C&SP17: NMJ Calcium Signaling

A. Collaborating Investigators: Stephen D. Meriney,¹ Tom M. Bartol,² Terry J. Sejnowski,² Ivet Bahar,¹ Mary H. Cheng, ¹ Rozita Lahgaei³ and Greg Hood ³

B. Institutions: ¹University of Pittsburgh, ²Salk Institute, ³Pittsburgh Supercomputing Center

C. Funding Status: NINDS5R01-NS090644-02, CRCNS: Transmitter Release Site Organization in Plasticity and Disease at the NMJ (Meriney) 8/1/14-5/31/19

D. Biomedical Research Problem: A significant number of neurological diseases are known to affect synaptic transmission by targeting synaptic organization.^19,20 While most studies on this topic focus on postsynaptic adaptations,²¹ it has become increasingly clear that presynaptic homeostatic changes also play a major role. In particular, presynaptic homeostatic plasticity under normal and disease conditions can be mediated by mechanisms²² that influence the structure and function of the active zone (AZ), the nerve terminal specialization that controls transmitter release.²³ Ca²⁺ influx into AZs is critical to triggering neurotransmitter (NT) release, and alterations in Ca²⁺ influx are a target for presynaptic homeostatic plasticity.^24,25 Thus, a better understanding of the role of presynaptic structure in synaptic function is needed. LEMS (Lambert-Eaton Myasthenic Syndrome) is one of a number of devastating Ca²⁺ channelopathies^26,27 known to alter presynaptic Ca²⁺ channel number and AZ organization at the NMJ.^28,29 The presynaptic plasticity in normal and diseased NMJs provides an excellent opportunity to study the impact of Ca²⁺ channel-NT release site organization at a model synapse. We hypothesize that major aspects of synaptic function and presynaptic homeostatic plasticity (in control and disease states) can be explained by changes in the number and organization of Ca²⁺ channels within transmitter release sites.

E. Methods and Procedures Aim 1. Presynaptic activity-dependent homeostatic regulation of structure and function within the NMJ. We hypothesize that homeostatic changes serve to fine-tune the strength of communication with muscle cells as activity patterns and muscle sizes change over time. Using MCell³⁰ modeling and experimental techniques, we will evaluate the impact of homeostatically driven changes in the organization of individual single-vesicle release sites on the probability of release, synaptic latency, and short-term synaptic plasticity. This approach will be an extension of our recently published development of MCell (TR&D2) models to evaluate synaptic function and plasticity.^4,6

Aim 2. Presynaptic changes in AZ structure and function in a mouse model of LEMS. LEMS is known to disrupt the normally well-organized AZ at the NMJ (Fig VIII.1). These changes reduce total Ca²⁺ flux into motor nerve terminals, which results in reduced NT release, and altered short-term synaptic plasticity. Using a combination of physiological, anatomical, and MCell computational approaches, we will elucidate the number and distribution of Ca²⁺ channels in diseased mouse model LEMS AZs, their probability of opening during an action potential, and the functional coupling between these Ca²⁺ channels and synaptic vesicle release sites. We will test the hypothesis that LEMS-induced changes in AZ organization underlie functional consequences. This approach expands upon our studies of LEMS³¹ and will aid in LEMS treatment, and advance our understanding of the functional impact of AZ organization

Fig VIII.1. Mouse NMJ anatomy, AZ organization, synaptic function, and MCell modeling. A. Mouse NMJ stained with bassoon antibodies (green) to identify the location of AZs. Postsynaptic ACh receptors are stained red. B. Mouse AZ in normal and LEMS disease states. C. Tans-mitter release is greatly reduced in LEMS. D. MCell model of mouse AZ.

Aim 3. Integration of presynaptic Ca²⁺ channel function with the spatio-temporal dynamics of Ca²⁺ ion action within a multi-active zone MCell model. In collaboration with Dr. Bahar’s group (TR&D1) we will use molecular computational tools for studying normal and drug-modulated Ca²⁺ channel gating. The structure-based data on functional dynamics will be combined with MCell modeling of spatiotemporal dynamics of intra-terminal Ca²⁺ ions, to take a multi-scale approach to increasing our understanding of how Ca²⁺ channel number and gating function can influence synaptic transmitter release within complex nerve terminals. The approach at both molecular and cell levels will allow a more detailed understanding of how ion channel biophysics, our newly developed gating modifier drugs,^32,33 and stochastic diffusion and reaction biochemistry combine to regulate the function of motor nerve terminals.