Axonal connections within the white matter of the central nervous system play the crucial role of transmitting electrical signals. Common diseases such as stroke, spinal cord injury and multiple sclerosis cause damage to white matter structures, yet white matter pathophysiology is not well understood. We hypothesize that in CNS white matter, release of Ca from intracellular stores, triggered by physiological (eg, activity) or pathological (eg, ischemia, trauma) depolarization, is triggered by gating of L-type Ca channels which in turn activate ryanodine receptors on endoplasmic reticulum.
The Specific Aims i nclude: 1) To study the mechanisms of Ca release from endoplasmic reticulum (ER) in myelinated axons and glia under physiological conditions, with a specific focus on the interaction between L-type Ca channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs). 2) To study the mechanisms of Ca release from ER in myelinated axons and glia under pathological conditions (eg, anoxia, ischemia), with a focus on the interaction between DHPRs and RyRs. 3) To study the insertion of DHPRs into axon membranes and the effect of increased Ca channel density on gating of RyRs and Ca release. 4) To study the distribution and interactions of various DHPR and RyR isoforms in axons and glia. Myelinated fibers will be studied using electrophysiology, imaging of ionized Ca, measurement of total subcellular Ca (which will better reflect stored, non-ionized Ca). Initial studies will examine how axonal Ca levels can be altered by modulation of the Cav-RyR interaction under physiological conditions. Then, we will examine in greater detail how excess release from internal stores contributes to axonal injury, with a focus on the role played by axonal L-type Ca channels that we have implicated in activating RyRs and release of Ca in dorsal column axons. The physiological purpose of such a mechanism will be explored, and the contribution to ischemic injury analyzed in greater depth, with a goal to inhibit this potent Ca sourcing machinery during ischemia (and possibly trauma and inflammatory attack as well). We will explore whether Ca channel densities are increased in anoxic/ischemic axons, thereby promoting pathological """"""""excitation-contraction coupling""""""""-Iike Ca release. Combinations of techniques including electrophysiology, 2-photon confocal ion imaging, energy-dispersive X-ray microanalysis and immunohistochemistry, together with manipulations of internal stores using pharmacology and molecular biology, will be applied to address the four Specific Aims.
