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.

Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function.

Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. Here, we generate null alleles of TRMT9B and find it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, we find that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

A CRISPR view of development.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is poised to transform developmental biology by providing a simple, efficient method to precisely manipulate the genome of virtually any developing organism. This RNA-guided nuclease (RGN)-based approach already has been effectively used to induce targeted mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. Illustrating the adaptability of RGNs, the genomes of >20 different plant and animal species as well as multiple cell lines and primary cells have been successfully modified. Here we review the current and potential uses of RGNs to investigate genome function during development.

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.

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.

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.

PDZD8 promotes autophagy at ER-Lysosome contact sites to regulate synaptogenesis.

Building synaptic connections, which are often far from the soma, requires coordinating a host of cellular activities from transcription to protein turnover, placing a high demand on intracellular communication. Membrane contact sites (MCSs) formed between cellular organelles have emerged as key signaling hubs for coordinating an array of cellular activities. We have found that the endoplasmic reticulum (ER) MCS tethering protein PDZD8 is required for activity-dependent synaptogenesis. PDZD8 is sufficient to drive ectopic synaptic bouton formation through an autophagy-dependent mechanism and required for basal synapse formation when autophagy biogenesis is limited. PDZD8 functions at ER-late endosome/lysosome (LEL) MCSs to promote lysosome maturation and accelerate autophagic flux. Mutational analysis of PDZD8's SMP domain further suggests a role for lipid transfer at ER-LEL MCSs. We propose that PDZD8-dependent lipid transfer from ER to LELs promotes lysosome maturation to increase autophagic flux during periods of high demand, including activity-dependent synapse formation.

Introduction to CRISPR and Its Use in Drosophila.

The preeminence of Drosophila genetics has led to key discoveries in biology across a variety of fields and disciplines. The advent of CRISPR gene editing has expanded the toolkit of genetic reagents that can be applied to manipulate and observe genes, RNAs, and proteins in an in vivo context. This review describes CRISPR and its use as a transformative gene editing tool in Drosophila We focus on the canonical pathway in which the Cas9 nuclease is directed to specific sequences by guide RNA (gRNA), where cleavage leads to DNA repair by one of two main cellular pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway can be appropriated to disrupt targeted sequences, enabling a variety of loss-of-function studies. Induction of the HDR pathway allows precise editing, including defined deletions, the introduction of specific sequence changes, and the incorporation of fluorescent and epitope tags. These approaches have increased the power of Drosophila genetics and been successfully used to conduct in vivo structure-function studies, study disease-associated variants, and follow protein dynamics.

Generating CRISPR Alleles in Drosophila.

CRISPR gene editing is a versatile and efficient approach for generating a wide variety of genetic reagents in flies. This unparalleled ability to manipulate the genome has revolutionized neuroscience, allowing Drosophila neurobiologists to readily generate new alleles to probe gene function, investigate the functional consequences of disease-associated variants, tag endogenous proteins to follow their dynamic localization in neurons and glia, and much more. Here, we provide a comprehensive protocol for generating heritable mutations in Drosophila We particularly focus on design considerations and tips for avoiding common errors to maximize the likelihood of successful gene editing.

Molecular and organizational diversity intersect to generate functional synaptic heterogeneity within and between excitatory neuronal subtypes.

Synaptic heterogeneity is a hallmark of complex nervous systems that enables reliable and responsive communication in neural circuits. In this study, we investigated the contributions of voltage-gated calcium channels (VGCCs) to synaptic heterogeneity at two closely related Drosophila glutamatergic motor neurons, one low- and one high-Pr. We find that VGCC levels are highly predictive of heterogeneous release probability among individual active zones (AZs) of low- or high-Pr inputs, but not between neuronal subtypes. Underlying organizational differences in the AZ cytomatrix, VGCC composition, and a more compact arrangement of VGCCs alter the relationship between VGCC levels and Pr at AZs of low- vs. high -Pr inputs, explaining this apparent paradox. We further find that the CAST/ELKS AZ scaffolding protein Bruchpilot differentially regulates VGCC levels at low- and high-Pr AZs following acute glutamate receptor inhibition, indicating that synapse-specific organization also impacts adaptive plasticity. These findings reveal intersecting levels of molecular and spatial diversity with context-specific effects on heterogeneity in synaptic strength and plasticity.

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.

tRNA modification enzyme-dependent redox homeostasis regulates synapse formation and memory.

Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA modifying enzyme that methylates wobble uridines in specific tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila, we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit a global reduction in protein synthesis, including a specific decrease in selenoprotein levels. Loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 animals also exhibit associative learning and memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA modification in redox homeostasis in the nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability.

Sanpodo: a context-dependent activator and inhibitor of Notch signaling during asymmetric divisions.

Asymmetric cell divisions generate sibling cells of distinct fates ('A', 'B') and constitute a fundamental mechanism that creates cell-type diversity in multicellular organisms. Antagonistic interactions between the Notch pathway and the intrinsic cell-fate determinant Numb appear to regulate asymmetric divisions in flies and vertebrates. During these divisions, productive Notch signaling requires sanpodo, which encodes a novel transmembrane protein. Here, we demonstrate that Drosophila sanpodo plays a dual role to regulate Notch signaling during asymmetric divisions - amplifying Notch signaling in the absence of Numb in the 'A' daughter cell and inhibiting Notch signaling in the presence of Numb in the 'B' daughter cell. In so doing, sanpodo ensures the asymmetry in Notch signaling levels necessary for the acquisition of distinct fates by the two daughter cells. These findings answer long-standing questions about the restricted ability of Numb and Sanpodo to inhibit and to promote, respectively, Notch signaling during asymmetric divisions.

The continuum of Drosophila embryonic development at single-cell resolution.

Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. We profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, we applied deep neural networks to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. We identify cell lineages; infer their developmental relationships; and link dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs' cognate motifs. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation.

Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila.

Cellular diversity is a fundamental characteristic of complex organisms, and the Drosophila CNS has proved an informative paradigm for understanding the mechanisms that create cellular diversity. One such mechanism is the asymmetric localization of Numb to ensure that sibling cells respond differently to the extrinsic Notch signal and, thus, adopt distinct fates (A and B). Here we focus on the only genes known to function specifically to regulate Notch-dependent asymmetric divisions: sanpodo and numb. We demonstrate that sanpodo, which specifies the Notch-dependent fate (A), encodes a four-pass transmembrane protein that localizes to the cell membrane in the A cell and physically interacts with the Notch receptor. We also show that Numb, which inhibits Notch signaling to specify the default fate (B), physically associates with Sanpodo and inhibits Sanpodo membrane localization in the B cell. Our findings suggest a model in which Numb inhibits Notch signaling through the regulation of Sanpodo membrane localization.

The calcium channel subunit α2δ-3 organizes synapses via an activity-dependent and autocrine BMP signaling pathway.

Synapses are highly specialized for neurotransmitter signaling, yet activity-dependent growth factor release also plays critical roles at synapses. While efficient neurotransmitter signaling relies on precise apposition of release sites and neurotransmitter receptors, molecular mechanisms enabling high-fidelity growth factor signaling within the synaptic microenvironment remain obscure. Here we show that the auxiliary calcium channel subunit α2δ-3 promotes the function of an activity-dependent autocrine Bone Morphogenetic Protein (BMP) signaling pathway at the Drosophila neuromuscular junction (NMJ). α2δ proteins have conserved synaptogenic activity, although how they execute this function has remained elusive. We find that α2δ-3 provides an extracellular scaffold for an autocrine BMP signal, suggesting a mechanistic framework for understanding α2δ's conserved role in synapse organization. We further establish a transcriptional requirement for activity-dependent, autocrine BMP signaling in determining synapse density, structure, and function. We propose that activity-dependent, autocrine signals provide neurons with continuous feedback on their activity state for modulating both synapse structure and function.

Advances in Engineering the Fly Genome with the CRISPR-Cas System.

Drosophila has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in Drosophila and highlight key areas for future advances.

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.

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.