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Research

Calcium Control of Vesicular Release Machinery

We aim to develop mechanistic insights into how nerve terminals precisely control Ca²⁺-evoked neurotransmitter release across varying time scales and modulate the strength/efficacy of this process in an activity-dependent manner. Recent structural and functional studies have provided critical insights into the molecular organization of presynaptic vesicular release machinery, leading to several molecular models (Rothman, Krishnakumar et al., FEBS Letters 2017; Volynski & Krishnakumar, Curr Opin Neurobiol 2018) that could potentially explain the mechanics of synaptic vesicle priming, calcium-evoked release, and short-term plasticity. Building on these models, our current focus is to delineate the molecular mechanisms of fast synchronous and delayed asynchronous release and understand how distinct calcium sensors synergistically regulate these different modes of evoked exocytosis; and determine how presynaptic Ca²⁺ dynamics modulate vesicle docking, priming, and fusion processes to drive plasticity in neurotransmitter release.

To achieve these goals, we employ a multidisciplinary approach, integrating biochemical, biophysical, and high-resolution structural methods, with a strong emphasis on systematic reconstitution strategies. By leveraging a recently-developed in vitro single-vesicle fusion setup (Ramakrishnan et al., Elife 2020; Kalyana Sundaram et al. PNAS, 2023) in combination with calcium uncaging paradigms, we are reconstituting different modes of calcium-evoked release and short-term plasticity with millisecond precision under cell-free conditions. Our in vitro functional analyses with isolated proteins guide physiological studies in cultured neurons, in collaboration with Kirill Volynski, PhD, University College London (UCL) Queen Square Institute of Neurology.

Molecular Characterization of Presynaptic Synaptopathies

This collaborative research program aims to uncover the molecular and mechanistic basis of pre-synaptic synaptopathies. We employ reconstituted fusion systems combined with biochemical tools to examine the functional impact of de novo mutations and neuropathy-associated genetic variants in core pre-synaptic proteins (Coleman et al. Cell Reports, 2018; Salpietro et al. American Journal of Human Genetics, 2019). This approach allows us to rigorously and rapidly assess the consequences of mutations or alterations in individual synaptic proteins (as well as any epistatic effects) on neurotransmitter release. These “experiments-of-nature” mutations provide crucial insights into the mechanisms of synaptic transmission. We aim to translate our in vitro findings to neuronal cultures and animal models to establish physiological relevance, with the ultimate goal of developing new treatment strategies, including gene therapies.

We are currently focusing on mutations in SNAREs and associated regulatory proteins that have been implicated in a spectrum of neurological disorders collectively referred to as "SNAREopathies." These disorders, which often present at a young age, include epilepsy, movement disorders, intellectual disabilities, and neurodevelopmental conditions such as autism spectrum disorder. This project is conducted in collaboration with researchers at the UCL Queen Square Institute of Neurology, who bring complementary expertise in clinical genetics and deep phenotyping (Henry Houlden, PhD); biophysics of presynaptic vesicle trafficking and exocytosis in vivo (Dr. Volynski); and gene therapy approaches for neurological diseases (Dimitri Kullmann, DPhil, MAE). We are also exploring new collaborative opportunities both within and outside of Yale to extend the analysis to other associated neurological, neurodevelopmental, and psychiatric disorders.