Adam J. Bloom, Ph.D. - US grants

Project Summary/Abstract: Disease associated with cigarette smoking remains the largest cause of preventable death. A large margin for improvement in smoking cessation treatment exists, with great potential to benefit public health: more than half of American smokers attempt to quit every year, but only ~6% succeed annually. Differences in smoking phenotypes, including cessation, have significant genetic components, but the large majority of this influence is unexplained. Genetic studies of smoking behavior that reveal the mechanisms underlying variation in these traits will identify further targets for pharmacotherapy and aid in improving personalized cessation treatment. Utilizing measurements from an in vivo nicotine metabolism experiment, I developed a predictive genetic model that explains >70% of the variation in oral nicotine C-oxidation (the primary nicotine metabolism pathway), based on CYP2A6 genotype. Model predictions were significantly associated with different measures of cigarette consumption and smoking cessation success in further subjects. The model also allowed me to demonstrate two key novel findings: the independent influences upon smoking behaviors of polymorphisms in 1) EGLN2, a.k.a. Hypoxia Inducible Factor Prolyl Hydroxylase, which initiates a transcriptional cascade in response to cellular hypoxia, and 2) in FMO3 and CYP2B6, further nicotine metabolism genes that may have important extra-hepatic activity. The specific aims of this grant are: 1) Develop a comprehensive predictive genetic model of nicotine metabolism incorporating all three nicotine metabolism pathways and their associated genes, focusing on CYP2A6, the UGTs and FMOs. The improved model will then be applied in further samples to determine the influence of heritable differences in nicotine metabolism upon smoking phenotypes; 2) Identify variants that alter nicotine metabolism gene function and demonstrate the mechanisms of their effects. I will focus especially on protein and mRNA expression, and splicing, in human brain and liver samples; 3) Identify variants in EGLN2 and other hypoxia-response candidate genes, and determine the mechanisms of their effects on smoking phenotypes. My preliminary data indicate an EGLN2 variant associated with nicotine dependence and cigarette consumption alters the relative expression of different EGLN2 5'UTR mRNA splice-variants. I will identify differences in gene and protein expression influenced by EGLN2 genotype in cells cultured under normal and hypoxic conditions. The goal of this K01 proposal is to obtain further training in statistical human genetics and cell culture under special conditions and to apply this expertise to problems of substance abuse.

Motor axon loss is a cardinal symptom of amyotrophic lateral sclerosis (ALS). Axon loss can be driven by a genetically encoded program in which the axon survival factors NMNAT2 and STMN2 inhibit the activity of the axon destruction factor SARM1. Recent data suggest that this program of axon self-destruction may contribute to pathology in ALS. First, aggregation of TDP-43, a hallmark of most ALS cases, results in the selective loss of mRNA encoding functional STMN2, a key axon survival factor. Second, loss of SARM1 suppresses some neurodegenerative phenotypes in a mouse ALS model that expresses pathogenic human TDP-43. Here we investigate the contribution of this axon degeneration pathway to ALS. We have defined the mechanism of action of SARM1, demonstrating that it is the founding member of a new class of NAD-cleaving enzymes. SARM1 enzyme activity is normally held in check via an autoinhibitory domain. Injury- or disease- induced loss of NMNAT2 and STMN2 disinhibits SARM1, leading to rapid NAD+ depletion, metabolic catastrophe, and axon fragmentation. Our structure-function studies of the SARM1 protein have identified mutations with a range of consequences, from constitutively active variants that promote cell death and axon loss, to dominant negative variants that are neuroprotective. These findings imply that human variants may exist that either promote or protect against neurodegeneration, and that understanding the phenotypic consequences of genetic variation requires functional studies of enzyme activity. In support of this hypothesis, we have identified several rare SARM1 variants in ALS patients, but not in controls, that have constitutive NADase activity and promote neuron death and axon loss. These variants also cause motor dysfunction and paralysis when expressed in the mouse CNS, suggesting that activating SARM1 mutations may contribute to ALS pathogenesis. Here we propose to define the function of SARM1 variants from ALS patients, controls, and the general population. These studies will allow us to categorize SARM1 variants as putatively pro-degenerative, neuroprotective, or neutral. In parallel, we will dissect the contribution of variation in components of the programmed axon destruction pathway to ALS phenotypes, alone and in combination with known ALS genetic risk-factors, in motor neurons differentiated from human induced pluripotent stem cells (iPSCs). Finally, we will investigate neurodegeneration in a mouse knock-in model carrying a Sarm1 allele equivalent to a pro- degenerative allele found in ALS patients, alone and in combination with a SOD1 model, based on a specific patient genotype that we identified. We will attempt to suppress ALS phenotypes with SARM1 inhibition via a proven gene therapy approach and with experimental small molecule inhibitors. Results of these studies will establish the relationship between the SARM1-mediated axon destruction program and ALS, and build the foundation to develop axoprotective therapeutics to treat this devastating disease.