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.

Synaptic Development in Diverse Olfactory Neuron Classes Uses Distinct Temporal and Activity-Related Programs.

Developing neurons must meet core molecular, cellular, and temporal requirements to ensure the correct formation of synapses, resulting in functional circuits. However, because of the vast diversity in neuronal class and function, it is unclear whether or not all neurons use the same organizational mechanisms to form synaptic connections and achieve functional and morphologic maturation. Moreover, it remains unknown whether neurons united in a common goal and comprising the same sensory circuit develop on similar timescales and use identical molecular approaches to ensure the formation of the correct number of synapses. To begin to answer these questions, we took advantage of the Drosophila antennal lobe (AL), a model olfactory circuit with remarkable genetic access and synapse-level resolution. Using tissue-specific genetic labeling of active zones, we performed a quantitative analysis of synapse formation in multiple classes of neurons of both sexes throughout development and adulthood. We found that olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs) each have unique time courses of synaptic development, addition, and refinement, demonstrating that each class follows a distinct developmental program. This raised the possibility that these classes may also have distinct molecular requirements for synapse formation. We genetically altered neuronal activity in each neuronal subtype and observed differing effects on synapse number based on the neuronal class examined. Silencing neuronal activity in ORNs, PNs, and LNs impaired synaptic development but only in ORNs did enhancing neuronal activity influence synapse formation. ORNs and LNs demonstrated similar impairment of synaptic development with enhanced activity of a master kinase, GSK-3ß, suggesting that neuronal activity and GSK-3ß kinase activity function in a common pathway. ORNs also, however, demonstrated impaired synaptic development with GSK-3ß loss-of-function, suggesting additional activity-independent roles in development. Ultimately, our results suggest that the requirements for synaptic development are not uniform across all neuronal classes with considerable diversity existing in both their developmental time frames and molecular requirements. These findings provide novel insights into the mechanisms of synaptic development and lay the foundation for future work determining their underlying etiologies.SIGNIFICANCE STATEMENT Distinct olfactory neuron classes in Drosophila develop a mature synaptic complement over unique timelines and using distinct activity-dependent and molecular programs, despite having the same generalized goal of olfactory sensation.

γ-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor.

Developing synapses mature through the recruitment of specific proteins that stabilize presynaptic and postsynaptic structure and function. Wnt ligands signaling via Frizzled (Fz) receptors play many crucial roles in neuronal and synaptic development, but whether and how Wnt and Fz influence synaptic maturation is incompletely understood. Here, we show that Fz2 receptor cleavage via the γ-secretase complex is required for postsynaptic development and maturation. In the absence of γ-secretase, Drosophila neuromuscular synapses fail to recruit postsynaptic scaffolding and cytoskeletal proteins, leading to behavioral deficits. Introducing presenilin mutations linked to familial early-onset Alzheimer's disease into flies leads to synaptic maturation phenotypes that are identical to those seen in null alleles. This conserved role for γ-secretase in synaptic maturation and postsynaptic development highlights the importance of Fz2 cleavage and suggests that receptor processing by proteins linked to neurodegeneration may be a shared mechanism with aspects of synaptic development.

Conservation and Innovation: Versatile Roles for LRP4 in Nervous System Development.

As the nervous system develops, connections between neurons must form to enable efficient communication. This complex process of synaptic development requires the coordination of a series of intricate mechanisms between partner neurons to ensure pre- and postsynaptic differentiation. Many of these mechanisms employ transsynaptic signaling via essential secreted factors and cell surface receptors to promote each step of synaptic development. One such cell surface receptor, LRP4, has emerged as a synaptic organizer, playing a critical role in conveying extracellular signals to initiate diverse intracellular events during development. To date, LRP4 is largely known for its role in development of the mammalian neuromuscular junction, where it functions as a receptor for the synaptogenic signal Agrin to regulate synapse development. Recently however, LRP4 has emerged as a synapse organizer in the brain, where new functions for the protein continue to arise, adding further complexity to its already versatile roles. Additional findings indicate that LRP4 plays a role in disorders of the nervous system, including myasthenia gravis, amyotrophic lateral sclerosis, and Alzheimer's disease, demonstrating the need for further study to understand disease etiology. This review will highlight our current knowledge of how LRP4 functions in the nervous system, focusing on the diverse developmental roles and different modes this essential cell surface protein uses to ensure the formation of robust synaptic connections.

The Tenets of Teneurin: Conserved Mechanisms Regulate Diverse Developmental Processes in the Drosophila Nervous System.

To successfully integrate a neuron into a circuit, a myriad of developmental events must occur correctly and in the correct order. Neurons must be born and grow out toward a destination, responding to guidance cues to direct their path. Once arrived, each neuron must segregate to the correct sub-region before sorting through a milieu of incorrect partners to identify the correct partner with which they can connect. Finally, the neuron must make a synaptic connection with their correct partner; a connection that needs to be broadly maintained throughout the life of the animal while remaining responsive to modes of plasticity and pruning. Though many intricate molecular mechanisms have been discovered to regulate each step, recent work showed that a single family of proteins, the Teneurins, regulates a host of these developmental steps in Drosophila - an example of biological adaptive reuse. Teneurins first influence axon guidance during early development. Once neurons arrive in their target regions, Teneurins enable partner matching and synapse formation in both the central and peripheral nervous systems. Despite these diverse processes and systems, the Teneurins use conserved mechanisms to achieve these goals, as defined by three tenets: (1) transsynaptic interactions with each other, (2) membrane stabilization via an interaction with and regulation of the cytoskeleton, and (3) a role for presynaptic Ten-a in regulating synaptic function. These processes are further distinguished by (1) the nature of the transsynaptic interaction - homophilic interactions (between the same Teneurins) to engage partner matching and heterophilic interactions (between different Teneurins) to enable synaptic connectivity and the proper apposition of pre- and postsynaptic sites and (2) the location of cytoskeletal regulation (presynaptic cytoskeletal regulation in the CNS and postsynaptic regulation of the cytoskeleton at the NMJ). Thus, both the roles and the mechanisms governing them are conserved across processes and synapses. Here, we will highlight the contributions of Drosophila synaptic biology to our understanding of the Teneurins, discuss the mechanistic conservation that allows the Teneurins to achieve common neurodevelopmental goals, and present new data in support of these points. Finally, we will posit the next steps for understanding how this remarkably versatile family of proteins functions to control multiple distinct events in the creation of a nervous system.