Neuronal LRP4 directs the development, maturation and cytoskeletal organization of Drosophila peripheral synapses.

Synaptic development requires multiple signaling pathways to ensure successful connections. Transmembrane receptors are optimally positioned to connect the synapse and the rest of the neuron, often acting as synaptic organizers to synchronize downstream events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor that has been most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, identified emerging roles, but how LRP4 acts as a presynaptic organizer and the downstream mechanisms of LRP4 are not well understood. Here, we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motoneurons to instruct pre- and postsynaptic development. Loss of presynaptic LRP4 results in multiple defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. These data demonstrate a function for presynaptic LRP4 as a peripheral synaptic organizer, highlight a downstream mechanism conserved with its CNS function in Drosophila, and underscore previously unappreciated but important developmental roles for LRP4 in cytoskeletal organization, synapse maturation and active zone organization.

Neuronal LRP4 directs the development, maturation, and cytoskeletal organization of peripheral synapses.

Synapse development requires multiple signaling pathways to accomplish the myriad of steps needed to ensure a successful connection. Transmembrane receptors on the cell surface are optimally positioned to facilitate communication between the synapse and the rest of the neuron and often function as synaptic organizers to synchronize downstream signaling events. One such organizer, the LDL receptor-related protein LRP4, is a cell surface receptor most well-studied postsynaptically at mammalian neuromuscular junctions. Recent work, however, has identified emerging roles for LRP4 as a presynaptic molecule, but how LRP4 acts as a presynaptic organizer, what roles LRP4 plays in organizing presynaptic biology, and the downstream mechanisms of LRP4 are not well understood. Here we show that LRP4 functions presynaptically at Drosophila neuromuscular synapses, acting in motor neurons to instruct multiple aspects of pre- and postsynaptic development. Loss of presynaptic LRP4 results in a range of developmental defects, impairing active zone organization, synapse growth, physiological function, microtubule organization, synaptic ultrastructure, and synapse maturation. We further demonstrate that LRP4 promotes most aspects of presynaptic development via a downstream SR-protein kinase, SRPK79D. SRPK79D overexpression suppresses synaptic defects associated with loss of lrp4. These data demonstrate a function for LRP4 as a peripheral synaptic organizer acting presynaptically, highlight a downstream mechanism conserved with its CNS function, and indicate previously unappreciated roles for LRP4 in cytoskeletal organization, synapse maturation, and active zone organization, underscoring its developmental importance.

The microbiota promotes social behavior by modulating microglial remodeling of forebrain neurons.

Host-associated microbiotas guide the trajectory of developmental programs, and altered microbiota composition is linked to neurodevelopmental conditions such as autism spectrum disorder. Recent work suggests that microbiotas modulate behavioral phenotypes associated with these disorders. We discovered that the zebrafish microbiota is required for normal social behavior and reveal a molecular pathway linking the microbiota, microglial remodeling of neural circuits, and social behavior in this experimentally tractable model vertebrate. Examining neuronal correlates of behavior, we found that the microbiota restrains neurite complexity and targeting of forebrain neurons required for normal social behavior and is necessary for localization of forebrain microglia, brain-resident phagocytes that remodel neuronal arbors. The microbiota also influences microglial molecular functions, including promoting expression of the complement signaling pathway and the synaptic remodeling factor c1q. Several distinct bacterial taxa are individually sufficient for normal microglial and neuronal phenotypes, suggesting that host neuroimmune development is sensitive to a feature common among many bacteria. Our results demonstrate that the microbiota influences zebrafish social behavior by stimulating microglial remodeling of forebrain circuits during early neurodevelopment and suggest pathways for new interventions in multiple neurodevelopmental disorders.

Endogenous Tagging Reveals Differential Regulation of Ca2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression.

Neurons communicate through Ca2+-dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca2+ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca2+ channels during homeostatic plasticity. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca2+ influx during PHD is achieved through functional adaptions to pre-existing Ca2+ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.SIGNIFICANCE STATEMENT Presynaptic Ca2+ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca2+ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca2+ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca2+ channels at individual active zones within 10 min. We propose that differential regulation of Ca2+ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication.

Fife organizes synaptic vesicles and calcium channels for high-probability neurotransmitter release.

The strength of synaptic connections varies significantly and is a key determinant of communication within neural circuits. Mechanistic insight into presynaptic factors that establish and modulate neurotransmitter release properties is crucial to understanding synapse strength, circuit function, and neural plasticity. We previously identified Drosophila Piccolo-RIM-related Fife, which regulates neurotransmission and motor behavior through an unknown mechanism. Here, we demonstrate that Fife localizes and interacts with RIM at the active zone cytomatrix to promote neurotransmitter release. Loss of Fife results in the severe disruption of active zone cytomatrix architecture and molecular organization. Through electron tomographic and electrophysiological studies, we find a decrease in the accumulation of release-ready synaptic vesicles and their release probability caused by impaired coupling to Ca2+ channels. Finally, we find that Fife is essential for the homeostatic modulation of neurotransmission. We propose that Fife organizes active zones to create synaptic vesicle release sites within nanometer distance of Ca2+ channel clusters for reliable and modifiable neurotransmitter release.

BAR-SH3 sorting nexins are conserved interacting proteins of Nervous wreck that organize synapses and promote neurotransmission.

Nervous wreck (Nwk) is a conserved F-BAR protein that attenuates synaptic growth and promotes synaptic function in Drosophila. In an effort to understand how Nwk carries out its dual roles, we isolated interacting proteins using mass spectrometry. We report a conserved interaction between Nwk proteins and BAR-SH3 sorting nexins, a family of membrane-binding proteins implicated in diverse intracellular trafficking processes. In mammalian cells, BAR-SH3 sorting nexins induce plasma membrane tubules that localize NWK2, consistent with a possible functional interaction during the early stages of endocytic trafficking. To study the role of BAR-SH3 sorting nexins in vivo, we took advantage of the lack of genetic redundancy in Drosophila and employed CRISPR-based genome engineering to generate null and endogenously tagged alleles of SH3PX1. SH3PX1 localizes to neuromuscular junctions where it regulates synaptic ultrastructure, but not synapse number. Consistently, neurotransmitter release was significantly diminished in SH3PX1 mutants. Double-mutant and tissue-specific-rescue experiments indicate that SH3PX1 promotes neurotransmitter release presynaptically, at least in part through functional interactions with Nwk, and might act to distinguish the roles of Nwk in regulating synaptic growth and function.

Advances in imaging ultrastructure yield new insights into presynaptic biology.

Synapses are the fundamental functional units of neural circuits, and their dysregulation has been implicated in diverse neurological disorders. At presynaptic terminals, neurotransmitter-filled synaptic vesicles are released in response to calcium influx through voltage-gated calcium channels activated by the arrival of an action potential. Decades of electrophysiological, biochemical, and genetic studies have contributed to a growing understanding of presynaptic biology. Imaging studies are yielding new insights into how synapses are organized to carry out their critical functions. The development of techniques for rapid immobilization and preservation of neuronal tissues for electron microscopy (EM) has led to a new renaissance in ultrastructural imaging that is rapidly advancing our understanding of synapse structure and function.

CD4 T-cell memory responses to viral infections of humans show pronounced immunodominance independent of duration or viral persistence.

Little is known concerning immunodominance within the CD4 T-cell response to viral infections and its persistence into long-term memory. We tested CD4 T-cell reactivity against each viral protein in persons immunized with vaccinia virus (VV), either recently or more than 40 years ago, as a model self-limited viral infection. Similar tests were done with persons with herpes simplex virus 1 (HSV-1) infection as a model chronic infection. We used an indirect method capable of counting the CD4 T cells in blood reactive with each individual viral protein. Each person had a clear CD4 T-cell dominance hierarchy. The top four open reading frames accounted for about 40% of CD4 virus-specific T cells. Early and long-term memory CD4 T-cell responses to vaccinia virus were mathematically indistinguishable for antigen breadth and immunodominance. Despite the chronic intermittent presence of HSV-1 antigen, the CD4 T-cell dominance and diversity patterns for HSV-1 were identical to those observed for vaccinia virus. The immunodominant CD4 T-cell antigens included both long proteins abundantly present in virions and shorter, nonstructural proteins. Limited epitope level and direct ex vivo data were also consistent with pronounced CD4 T-cell immunodominance. We conclude that human memory CD4 T-cell responses show a pattern of pronounced immunodominance for both chronic and self-limited viral infections and that this pattern can persist over several decades in the absence of antigen.

Fife, a Drosophila Piccolo-RIM homolog, promotes active zone organization and neurotransmitter release.

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. Here, we present the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, we find that the Drosophila homolog, Fife, is neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, we generated a deletion of the fife locus. We find that evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, we identify underlying AZ abnormalities including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. Our data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems.

Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine.

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8+ T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8+ and CD4+ T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4+ T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8+ and CD4+ T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods - also demonstrated in principle for vaccinia virus for both CD8+ and CD4+ T cells - should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens.