Summary
Our nervous system is the primary organ by which we sense, interpret, remember, and respond to the outside world and to our own internal physiology. This elaborate system of neurons functions as a communication network, with vast arrays of chemical and electrical synapses between individual neuronal cells. The nervous system also interfaces with other tissues of the body, either directly (e.g., neuromuscular junctions at skeletal muscles) or indirectly (e.g., the release of hormones, biogenic amine neurotransmitters, and neuropeptides into the blood stream), to regulate physiology and behavior, as well as maintain overall body homeostasis. Unlike many bodily tissues, the nervous system is largely incapable of replacing damaged cells once development is complete, making it susceptible to traumatic injury and age-associated decline. The high energy demands of electrochemical signaling, combined with the inability to store energy in the form of glycogen reserves, makes neurons highly dependent on oxygen, oxidative phosphorylation, and mitochondria. The nervous system has evolved multiple mechanisms to maximize mitochondrial function and prevent damage from acute oxygen starvation. Indeed, the underlying etiology of many neurological disorders and diseases, including ischemic stroke, Parkinson’s Disease, and Alzheimer’s Disease, are due to defects in one or more of these key neurophysiological processes. A more complete understanding of these processes will facilitate better diagnosis and treatment of multiple neurological disorders.
We focus on understanding three areas of neurophysiology. First, we are interested in understanding how the transport and dynamics of mitochondria are mediated along axons and dendrites, as well as at synapses. Second, we are interested in understanding how neurons, synapses, and neuronal mitochondria respond to hypoxic stress (e.g., ischemic stroke). Finally, we are interested in understanding the function of the Ubiquitin Proteasome System (UPS) and its role in cellular aging, including the function of the UPS in neurons, as well as how neurons can regulate the UPS and proteostasis in distal tissues.
We use C. elegans to study these areas of neurophysiology because the nematode has a simple nervous system, which is easily visualized through its transparent body, allowing us to observe mitochondria and other structures within neurons in an intact and behaving animal. My lab has used the rich genetic and genomic tools of this organism, and both forward and reverse genetic approaches, to identify multiple genes that function in mitochondrial, hypoxic stress, and UPS biology. All of the genes we have identified have human equivalents that seem to be playing similar or identical roles in the human brain, suggesting that our findings are likely to be applicable to human health.
The Response Of Neurons To Low Oxygen Levels (Hypoxia And Anoxia)
Environment can impact nervous system function, and neurons can respond to accommodate a changing environment. Specifically, oxygen influences behavior in many organisms, and low oxygen levels (hypoxia) can have devastating consequences for neuron survival due to excitotoxicity from overactivated neurotransmitter receptors and impaired mitochondrial function. We have shown that hypoxia blocks the membrane recycling of glutamate-gated ion channels to synapses, thereby depressing glutamatergic signaling. Surprisingly, the canonical transcriptional factor that mediates most cellular hypoxia responses is not required for this effect. Instead, a specific isoform of the prolyl hydroxylase EGL-9, a key sensor for oxygen, recruits LIN-10, a known PDZ scaffolding protein, to endosomes, where together the two proteins promote glutamate receptor recycling. Our discovery demonstrates a novel way by which animals can sense and respond behaviorally to oxygen levels. It identifies a novel substrate of the EGL-9 prolyl hydroxylase. Finally, it indicates that neurons have signaling pathways that play a neuroprotective function to help minimize damage during ischemic events by using molecular and cellular mechanisms more diverse than originally appreciated.
It is also important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. We have shown that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid functional recovery of the nervous system during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. This conserved anoxic stress response thus changes mitochondrial fission and fusion to help neurons survive the oxidative damage resulting from oxygen deprivation.
A complete understanding of the hypoxia response pathway (i.e., EGL-9 and HIF-1) is important for understanding ischemic stroke. In addition, this pathway has become a target of interest for new chemotherapeutics, as HIF-1 is activated and plays a key role in cancer progression and metastasis. Therefore, we have broadened our studies of this pathway, and we are now conducting RNA-seq and ChIP-seq experiments to identify both HIF-1-dependent and HIF-1-independent targets of hypoxia-induced gene regulation.
Regulators Of Mitochondrial Dynamics In Neurons
In addition to being the “powerhouse of the cell,” mitochondria play critical roles in mediating calcium buffering, apoptosis, and necrosis. They are also a major source of reactive oxygen species (ROS), which can have both a signaling role and be damaging to cells. Mitochondria are actively transported within neurons to synapses, and damaged mitochondria – a potential threat to the cell – are transported back to the cell body for removal by mitophagy. Mitochondria are also dynamic, undergoing fusion and fission. Fusion is thought to be a mechanism for boosting mitochondrial output and protecting mitochondrial health, whereas fission is thought to be the first step on the way to mitophagy and the removal of damaged mitochondria. Defects in mitochondrial dynamics have a clear role in Parkinson’s Disease. Defects in mitochondrial transport have a clear role in Alzheimer’s Disease. Thus, an understanding of mitochondrial dynamics and transport is important for our understanding of neurological disorders with mitochondrial etiology, as well as our understanding of aging and age-associated diseases.
Mitochondrial dynamics as a field has largely been studied in single celled yeast; thus, little is known about the machinery that conducts mitochondrial fission and fusion in specialized tissues like neurons. We are studying mitochondrial dynamics in C. elegans neurons using a mitochondrially-localized GFP reporter, which makes it easy to visualize individual mitochondria in axons and dendrites of live animals. Using this tool, we are performing a forward genetic screen for mutants with defects in mitochondrial transport, dynamics, and mitophagy. Our goal is to clone and characterize the underlying genes so as to have a complete understanding of the factors that mediate and regulate mitochondrial biology in neurons.
Dopamine Signaling Activates The UPS In Distal Epithelial Tissues
Our genetic screens led us to explore more multicellular functions of the Ubiquitin Proteasome System (UPS). The UPS comprises many ubiquitin ligases, which tag individual proteins for degradation by the 26S Proteasome. The UPS is a key mechanism by which cells maintain protein homeostasis (proteostasis) by removing misfolded and oxidized proteins. As cells age, UPS activity becomes impaired, resulting in the accumulation of damaged proteins and age-associated physiological decline. By understanding how UPS activity is regulated in neurons and in non-neuronal tissue by neurons, we should be able to provide new therapeutic targets for diseases that involve protein aggregates and disrupted proteostasis.
We previously generated a GFP-based reporter system for UPS activity in C. elegans, allowing us to query UPS activity in specific tissues and at specific points along development. We found that epithelial cells undergo a dramatic increase in UPS activity as animals mature. We have also found that the humoral neurohormone/biogenic amine neurotransmitter dopamine promotes UPS activity in epithelia. In C. elegans, mechanosensory neurons release dopamine when nematodes encounter a potential bacterial food source. Dopamine in turn inhibits motoneuron activity through the dopamine receptors DOP-2 and DOP-3, resulting in a behavioral change that slows the animal down so that it can feed. We found that this released dopamine also activates the UPS in epithelial tissues, including the intestine and epidermis, through the dopamine receptors DOP-1 and DOP-4, and the cAMP-Response Element Binding Protein (CREB) transcription factor. This signaling pathway activates the expression of enzymes involved in xenobiotic detoxification (e.g., cytochrome P450 enzymes) and innate immunity, which in turn promote protein polyubiquitin. While we do not yet understand exactly how xenobiotic detoxification activates the UPS, our results show that dopamine signaling is essential for nematodes to survive xenobiotic stress and to maintain normal proteostasis. Taken together, our results suggest that dopaminergic sensory neurons, in addition to slowing down locomotion upon sensing a potential bacterial feeding source, also signal to epithelial tissues to prepare for infection in case that potential bacterial food source turns out to be pathogenic.
