Ege T. Kavalali

Chemical synapses are fundamental computational units of the nervous system that enable rapid and precise information transfer through the nanoscale organization of presynaptic and postsynaptic components. Despite hundreds of millions of years of animal evolution and extraordinary diversity in nervous system architectures, the core physiological principles of chemical synaptic transmission remain remarkably conserved. Studies across Drosophila melanogaster , zebrafish, Caenorhabditis elegans , frogs and mammals reveal recurring principles governing active‐zone organization, synaptic vesicle exocytosis, calcium signalling and postsynaptic specialization. Here, we examine the evolution of chemical synapses across the animal kingdom, focusing on the relationship between molecular diversification, nanoscale organization and functional conservation. We propose that the evolutionary stability of synapses reflects, in part, a stringent biophysical constraint imposed by the requirements for rapid chemical communication. We further consider how these conserved synaptic operations are assembled into neural circuits of dramatically different complexities, as well as the ctenophore nervous system as a critical test case for distinguishing molecular conservation from architectural and functional convergence. Across species, the emerging picture is that evolution has adhered to conserving the geometry and organizational principles of chemical synapses more stringently than a precise molecular composition. Synapses thus represent stable yet adaptable information‐processing architectures in which conserved nanoscale constraints coexist with molecular flexibility and extensive diversification of neural circuits. image

Synaptic vesicle fusion is orchestrated by the soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) complex. The SNARE complex includes SNAP25, synaptobrevin-2, and syntaxin-1. This machinery drives action potential-evoked release, as well as the majority of spontaneous release. Mutations in components of the SNARE machinery can differentially affect spontaneous or evoked release and result in severe neurodevelopmental phenotypes, as exemplified by SNAP25 encephalopathies that arise from mutations in the SNAP25 gene. Among various pathogenic SNAP25 variants, L50S, V48F, and D166Y increase spontaneous glutamate release that destabilizes network activity. Patients who carry these variants develop recurrent seizures that are refractory to conventional antiepileptic drugs, which primarily act on components of evoked neurotransmission and leave spontaneous release abnormalities unaddressed. To surmount this limitation, we explored a strategy targeting homeostatic plasticity mechanisms with lithium, which has been shown to downscale postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). In vitro, lithium corrected cellular phenotypes in neurons expressing SNAP25 variants. In vivo, we generated a knock-in mouse model recapitulating the SNAP25 L50S mutation to test these results. These mice displayed increased neonatal mortality, infertility, hypomobility, frequent seizure activity, anxiety-like behavior, social deficits, and impaired learning and memory. Chronic lithium treatment reduced seizure frequency and ameliorated long-term memory and locomotor activity. Taken together, lithium alleviated synaptic and behavioral disturbances driven by pathological spontaneous glutamate release by inducing homeostatic AMPAR downscaling. Thus, this work provides a mechanistically grounded therapeutic strategy for SNAP25 encephalopathies and other disorders characterized by elevated spontaneous glutamate release.

Proteostasis, or protein homeostasis, is a tightly regulated network of cellular pathways essential for maintaining proper protein folding, trafficking, and degradation. Neurons are particularly vulnerable to proteostasis collapse due to their postmitotic and long-lived nature and thus represent a unique cell type to understand the dynamics of proteostasis throughout development and maturation. Here, we utilized a dual-species co-culture model of human excitatory neurons and mouse glia to recapitulate and investigate cell type–specific, maturation-related changes in the proteostasis network using data-independent acquisition LC–MS/MS proteomics. We quantified branch-specific unfolded protein response (UPR) activation by monitoring curated effector proteins downstream of the ATF6, IRE1/XBP1s, and PERK pathways, enabling a comprehensive, unbiased evaluation of UPR dynamics during in vitro neuronal maturation between 30 d and 60 d. Species-specific analysis revealed that mature neurons largely preserved proteostasis, although they showed some signs of collapse, primarily in endoplasmic reticulum (ER)-to-Golgi transport mechanisms. However, these changes were accompanied by upregulation of proteostasis-related machinery and activation of the ATF6 branch, as well as maintenance of the XBP1s and PERK branches of the UPR over time. In contrast, glia exhibited broad downregulation of proteostasis factors and UPR components, independent of neuronal presence. Furthermore, we quantified stimulus-specific modulation of select UPR branches in matured neurons exposed to pharmacologic ER stressors. These findings highlight distinct, cell-type-specific stress adaptations during in vitro maturation and provide a valuable proteomic resource for dissecting proteostasis and UPR regulation in human neurons.

Several studies have revealed deleterious synapse formation onto cancer cells within the brain tumor microenvironment, yet these synapses are ∼100-fold weaker in presynaptic release rates and postsynaptic strength relative to bona fide synapses formed between neurons. Here, we find that most of the functional synapses on tumor cells are kept dormant and can be unlocked by overcoming GABA _B receptor-mediated metabotropic signaling in neurons. Scavenging G _βγ signaling in neurons increased presynaptic release probability on tumor cells and augmented cancer cell proliferation. Optical analysis of the tumor microenvironment revealed regulated secretion of neurotransmitters from tumor cells in response to GABA _B receptor inhibition or electrical stimulation. These results reveal how cancer cells with a high propensity for brain metastasis leverage precise moments of aberrant excitation between neurons to engage reciprocal interactions that ultimately fuel cancer proliferation.

De novo mutations in synaptotagmin-1 (syt1) cause a rare neurodevelopmental disorder, manifesting in global developmental delay, ophthalmic abnormalities, infantile hypotonia, facial dysmorphisms, absent speech, EEG abnormalities, and hyperkinetic movements, ranging from moderate to severe. Here, we evaluate eleven patient-relevant mutations spanning the Ca ²⁺ binding domains of syt1—C2A and -C2B impact neurotransmission. We found that the mutation causing the most severe impact on neurotransmission, p.N341S, triggers aberrant spontaneous neurotransmission and occludes homeostatic synaptic plasticity signaling pathways. Our results suggest that potential phosphorylation of this newly introduced Ser residue underlies the functional change. A serine missense mutation creates a novel phosphorylation site as a broad spectrum protein kinase inhibitor rescues spontaneous neurotransmission. We identify key residues, localized to the primary interface between syt1 and SNAP-25, responsible for this shift in syt1 function in synaptic vesicle release. Substituting neutral amino acids at residue 341 alters the interaction of the Ser mutation, with double mutations in the surrounding amino acids in the primary interface rescuing synaptic function. These results provide a framework for how a syt1 point mutation introduces a substrate for phosphorylation and disrupts intermolecular interactions at the primary interface with SNAP-25 altering spontaneous neurotransmission and homeostatic plasticity. AUTHOR SUMMARY: Mutations in synaptotagmin-1 ( SYT1 ), a protein essential for communication between neurons, cause a rare neurodevelopmental disorder marked by developmental delay, low muscle tone, abnormal movements, vision problems, and disrupted brain electrical activity. Disease severity varies, and how specific syt1 mutations alter brain signaling remains unclear. In this study, we examined 11 disease-associated syt1 mutations that affect regions of the protein responsible for sensing calcium, a key trigger for neurotransmitter release. Using a range of electrophysiological approaches, we measured how these mutations influence different modes of synaptic communication within neuronal networks. We found that one mutation, N341S, produced the most severe disruption. Neurons carrying this mutation released neurotransmitters abnormally at rest and were unable to engage normal homeostatic plasticity mechanisms that stabilize brain activity. These effects suggest a fundamental breakdown in how synapses regulate signaling strength. We investigated the molecular basis of this dysfunction and identified a likely explanation: the N341S mutation introduces a new serine residue that can be phosphorylated, a common regulatory modification in cells. Our data indicate that this newly created phosphorylation site alters syt1 function, as blocking phosphorylation pathways could modify the mutant’s effects. Importantly, we also show that N341 residue lies within a critical interaction interface between syt1 and another synaptic protein, SNAP-25. Adjusting nearby amino acids to neutralize this interaction restores wild-type levels of synaptic signaling. Together, these findings reveal how a single disease-associated mutation can rewire synaptic regulation by introducing a phosphorylation site, offering new insight into the underpinning of syt1-related neurodevelopmental disorders and potential therapeutic targets.

Within the single micron of the synapse, three distinct modes of neurotransmission, driven by synchronous, asynchronous, and spontaneous neurotransmitter release, occur concurrently. In this chapter, we discuss the synaptic nano-organization comprised of neurotransmitter release machinery, molecular platforms, scaffolding proteins, and liquid complexes that support the discrete signaling of these three modes of neurotransmission. This robust nano-organization supports unique functional roles for each discrete mode at both excitatory glutamatergic and inhibitory GABAergic synapses. Modular nanocolumn organization of excitatory synapses and largely single-domain organization of inhibitory synapses maintain homeostatic plasticity within neural circuits. These recent findings support a basic design principle where the single synapse is a highly ordered and compartmentalized unit that by the functional nano-segregation of distinct forms of neurotransmission shapes synaptic efficacy, determines neurotransmission reliability, and tunes plasticity. The development of novel tools will be instrumental in further elucidating the nano-environment of the synapse, essential to both uncovering mechanisms underlying neurological disorders as well as their treatment.

Synapses are the fundamental units of communication and information processing in the nervous system. They show remarkable functional autonomy, as well as speed of signaling, bidirectional plasticity, and diversity. Synapses shape complex network functions, such as learning, memory, pattern separation, pattern completion, and many others. Synaptic dysfunction is responsible for a wide range of neurological and psychiatric diseases. Many of these diseases are emerging as synaptopathies, but the precise disease mechanisms are unknown. Given their micron-scale small size and seemingly compact structure, synapses were generally thought to be functionally indivisible structures. However, with the advent of high-resolution electrophysiology and imaging techniques, it is increasingly clear that synapses harbor functionally specialized nanomodules. This expanding nanobiology of the synapse provides a new perspective on synaptic signaling. The novel insight gained from this work is critical to understand disease mechanisms and to guide the development of appropriate therapeutic strategies for major brain diseases ranging from neurodegenerative and neurodevelopmental disorders to neuropsychiatric disorders such as depression and schizophrenia.

Dopamine is a monoamine neurotransmitter that regulates neuronal activity and synaptic transmission. While dopaminergic activity is known to suppress action potential-dependent glutamate release in certain brain regions, the modulatory effect of dopaminergic tone on spontaneous glutamate release is unclear. Here, we used primary rat ventral tegmental area-cortex co-cultures to assess how decreased dopaminergic tone affects spontaneous synaptic glutamate release using whole-cell patch-clamp electrophysiology. We found that an acute decrease in dopaminergic tone increases the frequency of spontaneous glutamate release, driven by a surge in basal presynaptic calcium. This presynaptic calcium surge results from disinhibition of voltage-gated calcium channels (VGCCs) due to reduced Gβγ subunit activity downstream of D2 receptor signaling. While acute reduction in dopaminergic tone has robust presynaptic effects, chronic reduction results in homeostatic synaptic plasticity, characterized by postsynaptic insertion of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, a process known as synaptic upscaling. Notably, chronic antagonism of both D1 and D2 receptors using selective antagonists, as well as long-term treatment with first- and second-generation antipsychotics haloperidol, chlorpromazine, olanzapine, clozapine, and aripiprazole, promoted robust synaptic upscaling. These findings reveal a novel mechanism of action for antipsychotic medications and suggest that antipsychotics do not solely act on counteracting hyperdopaminergia, but also tune glutamatergic neurotransmission by activating homeostatic plasticity mechanisms.

Neuronal communication is governed by a diverse repertoire of neurotransmitter release modes, each with distinct molecular machinery and functional roles. Beyond rapid, high-fidelity synchronous release, asynchronous release supports sustained neurotransmitter output, while spontaneous, action-potential-independent release plays a critical role in synaptic development, homeostasis, and plasticity. Complementing these forms of release, slow neurotransmission mediated by monoamines and neuropeptides acts over longer timescales to shape network-wide activity. This review explores the unique mechanisms of each mode, highlighting compelling evidence that spontaneous and evoked release are functionally segregated through separate vesicle pools and distinct postsynaptic receptors. We also examine the molecular complexity and debated physiological roles of asynchronous release, particularly at excitatory synapses, and the specialized machinery of neuromodulatory systems. A comprehensive understanding of these varied release mechanisms is fundamental to neuroscience and opens novel therapeutic avenues. Targeting the unique molecular components of each release mode offers a promising strategy for developing more precise treatments for neurological and psychiatric disorders.

Tumor cell plasticity in novel microenvironments is central to the integration and subsequent growth of metastatic cells. However, the functional consequences of tumor cell integration with central neurons remains understudied. Here, we address this question using small cell lung cancer (SCLC), which has an extraordinary propensity to metastasize to the brain in humans. Transcriptomic and electrophysiological analysis of SCLC cells in neuronal microenvironments reveal a heterogeneous population of synapse-forming SCLC cells with neurons. While a proportion of neuron-SCLC synapses are blocked by AMPA receptor antagonists, we also find a sensitivity of these synapses to GABA_A receptor inhibition. The functional integration of SCLC with central neurons induced multiplicative synaptic upscaling between neurons and dysregulated neuronal excitability. Aberrant excitation in human neurons with SCLC was sustained by synaptic NMDA receptor activation and can be reduced by the FDA approved NMDA receptor blocker memantine. These findings reveal strategies to normalize tumor-induced exacerbation of aberrant neuronal activity.