Kendal Broadie

Experience-dependent brain circuit optimization choreographed by environmental sensory input activity involves synapse formation, pruning, and remodeling to sculpt appropriate connectivity. The serotonin (5-HT) neuromodulator acts as a core regulator of this circuit plasticity. Classically, serotonergic control has been understood solely through neuronal mechanisms, however new evidence reveals glial 5-HT signaling roles. This review focuses on recent studies in Drosophila with reference to foundational mammalian work to discuss 5-HT functions in both neurons and glia, particularly experience-dependent extracellular matrix remodeling, glial infiltration, and synapse elimination in early-life critical periods. Disruption of serotonergic regulation is proposed to contribute to a spectrum of neurodevelopmental disorders, including Fragile X syndrome, in which failure to prune and persistence of immature connectivity cause severe life-long behavioral impairments. Recent discoveries further reveal targeted induction of glial serotonergic signaling can re-open “critical period-like” synapse pruning at maturity. Enabling large-scale connectivity changes has broad potential therapeutic applications for disease, injury, trauma, and cognitive dysfunction. A key advance is the emerging evidence that glia—not just neurons—are serotonergic mediators of synaptic remodeling: glial 5-HT biosynthesis, 5-HT _2A receptor activation, and matrix metalloprotease-mediated function together allow access for experience-driven synapse elimination. We propose glia-to-glia class serotonergic signaling—linking sensory experience to synapse pruning—may represent a conserved plasticity gating mechanism that determines whether circuitry is permissive or resistant to synaptic connectivity modification. Harnessing glial class-specific serotonergic control of experience-dependent brain circuit remodeling may enable new targeted therapies to correct brain function while avoiding the negative side effects of global serotonin elevation.

Serotonin (5-hydroxytryptamine, 5-HT) has central roles enabling learning and memory, particularly via serotonin receptor 2A (5-HT _2A R) signaling. Drosophila Fragile X syndrome model ( dfmr1 null mutant) studies reveal impaired learning and memory, which may reflect serotonergic signaling deficits. Here, we use classical olfactory T-maze conditioning to assess behavior, combined with imaging to assess 5-HT and 5-HT _2A R levels within the underlying Mushroom Body (MB) brain circuitry. Null dfmr1 mutants exhibit learning and memory deficits that are corrected by elevating 5-HT signaling via 1) overexpression of the serotonin biosynthetic enzyme tryptophan hydroxylase (Trhn) or 2) knockdown of the serotonin reuptake transporter (SERT). Direct comparisons reveal both Trhn and SERT manipulations equally restore learning and memory in dfmr1 null mutants. 5-HT _2A R levels in the MB circuit are reduced relative to controls in dfmr1 mutants, and 5-HT _2A R RNAi phenocopies dfmr1 null behavioral deficits, suggesting these phenotypes are primarily caused by the loss of 5-HT _2A R signaling. Consistently, 5-HT _2A R overexpression in dfmr1 nulls restores normal learning and memory compared to controls. These findings suggest loss of 5-HT _2A R signaling causes learning and memory deficits in this Fragile X syndrome model, and that rectifying this signaling impairment can restore learning and memory, providing a framework for serotonergic intervention strategies.

Synapse connectivity is optimized in response to environmental input in critical periods, characterized by experience-dependent, temporally-restricted, and transiently-reversible synapse pruning by glial phagocytes. This precise, targeted synaptic elimination process requires glial serotonergic intercellular signaling. We discover glia-to-glia communication between different glial classes is essential for experience-dependent synaptic pruning in a well-defined Drosophila juvenile brain olfactory critical period. We find ensheathing glia infiltrate specific target synaptic glomeruli in response to guiding odorant experience via 5-HT _2A receptor (5-HT _2A R) signaling. Using cell-targeted tryptophan hydroxylase (Trhn) RNAi to block serotonin production, we discover serotonin signaling from ensheathing glia is required for experience-dependent synapse pruning. Using cell-targeted 5-HT _2A R RNAi, we find the serotonin receptor is required exclusively in astrocyte-like glia (ALG). Using cell-targeted 5-HT _2A R rescue in 5-HT _2A R null mutants, we discover the serotonin receptor mediates experience-dependent synapse pruning. Thus, glia-to-glia serotonin signaling between different glial classes mediated by 5-HT _2A receptors is necessary and sufficient for synapse elimination. We discover that ALG-targeted conditional 5-HT _2A R in mature adults induces experience-dependent synapse pruning indistinguishable from the critical period mechanism. Thus, astrocyte 5-HT _2A R signaling is sufficient to ‘re-open’ this characteristic critical period remodeling capability at maturity. We find astrocytic matrix metalloproteinase-1 (MMP-1) induced by critical period odorant experience is required for experience-dependent synapse pruning downstream of 5HT _2A R activation. We discover that ALG-targeted MMP-1 induction restores synapse pruning in the absence of 5HT _2A R signaling. Taken together, we conclude that glia-to-glia serotonergic 5HT _2A R signaling drives MMP-1 for experience-dependent infiltration phagocytosis synapse pruning, and can rekindle this remodeling capacity at adult maturity.

A classic human patient comorbidity of fragile X syndrome and glycogen storage disease type IX has symptoms far more severe than those for either disease alone. Causal variants result in loss of the translational regulator fragile X ribonucleoprotein 1 (FMRP) and metabolic regulator phosphorylase kinase regulatory subunit alpha 2 (PHKA2), respectively. We hypothesized FMRP-PHKA2 interaction based on unsustainably elevated metabolic demand. In Drosophila disease models, single null mutants were viable, but double knockout (DKO) animals exhibited 100% synthetic lethality, showing an essential interaction. Metabolically, dFMRP and dPHKA2 loss alone caused opposing changes in glycogen and fat stores, but DKO animals had both energy stores returned to normal. Regulatory Fat body protein 1 was elevated in single disease models but likewise returned to normal in the DKO animals. In tests of neurological phenotypes, neuromuscular junction mitochondrial function, synapse architecture and neurotransmission strength were all elevated by dFMRP loss, but these synaptic properties were restored to normal levels by co-removal of dPHKA2 in DKO animals. Thus, dFMRP and dPHKA2 strongly interact in metabolic and neuromuscular mechanisms, without explaining the DKO lethal interaction.

Critical periods enable early-life synaptic connectivity optimization whereby initial sensory experience remodels circuits to a variable environment. In the Drosophila juvenile brain, synapse remodeling occurs within the precisely-mapped olfactory circuit, which has an extensively characterized, manageably short (< 1 week) critical period. In this brain circuit, single receptor olfactory sensory neuron (OSN) classes synapse onto single projection neurons extending to the central mushroom body learning/memory center. Critical period odorant experience drives OSN synapse remodeling, which can only be reversed during this brief interval. Our objective is to dissect intercellular signaling pathways from neurons to glial phagocytes sculpting synapse elimination in response to critical period experience. We find critical period experience causes externalized phosphatidylserine (PS) exposure in activated OSN synaptic glomeruli in an experiential dose-dependent mechanism. We discover that genetic knockdown of phosphatidylserine synthase inhibits critical period experience-dependent pruning of these synaptic glomeruli. We show a genetic interaction in trans-heterozygous mutants of phosphatidylserine synthase and Draper (mammalian MEGF10), the well-conserved glial engulfment receptor that binds phosphatidylserine, with double trans-heterozygotes blocking critical period experience-dependent pruning. This interaction mechanistically links phosphatidylserine signaling to glial phagocytosis synapse elimination. We identify the OSN scramblase that transports phosphatidylserine from the synaptic membrane inner to outer leaflet, and demonstrate phosphatidylserine externalization is rate-limiting for experience-dependent synaptic glomeruli pruning. We discover glial insulin receptors direct experience-dependent glial infiltration phagocytosis. We find activated glial insulin receptor signaling elevates critical period synapse pruning. Together this work identifies coupled intercellular signaling pathways from target neurons to glial phagocytes orchestrating experience-dependent synapse elimination.

The optimization of brain circuit connectivity based on initial environmental input occurs during critical periods characterized by sensory experience-dependent, temporally restricted, and transiently reversible synapse elimination. This precise, targeted synaptic pruning mechanism is mediated by glial phagocytosis. Serotonin signaling has prominent, foundational roles in the brain, but functions in glia, or in experience-dependent brain circuit synaptic connectivity remodeling, have been relatively unknown. Here, we discover that serotonergic signaling between glia is essential for olfactory experience-dependent synaptic glomerulus pruning restricted to a well-defined Drosophila critical period. We find that experience-dependent serotonin signaling is restricted to the critical period, with both (1) serotonin production and (2) 5-HT_2A receptors specifically in glia, but not neurons, absolutely required for targeted synaptic glomerulus pruning. We discover that glial 5-HT_2A receptor signaling limits the experience-dependent synaptic connectivity pruning in the critical period and that conditional reexpression of 5-HT_2A receptors within adult glia reestablishes “critical period-like” experience-dependent synaptic glomerulus pruning at maturity. These results reveal an essential requirement for glial serotonergic signaling mediated by 5-HT_2A receptors for experience-dependent synapse elimination.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

[This corrects the article DOI: 10.3389/fcell.2022.934662.].

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.