early days, the continued development of novel imaging technologies - both on light and electron microscopic level - has driven the auditory research field and now enables the visualization of cochlear structures across multiple scales with unprecedented clarity and exquisite detail. To honor these achievements, this review aims to provide a concise overview of current multi-scale imaging methodologies to investigate cochlear anatomy and cellular function in the peripheral auditory pathway. For this purpose, we will outline the technological concepts underlying these techniques - ranging from label-free to label-containing approaches - highlight their respective strengths and limitations and provide specific examples of their use in modern auditory research. We will focus on traditional as well as less conventional imaging techniques that present essential tools for unraveling the protein composition, nanoscale assembly, and physiology of the first auditory synapse and associated structures. In addition, we will introduce novel non-invasive large-scale methodologies that allow for high-resolution in situ imaging of the structurally-unperturbed cochlea and point out potential future applications. In combination, these techniques allow for a comprehensive multi-scale analysis of cochlear structure and function.

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.

AP180 promotes release site clearance and clathrin-dependent vesicle reformation in mouse cochlear inner hair cells.

High-throughput neurotransmission at ribbon synapses of cochlear inner hair cells (IHCs) requires tight coupling of neurotransmitter release and balanced recycling of synaptic vesicles (SVs) as well as rapid restoration of release sites. Here, we examined the role of the adaptor protein AP180 (also known as SNAP91) for IHC synaptic transmission by comparing AP180-knockout (KO) and wild-type mice using high-pressure freezing and electron tomography, confocal microscopy, patch-clamp membrane capacitance measurements and systems physiology. AP180 was found predominantly at the synaptic pole of IHCs. AP180-deficient IHCs had severely reduced SV numbers, slowed endocytic membrane retrieval and accumulated endocytic intermediates near ribbon synapses, indicating that AP180 is required for clathrin-dependent endocytosis and SV reformation in IHCs. Moreover, AP180 deletion led to a high prevalence of SVs in a multi-tethered or docked state after stimulation, a reduced rate of SV replenishment and a hearing impairment. We conclude that, in addition to its role in clathrin recruitment, AP180 contributes to release site clearance in IHCs.This article has an associated First Person interview with the first author of the paper.

Mapping developmental maturation of inner hair cell ribbon synapses in the apical mouse cochlea.

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size.

Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants.

The multi-C2 domain protein otoferlin is required for hearing and mutated in human deafness. Some OTOF mutations cause a mild elevation of auditory thresholds but strong impairment of speech perception. At elevated body temperature, hearing is lost. Mice homozygous for one of these mutations, OtofI515T/I515T, exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In OtofI515T/I515T inner hair cells (IHCs), otoferlin levels are diminished by 65%, and synaptic vesicles are enlarged. Exocytosis during prolonged stimulation is strongly reduced. This indicates that otoferlin is critical for the reformation of properly sized and fusion-competent synaptic vesicles. Moreover, we found sustained exocytosis and sound encoding to scale with the amount of otoferlin at the plasma membrane. We identified a 20 amino acid motif including an RXR motif, presumably present in human but not in mouse otoferlin, which reduces the plasma membrane abundance of Ile515Thr-otoferlin. Together, this likely explains the auditory synaptopathy at normal temperature and the temperature-sensitive deafness in humans carrying the Ile515Thr mutation.

Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing.

The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca2+ channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.

Vesicle sub-pool organization at inner hair cell ribbon synapses.

The afferent inner hair cell synapse harbors the synaptic ribbon, which ensures a constant vesicle supply. Synaptic vesicles (SVs) are arranged in morphologically discernable pools, linked via filaments to the ribbon or the presynaptic membrane. We propose that filaments play a major role in SV resupply and exocytosis at the ribbon. Using advanced electron microscopy, we demonstrate that SVs are organized in sub-pools defined by the filament number per vesicle and its connections. Upon stimulation, SVs increasingly linked to other vesicles and to the ribbon, whereas single-tethered SVs dominated at the membrane. Mutant mice for the hair cell protein otoferlin (pachanga, OtofPga/Pga ) are profoundly deaf with reduced sustained release, serving as a model to investigate the SV replenishment at IHCs. Upon stimulation, multiple-tethered and docked vesicles (rarely observed in wild-type) accumulated at OtofPga/Pga active zones due to an impairment downstream of docking. Conclusively, vesicles are organized in sub-pools at ribbon-type active zones by filaments to support vesicle supply, transport, and finally release.

Disruption of adaptor protein 2µ (AP-2µ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2µ (AP-2µ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2µ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2µ-deficient IHCs, indicating a further role of AP-2µ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

Nanomachinery Organizing Release at Neuronal and Ribbon Synapses.

A critical aim in neuroscience is to obtain a comprehensive view of how regulated neurotransmission is achieved. Our current understanding of synapses relies mainly on data from electrophysiological recordings, imaging, and molecular biology. Based on these methodologies, proteins involved in a synaptic vesicle (SV) formation, mobility, and fusion at the active zone (AZ) membrane have been identified. In the last decade, electron tomography (ET) combined with a rapid freezing immobilization of neuronal samples opened a window for understanding the structural machinery with the highest spatial resolution in situ. ET provides significant insights into the molecular architecture of the AZ and the organelles within the presynaptic nerve terminal. The specialized sensory ribbon synapses exhibit a distinct architecture from neuronal synapses due to the presence of the electron-dense synaptic ribbon. However, both synapse types share the filamentous structures, also commonly termed as tethers that are proposed to contribute to different steps of SV recruitment and exocytosis. In this review, we discuss the emerging views on the role of filamentous structures in SV exocytosis gained from ultrastructural studies of excitatory, mainly central neuronal compared to ribbon-type synapses with a focus on inner hair cell (IHC) ribbon synapses. Moreover, we will speculate on the molecular entities that may be involved in filament formation and hence play a crucial role in the SV cycle.

Bassoon-disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse.

Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse.

Developmental refinement of hair cell synapses tightens the coupling of Ca2+ influx to exocytosis.

Cochlear inner hair cells (IHCs) develop from pre-sensory pacemaker to sound transducer. Here, we report that this involves changes in structure and function of the ribbon synapses between IHCs and spiral ganglion neurons (SGNs) around hearing onset in mice. As synapses matured they changed from holding several small presynaptic active zones (AZs) and apposed postsynaptic densities (PSDs) to one large AZ/PSD complex per SGN bouton. After the onset of hearing (i) IHCs had fewer and larger ribbons; (ii) CaV1.3 channels formed stripe-like clusters rather than the smaller and round clusters at immature AZs; (iii) extrasynaptic CaV1.3-channels were selectively reduced, (iv) the intrinsic Ca(2)(+) dependence of fast exocytosis probed by Ca(2)(+) uncaging remained unchanged but (v) the apparent Ca(2)(+) dependence of exocytosis linearized, when assessed by progressive dihydropyridine block of Ca(2)(+) influx. Biophysical modeling of exocytosis at mature and immature AZ topographies suggests that Ca(2)(+) influx through an individual channel dominates the [Ca(2)(+)] driving exocytosis at each mature release site. We conclude that IHC synapses undergo major developmental refinements, resulting in tighter spatial coupling between Ca(2)(+) influx and exocytosis.

Rab3-interacting molecules 2α and 2ß promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones.

Ca(2+) influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)-interacting molecules 2α and ß (RIM2α and RIM2ß) in clustering voltage-gated CaV1.3 Ca(2+) channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2ß and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca(2+) imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic CaV1.3 Ca(2+) channels, resulting in reduced synaptic Ca(2+) influx. Using superresolution microscopy, we found that Ca(2+) channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca(2+) current, whereas the apparent Ca(2+) dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca(2+) influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2ß promote a large complement of synaptic Ca(2+) channels at IHC AZs and are required for normal hearing.

Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons.

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B' [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3ß kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot).

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.