Ryan Insolera - US grants

Project Summary/Abstract Our brains carry out complex cognitive tasks via the intricate electrical communication between neurons. The specialized site where this communication occurs is the synapse, and it is capable of various forms of plasticity that enable the constant and adaptive refinement of the communication between neurons. The amount of neuronal activity that is transmitted at the synapse typically dictates the type of modification, and raising the level of communication between neurons strengthens their synaptic connection through an adaptive increase in the number of connections. This process is collectively known as activity-dependent synaptic plasticity, and is thought to be the cellular foundation for learning and memory. The long term goal of this project is to better understand the basic cell biological roots of activity-dependent synaptic plasticity. In particular, I am interested in understanding the functional role of intracellular mitochondrial trafficking in supporting the growth of synapses adapting to increased neuronal activation. I will carry out this project using Drosophila Melanogaster larvae, or fruit fly maggots, as an experimental model system that was chosen due to the powerful genetic tools available. Like motor neurons in our spinal cord, the muscles in the body wall of the larvae are innervated by motoneurons that are responsible for sending the signal when to move. These motoneurons form a synapse onto the muscle, which is known as the neuromuscular junction (NMJ) synapse. The presynaptic terminal of the NMJ synapse will undergo activity- dependent plasticity upon increased activity of the motoneurons, from increased movement of the larvae. I can microscopically image this synapse directly through the cuticle (skin) of the larvae using technology developed in my lab; including its changing shape and intracellular dynamics (such as changes in mitochondria) that occur simultaneously with the growth when neuronal activity is increased. My central hypothesis is that neuronal activity induces the formation of acute synaptic growth that is eventually stabilized by the trafficking of mitochondria into this nascent growth, which facilitates its long-term maturation to becoming a mature synaptic connection. Using the tools I described, I will test this hypothesis with two specific aims: (1) I will use a genetic mutant larvae in which the trafficking of mitochondria to the NMJ is dysfunctional to see if any aspects of activity-dependent growth are successful, hence pinpointing a precise role for mitochondrial trafficking in the process. (2) I will seek to functionally characterize a molecular mediator responsible for promoting activity-dependent synaptic growth, and determine whether the driving force for its ability to promote the growth of synapses is in its regulation of mitochondrial trafficking. The insight gained from this work will uncover new knowledge on the cell biology of synaptic plasticity and mitochondrial trafficking in neurons, two processes that commonly result in neurodegenerative diseases when dysfunctional.

Neurons are among the most energetically-demanding cell types in the body, and as such, heavily rely on the output of mitochondria to fuel their basic functionality. Because of this, a disproportionate number of genes whose dysfunction is associated with neurodegenerative disease are related to mitochondrial function. Of particular importance to neuronal health and survival is the process of degrading mitochondria. Despite this link, little is known about the cell biological pathways that mediate the breakdown of mitochondria in neurons within their native, physiological environment in nervous tissue. The overall goal of this proposal is to gain a further understanding of the various pathways neurons utilize to break down their mitochondria. Within nervous tissue, neurons get support from a specialized cell type known as glial cells. It has recently been discovered that knock down of the ataxia-associated gene Vps13D leads to the strong inhibition of a neuron?s ability to break down their mitochondria via a pathway known as mitophagy. In response to this robust inhibition, neurons adaptively induce an alternative means of mitochondrial breakdown that involves the transfer of damaged mitochondria to supportive glial cells for their ultimate degradation. With the discovery of this new pathway of mitochondrial degradation, the mentored phase of this proposal will use the robust phenotype associated with loss of Vps13D to guide the development of tools for studying non-cell autonomous mitochondrial degradation in neurons in vivo. This work will also uncover the conditions that lead to the induction of this transcellular neuron to glia mitochondrial breakdown. In the independent phase of this proposal, the goal will be to use these newly developed tools to gain a mechanistic understanding of how mitochondria are released from neurons. Using the powerful genetic tools available in fruit fly research, this Aim will test out candidate molecules with a history of mediating intercellular mitochondrial trafficking in other cell types to determine whether they participate in neuron to glial mitochondrial transfer. Finally, the goal of this proposal in the independent phase will be to determine if this newly discovered, alternative form of mitochondrial degradation become adaptively more prevalent during the course of aging as levels of autophagy decrease. Then, this Aim will test the cellular and functional consequences of disrupting neuron-to-glial transfer of mitochondrial in this susceptible population of aged neurons. With the completion of the research proposed in this application, the applicant will have carved out a distinctive niche for an independent research career focusing on the cell biology of transcellular mitochondrial degradation in neurons.