Neurodegenerative diseases, while different in their presentation of symptoms and specific hallmarks of pathology, share the common denominator of extensive highly reactive oxygen molecule production. Currently, there are no known viable treatments to reduce the death of neurons in patients suffering from brain diseases, such as Alzheimer's, Parkinson's, Huntington's diseases, and ALS. The sheer number of people suffering from a neurodegenerative disease are staggering, with an estimated 35.6 million worldwide diagnosed with Alzheimer's (1), for example. The cost to the U.S. economy of Alzheimer's disease is projected to grow to $1.1 trillion dollars per year by the year 2050 (1), a figure that does not include the burden to patients and caregivers. Put simply, in terms of cost, one neurodegenerative disease will cost as much as twelve years of the ?War on Terror? (2) every single year. Taking a broader view, most neurodegenerative conditions, including Alzheimer's, Parkinson's, and Huntington's diseases, are characterized by similar pathological or inflammatory indicators, which include copious reactive oxygen species (ROS) production, microglial activation, inflammatory cytokine production, and mitochondrial dysfunction (1). Many of these factors are insufficiently understood under either healthy or degenerative conditions. The goal of my thesis work in Prof. Chris Chang's lab at UC Berkeley is to gain insight into the function of ROS under both normal synaptic and disease conditions in the brain. In neurons, ROS are produced in an activity dependent manner by NADPH Oxidase 2 (NOX2) downstream of neurotransmitter receptor activation, such as NMDA receptors, or by inflammatory stimuli(5). To date, the only way to endogenously activate NOX2, and thus, generate ROS in neurons is to stimulate an upstream receptor, such as the NMDA receptor or a growth factor receptor, which leads to activation of numerous other pathways and complicates isolating the functions of ROS produced specifically by NOX2. In order to stimulate NOX2 with a high degree of spatial and temporal precision, I have devised a new optogenetic tool, termed optically activated NOX2 (?OptoNOX?) that will turn on in the presence of 633nm light and turn off at 750nm, or an alternative tool that will turn on at 470nm light and off in the dark. Using this tool, I can activate NOX2 in subcellular regions, such as dendritic spines, soma, or axons with a high degree of spatial and temporal resolution. I also propose to develop this entirely genetically encoded tool into a floxed AAV based construct to be used in CRE selective circuits, such as dopaminergic, in vivo as an optogenetic neurodegenerative disease model. The objective of AIM1 is to develop and characterize the optogenetic NOX2, as well as use it to better understand the role of ROS at the synapse and to what degree NOX2 mediates pathology.
In AIM2, I will use a CRISPR library to selectively alter the cysteine residues in the target proteins of NOX2 mediated ROS, identified by proteomic screen in the Chang lab, and use a high throughput optical physiology screening method to determine which sulfenylated residues contribute greatly to synapse physiology. I will also look at neuron morphology and pathological markers to determine whether specific ROS mediated post-translational modifications effect the progression of a disease phenotype. The proposed project has tremendous significance to human health, as it will shed light on the intricate balance between health and disease mediated by ROS, with a molecular focus on NOX2, and will hopefully provide novel insight into specific pathway switches between physiology and pathology.
Excessive production of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are linked to major neurodegenerative diseases including Alzheimer's and Parkinson's, as well as can mediate physiological functions including plasticity and memory in a tightly controlled manner. This work proposes to develop an light activated disease model by creating an optogenetic NOX2 to allow direct, rapid, controllable activation of endogenously produced ROS, which will enable the dissection of the effects of ROS molecules on neuron physiology, downstream protein targets, cell morphology, as well as their contribution to the underpinnings of neurodegenerative disease.
