Date of Degree
PhD (Doctor of Philosophy)
Anatomy and Cell Biology
C. Andrew Frank
Neurons are specialized cells that communicate via electrical and chemical signaling. It is well-known that homeostatic mechanisms exist to potentiate neuronal output when activity falls. Likewise, while neurons rely on excitable states to function, these same excitable states must be kept in check for stable function. However, the identity of molecular factors and pathways regulating these pathways remain elusive.
Chapter 2 of this thesis reports the findings from an RNA interference- and electrophysiology-based screen to identify factors necessary for the long-term maintenance of homeostatic synaptic potentiation. Data is reported to resolve a long-standing question as to the role of presynaptic Cav2-type channels in homeostatic synaptic potentiation at the Drosophila NMJ. It is shown that reduction in Cav2 channel expression and resultant activity is not sufficient to occlude homeostatic potentiation. Thus, the homeostatic block of a amino-acid substituted Cav2-type calcium channel (cacS) channel is presumed to be due to loss of a specific signaling or binding activity, but not due to overall diminishment in channel function. It is also reported that both Drosophila homologs of phospholipase Cβ (PLCβ) and its putative activator Gαq were found to be necessary for a scaling up of neurotransmitter release upon genetic ablation of glutamate receptors. These factors are canonically involved in the activation of intracellular calcium stores through the inositol trisphosphate receptor (IP3R) and the closely related ryanodine receptor (RyR). Likewise, the Drosophila homolog of Cysteine String Protein (Csp) is identified as important for long-term homeostatic potentiation. CSP has also been reported to be involved in regulation of intracellular calcium. PLCβ, Gαq, and CSP are also known to regulate Cav2-type channels directly, and this possibility, as well as others, are discussed as mechanisms underlying their roles in homeostatic potentiation.
Chapter 3 of this thesis reports the extended findings from expression of a gain-of-function Cav2-type channel. The Cav2.1 channel in humans is known to cause a dominant, heritable form of migraine called familial hemiplegic migraine (FHM). Two amino-acid substitutions causative for migraine were cloned into their analogous residues of the Drosophila Cav2 homolog. Expression of these migraine-modeled channels gave rise to several forms of hyperexcitability. Hyperexcitability defects included abnormal evoked waveforms, generation of spontaneous action potential-like events, and multi-quantal release. It is shown that these forms of hyperexcitability can be mitigated through targeted down-regulation of the PLCβ-IP3R-RyR intracellular signaling pathway.
Chapter 4 presents an extended discussion as to the roles for presynaptic calcium channels, PLCβ, and CSP in homeostatic synaptic potentiation, and the mechanism underlying hyperexcitability downstream of gain-of-function Cav2-type channels. The proposed model aims to bridge the involvement of the PLCβ pathway in both homeostatic potentiation and neuronal excitability. Last, the implications for these findings on human disease conditions are elucidated.
Specialized cells called neurons are responsible for communication in the brain. These cells rely on calcium to drive release of chemicals called neurotransmitters from one cell to another. This process, called neurotransmission, must be highly regulated for normal neuronal communication. In the absence of normal function, neurological diseases such as migraine and epilepsy occur.
Neurons maintain normal function through a process called homeostasis. Homeostasis is the process of maintaining output, in this case neurotransmitter release, within a normal range, in spite of external perturbations. However, it is not clear what cellular components are needed for a cell to maintain homeostasis. Elucidation of such components has potential to inform our understanding of neurological disease.
This thesis used an experimental approach to identify novel factors necessary for homeostasis at sites of neuronal communication. It was found that a number of factors capable of regulating calcium inside the cell were necessary for homeostasis. By an alternative approach, cells were perturbed with an abnormal calcium channel. Calcium channels are responsible for regulating calcium entry to the cell for neurotransmission. The calcium channel used for these studies was modeled after abnormal channels known to cause migraine and epilepsy in humans. It was found that the same calcium regulation pathway identified as necessary for homeostasis could also be downregulated to block deviant effects of the abnormal calcium channel.
This thesis’ findings offer insight as to how neurons maintain normal function. They also reveal a novel role for a calcium-signaling pathway in processes related to human disease.
publicabstract, Drosophila, excitability, homeostasis, migraine, NMJ, synapse
Copyright 2015 Douglas J Brusich