Liquid-liquid phase separation in presynaptic nerve terminals.

The presynaptic nerve terminal is crucial for transmitting signals to the adjacent cell. To fulfill this role, specific proteins with distinct functions are concentrated in spatially confined areas within the nerve terminals. A recent concept termed liquid-liquid phase separation (LLPS) has provided new insights into how this process may occur. In this review, we aim to summarize the LLPS of proteins in different parts of the presynaptic nerve terminals, including synaptic vesicle (SV) clusters, the active zone (AZ), and the endocytic zone, with an additional focus on neurodegenerative diseases (NDDs), where the functional relevance of these properties is explored. Last, we propose new perspectives and future directions for the role of LLPS in presynaptic nerve terminals.

Presynaptic neurons self-tune by inversely coupling neurotransmitter release with the abundance of CaV2 voltage-gated Ca2+ channels.

The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans, revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability.

The intracellular C-terminus confers compartment-specific targeting of voltage-gated calcium channels.

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca2+ channels (CaVs) to distinct subcellular compartments. In hippocampal neurons, CaV2s trigger neurotransmitter release at the presynaptic active zone, and CaV1s localize somatodendritically. After knockout of all three CaV2s, expression of CaV2.1, but not CaV1.3, restores neurotransmitter release. We find that chimeric CaV1.3s with CaV2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to CaV1 blockers. This dominant targeting function of the CaV2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the CaV2.1 C-terminus with that of CaV1.3 abolishes CaV2.1 active zone localization and function. We conclude that CaV intracellular C-termini mediate compartment-specific targeting.

RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.

Different states of synaptic vesicle priming explain target cell type-dependent differences in neurotransmitter release.

Pronounced differences in neurotransmitter release from a given presynaptic neuron, depending on the synaptic target, are among the most intriguing features of cortical networks. Hippocampal pyramidal cells (PCs) release glutamate with low probability to somatostatin expressing oriens-lacunosum-moleculare (O-LM) interneurons (INs), and the postsynaptic responses show robust short-term facilitation, whereas the release from the same presynaptic axons onto fast-spiking INs (FSINs) is ~10-fold higher and the excitatory postsynaptic currents (EPSCs) display depression. The mechanisms underlying these vastly different synaptic behaviors have not been conclusively identified. Here, we applied a combined functional, pharmacological, and modeling approach to address whether the main difference lies in the action potential-evoked fusion or else in upstream priming processes of synaptic vesicles (SVs). A sequential two-step SV priming model was fitted to the peak amplitudes of unitary EPSCs recorded in response to complex trains of presynaptic stimuli in acute hippocampal slices of adult mice. At PC-FSIN connections, the fusion probability (Pfusion) of well-primed SVs is 0.6, and 44% of docked SVs are in a fusion-competent state. At PC-O-LM synapses, Pfusion is only 40% lower (0.36), whereas the fraction of well-primed SVs is 6.5-fold smaller. Pharmacological enhancement of fusion by 4-AP and priming by PDBU was recaptured by the model with a selective increase of Pfusion and the fraction of well-primed SVs, respectively. Our results demonstrate that the low fidelity of transmission at PC-O-LM synapses can be explained by a low occupancy of the release sites by well-primed SVs.

Paroxysmal dystonia results from the loss of RIM4 in Purkinje cells.

Full-length RIM1 and 2 are key components of the presynaptic active zone that ubiquitously control excitatory and inhibitory neurotransmitter release. Here, we report that the function of the small RIM isoform RIM4, consisting of a single C2 domain, is strikingly different from that of the long isoforms. RIM4 is dispensable for neurotransmitter release but plays a postsynaptic, cell type-specific role in cerebellar Purkinje cells that is essential for normal motor function. In the absence of RIM4, Purkinje cell intrinsic firing is reduced and caffeine-sensitive, and dendritic integration of climbing fibre input is disturbed. Mice lacking RIM4, but not mice lacking RIM1/2, selectively in Purkinje cells exhibit a severe, hours-long paroxysmal dystonia. These episodes can also be induced by caffeine, ethanol or stress and closely resemble the deficits seen with mutations of the PNKD (paroxysmal non-kinesigenic dystonia) gene. Our data reveal essential postsynaptic functions of RIM proteins and show non-overlapping specialized functions of a small isoform despite high homology to a single domain in the full-length proteins.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones.

In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin-dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small.

Ca2+ regulation of glutamate release from inner hair cells of hearing mice.

In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.

An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Presynaptic Cytomatrix Proteins.

The Cytomatrix Assembled at the active Zone (CAZ) of a presynaptic terminal displays electron-dense appearance and defines the center of the synaptic vesicle release. The protein constituents of CAZ are multiple-domain scaffolds that interact extensively with each other and also with an ensemble of synaptic vesicle proteins to ensure docking, fusion, and recycling. Reflecting the central roles of the active zone in synaptic transmission, CAZ proteins are highly conserved throughout evolution. As the nervous system increases complexity and diversity in types of neurons and synapses, CAZ proteins expand in the number of gene and protein isoforms and interacting partners. This chapter summarizes the discovery of the core CAZ proteins and current knowledge of their functions.

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.

The active zone protein CLA-1 (Clarinet) bridges two subsynaptic domains to regulate presynaptic sorting of ATG-9.

In neuronal synapses, autophagosome biogenesis is coupled with the activity-dependent synaptic vesicle cycle via ATG-9. How vesicles containing ATG-9 are sorted at the presynapse is unknown. We performed forward genetic screens at single synapses of C. elegans neurons for mutants that disrupt ATG-9 presynaptic localization, and identified the long isoform of the active zone protein CLA-1 (Clarinet; CLA-1 L). We find that disrupting CLA-1 L results in abnormal accumulation of ATG-9-containing vesicles enriched with clathrin. The adaptor protein complexes and proteins at the periactive zone genetically interact with CLA-1 L in ATG-9 sorting. Moreover, the phenotype of the ATG-9 protein in cla-1(L) mutants was not observed for integral synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic macroautophagy/autophagy.

The role of RIM in neurotransmitter release: promotion of synaptic vesicle docking, priming, and fusion.

There are many special sites at the end of a synapse called active zones (AZs). Synaptic vesicles (SVs) fuse with presynaptic membranes at these sites, and this fusion is an important step in neurotransmitter release. The cytomatrix in the active zone (CAZ) is made up of proteins such as the regulating synaptic membrane exocytosis protein (RIM), RIM-binding proteins (RIM-BPs), ELKS/CAST, Bassoon/Piccolo, Liprin-α, and Munc13-1. RIM is a scaffold protein that interacts with CAZ proteins and presynaptic functional components to affect the docking, priming, and fusion of SVs. RIM is believed to play an important role in regulating the release of neurotransmitters (NTs). In addition, abnormal expression of RIM has been detected in many diseases, such as retinal diseases, Asperger's syndrome (AS), and degenerative scoliosis. Therefore, we believe that studying the molecular structure of RIM and its role in neurotransmitter release will help to clarify the molecular mechanism of neurotransmitter release and identify targets for the diagnosis and treatment of the aforementioned diseases.

Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." Here, we used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated ∼2-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.SIGNIFICANCE STATEMENT "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how input-specific synaptic diversity is achieved, which could have implications for neurologic disorders that involve changes in synaptic function.

Structural basis of ELKS/Rab6B interaction and its role in vesicle capturing enhanced by liquid-liquid phase separation.

ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts with the vesicular traffic regulator, the Rab6 GTPase, the molecular basis governing ELKS-mediated trafficking of Rab6-coated vesicles, has remained unclear. In this study, we solved the Rab6B structure in complex with the Rab6-binding domain of ELKS1, revealing that a C-terminal segment of ELKS1 forms a helical hairpin to recognize Rab6B through a unique binding mode. We further showed that liquid-liquid phase separation (LLPS) of ELKS1 allows it to compete with other Rab6 effectors for binding to Rab6B and accumulate Rab6B-coated liposomes to the protein condensate formed by ELKS1. We also found that the ELKS1 condensate recruits Rab6B-coated vesicles to vesicle-releasing sites and promotes vesicle exocytosis. Together, our structural, biochemical, and cellular analyses suggest that ELKS1, via the LLPS-enhanced interaction with Rab6, captures Rab6-coated vesicles from the cargo transport machine for efficient vesicle release at exocytotic sites. These findings shed new light on the understanding of spatiotemporal regulation of vesicle trafficking through the interplay between membranous structures and membraneless condensates.

Two forms of asynchronous release with distinctive spatiotemporal dynamics in central synapses.

Asynchronous release is a ubiquitous form of neurotransmitter release that persists for tens to hundreds of milliseconds after an action potential. How asynchronous release is organized and regulated at the synaptic active zone (AZ) remains debatable. Using nanoscale-precision imaging of individual release events in rat hippocampal synapses, we observed two spatially distinct subpopulations of asynchronous events, ~75% of which occurred inside the AZ and with a bias towards the AZ center, while ~25% occurred outside of the functionally defined AZ, that is, ectopically. The two asynchronous event subpopulations also differed from each other in temporal properties, with ectopic events occurring at significantly longer time intervals from synchronous events than the asynchronous events inside the AZ. Both forms of asynchronous release did not, to a large extent, utilize the same release sites as synchronous events. The two asynchronous event subpopulations also differ from synchronous events in some aspects of exo-endocytosis coupling, particularly in the contribution from the fast calcium-dependent endocytosis. These results identify two subpopulations of asynchronous release events with distinctive organization and spatiotemporal dynamics.

C. elegans Clarinet/CLA-1 recruits RIMB-1/RIM-binding protein and UNC-13 to orchestrate presynaptic neurotransmitter release.

Synaptic transmission requires the coordinated activity of multiple synaptic proteins that are localized at the active zone (AZ). We previously identified a Caenorhabditis elegans protein named Clarinet (CLA-1) based on homology to the AZ proteins Piccolo, Rab3-interactingmolecule (RIM)/UNC-10 and Fife. At the neuromuscular junction (NMJ), cla-1 null mutants exhibit release defects that are greatly exacerbated in cla-1;unc-10 double mutants. To gain insights into the coordinated roles of CLA-1 and UNC-10, we examined the relative contributions of each to the function and organization of the AZ. Using a combination of electrophysiology, electron microscopy, and quantitative fluorescence imaging we explored the functional relationship of CLA-1 to other key AZ proteins including: RIM1, Cav2.1 channels, RIM1-binding protein, and Munc13 (C. elegans UNC-10, UNC-2, RIMB-1 and UNC-13, respectively). Our analyses show that CLA-1 acts in concert with UNC-10 to regulate UNC-2 calcium channel levels at the synapse via recruitment of RIMB-1. In addition, CLA-1 exerts a RIMB-1-independent role in the localization of the priming factor UNC-13. Thus C. elegans CLA-1/UNC-10 exhibit combinatorial effects that have overlapping design principles with other model organisms: RIM/RBP and RIM/ELKS in mouse and Fife/RIM and BRP/RBP in Drosophila. These data support a semiconserved arrangement of AZ scaffolding proteins that are necessary for the localization and activation of the fusion machinery within nanodomains for precise coupling to Ca2+ channels.