A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. Intracellular calcium signaling plays a fundamental role in the neuronal response to traumatic damage, yet the mechanisms that orchestrate this signaling pathway remain largely unknown. Following neuronal insult, elevation of cytoplasmic calcium levels can trigger degeneration but also is critical for efficient repair and regeneration. We will elucidate within an intact physiological context, the calcium signaling response in a damaged neuron and the molecular components that modulate it. Our work combines advanced biophotonic and genetic techniques within the nematode worm C. elegans to create a uniquely powerful experimental system. Combining advanced laser surgery techniques and time-lapse fluorescence imaging, we can selectively damage individual neurons within intact adult C. elegans and optically measure intracellular calcium signaling and outgrowth dynamics throughout the neuron. Employing the emerging technology of optogenetic photo- activation, we can stimulate the damaged neuron elevating cellular calcium signals to enhance regeneration. The genetic tractability of C. elegans allows us to pinpoint the roles of specific molecular components underlying these cellular events through analysis of genetic mutations.
The aims of this work are the following: first, to determine the molecular and cellular mechanisms that mediate spatially localized intracellular calcium dynamics and regeneration response in a damaged neuron, second to define the molecular details of a novel pathway by which an apoptotic caspace is activated by calcium to perform a beneficial role in early regeneration, and third to stimulate additional regeneration by artificially manipulating calcium physiology in a damaged neuron. As such, our study will generate a detailed understanding of the calcium signaling cascade within a damaged neuron that is critical for the advancement of neurotherapeutics. Numerous studies have suggested the inhibition of calcium through various means as a neuroprotective measure. This work will define new molecular targets and suggests novel therapeutic strategies for the modulation of calcium signaling to stimulate neuronal regeneration, establishing a detailed framework in which to interpret their effects. Our findings will put these possibilities into a comprehensive understanding of the cellular events that control neuronal regeneration.
Combining in vivo laser ablation, fluorescent imaging and optogenetic techniques with the powerful genetic tools of C. elegans, this work will fully characterize the intracellular calcium signaling in a damaged and regenerating neuron within an intact physiological context. By defining the molecular components that modulate this critical cellular signal in neuronal regeneration, we will identify novel targets and strategies for the advancement of neurotherapeutics.
|He, Siwei; Philbrook, Alison; McWhirter, Rebecca et al. (2015) Transcriptional Control of Synaptic Remodeling through Regulated Expression of an Immunoglobulin Superfamily Protein. Curr Biol 25:2541-8|
|Sun, Lin; Shay, James; McLoed, Melissa et al. (2014) Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation. J Neurosci 34:15947-56|