Deborah J. Good - US grants

neurogenetics; neurogenesis; neural degeneration; genetic regulation; aging; transcription factor; behavior test; laboratory mouse;

DESCRIPTION (provided by applicant): Although over 16 different hypothalamic transcription factors are implicated in controlling fertility, the molecular mechanisms of reproduction remain unclear. The hlh2 basic helix-loop-helix (bHLH) transcription factor is expressed throughout the developing nervous system. In adults, expression is limited to specific neurons in the hypothalamus, pre-optic area and hebenula. Nhlh2 knockout mice (N2KO) are obese and hypogonadal. In addition, they have reduced levels of follicle stimulating hormone and testosterone, and do not show normal male sexual behaviors. N2KO mice also have reduced sperm concentration in their caudal epididymus, but using in vitro fertilization, we have found that sperm from N2KO mice are fully capable of fertilizing oocytes. Thus, infertility in male N2KO mice is largely due to lack of sexual behavior and partially due to reduced sperm production. In other studies, we have found that levels of two neuropeptide processing enzymes, PC1 and PC2, are reduced up to 90% in N2KO mice. These processing enzymes are thought to process GnRH as well as other hypothalamic neuropeptides involved in both fertility and obesity. Based on these preliminary findings, we propose that the Nhlh2 transcription factor regulates male sexual behavior and fertility by up-regulating the expression of PC1and PC2 processing enzymes which are necessary for production of mature GnRH peptide in the pre-optic area of the brain. We will use both animal-based and molecular approaches to test this hypothesis. In the first aim, we will ask if Nhlh2 is co-expressed with GnRH, PC1 and PC2. We will also measure levels of PC1 and PC2 mRNA in pre-optic area neurons, and levels of mature GnRH peptide in N2KO and normal mice. In the second aim, we will supply male N2KO mice with testosterone and ask if this hormone is sufficient to restore male sexual behavior and spermatogenesis. To date, N2KO mice are the only knockout mice in which deletion of a neuronal bHLH transcription factor results in obesity, hypogonadism, and lack of male sexual behavior. Studies of these animals are key to understanding molecular control of male fertility.

Although over 20 neuronally-expressed genes have been implicated in the regulation of body weight, the transcriptional and post-transcriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Recent evidence from my laboratory suggests that the NhIh2 transcription factor plays a key role in the regulation of hypothalamic genes controlling body weight. NhIh2 knockout mice (N2KO) display adult-onset obesity and NhIh2 is expressed in the hypothalamic regions that control appetite-including the arcuate ncuelus (ARC) of the hypothalamus. In dual-label in situ hybridization studies, we found that nearly all pro- opiomelanocortin (POMC) neurons in the ARC expressed NhIh2. In these neurons, the POMC precursor protein is processed to alpha- melanocyte stimulating hormone (alphaMSH), a peptide that regulates food intake and energy expenditure. In studies comparing POMC mRNA and peptide levels in N2KO and normal mice, we found that while POMC mRNA levels were identical between the two groups, levels of two processed forms of POMC, beta-endorphin (betaEND) and adrenocorticotropin hormone (ACTH) were dramatically reduced, while levels of alphaMSH were modestly reduced in the N2KO animals. These findings suggest that N2KO animals could have a defect that reduces processing of the POMC precursor. Indeed, mRNA levels for two of the processing enzymes necessary for cleaving POMC into bioactive peptides prohormone convertases I (PC1) and II (PC2) were reduced up to 80 percent in N2KO animals. Thus, it is likely that PC1 and PC2 are direct gene regulatory targets of the NhIh2 transcription factor and that a reduction in fully processed neuropeptides contributes to obesity in the N2KO animals. In addition, we predict that NhIh2 is a direct target of leptin-signaling pathways, as it is expressed in the leptin-receptor containing POMC neurons and contains STAT motifs in its promoter. Based on these preliminary findings, we propose that positive energy balance increases the expression of NhIh2, which in turn increases the transcription rate of the PC1 and PC2 genes. To test this hypothesis, we will ask in the first two specific aims, if NhIh2, PC1 and PC2 are increased in response to a positive energy balance and if the of the PC1 and PC2 genes is lost in N2KO animals. In specific aim 3, we will switch to a molecular investigation of the NhIh2 promoter to determine if AP1 and STAT3 transcription factors can bind to and transactivate the NhIh2 gene. We will then ask in specific aim 4 if the NhIh2 transcription factor, in cooperation with activated STAT3, binds to and transactivates the PC1 promoter. The proposed experiments use both molecular and animal-based approaches to ask a fundamental question about transcriptional and post- transcriptional mechanisms controlling body weight.

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (06) Enabling Technologies and the specific Challenge Topic, 06-GM-105: Small RNAs. Nearly 60% of the US adult population is either obese or overweight. Clearly understanding the fundamental mechanisms of gene regulation in response to changes in energy availability is of clinical, pharmaceutical, and societal importance. The long-term objective of this research is to test the hypothesis that regulation of hypothalamic miRNA levels occurs with changes in energy availability targeting the expression of specific genes that control body weight. This multiple PI proposal is strengthened by the combined expertise of each individual laboratory. Dr. Good's lab has more than 10 years experience in studying the transcriptional control of hypothalamic genes in response to changes in energy availability. Dr. Jensen is world-renowned for his work on microarray quality and assessment of gene expression analyses. Dr. Helm's lab uses proteomic and metabolomic approaches to study cellular processes such as quiescence. Together, the three PIs will use miRNA and mRNA microarrays, proteomics and bioinformatics to ask specific and fundamental questions about the role of miRNA in body weight regulation including: (i) whether miRNA species are differentially-regulated in response to changes in energy availability (ii) if these miRNA species target specific hypothalamic genes that regulate body weight control. The co-regulated miRNA, mRNAs and proteins will be identified through a three-pronged bioinformatics approach to examine the expression levels of the putative targets of the identified miRNA species and compare these with possible discordance between the protein and mRNA levels for the target genes. This is an innovative method that has not yet been done for whole animal tissues. These studies specifically address the intent of the NIH Challenge Grants by (1) proposing to study the scientific challenge of the role of miRNA in obesity-a gap in our knowledge, especially as it relates to hypothalamic gene regulation;(2) proposing a study that is designed to benefit the scientific community through these 2-year stimulus funds, by generating at least 3 new datasets for us and others in the scientific community to use in future studies;and (3) proposing a study design with research methods that will benefit from an influx of funds to quickly advance the area of miRNA research in significant ways. With obesity at record proportions, the discovery of new mechanisms of gene regulation will lead to important new clinical, pharmaceutical and basic research directions and applications. PUBLIC HEALTH RELEVANCE: The proposed experiments will characterize a potentially new method for hypothalamic gene regulation during food intake, food deprivation or in obese individuals with high levels of circulating leptin. An understanding of these molecular pathways in the brain, and identification of the genes and proteins that are differentially regulated during changes in food availability should identify new therapeutic options for obese and overweight individuals, as well as give new information about the basic mechanisms that our brains use to control body weight.

PROJECT SUMMARY Undergraduate research opportunities are linked to improved outcomes for students, faculty, universities, and, ultimately, the scientific field of involving diabetes and digestive and kidney diseases. Virginia Tech (VT) promotes a culture of undergraduate research that moves students ?beyond boundaries? to best prepare students for their future careers. Notably, a) infrastructure is needed to recruit and retain students who may not typically seek out undergraduate research opportunities and b) knowledge and skills related to the translational spectrum are needed to improve students' abilities to contribute effectively to team science. The Translational Obesity Undergraduate Research (TOUR) Scholar program is an adapted version of the highly successful USDA Summer Scholars (2007-2010). For the TOUR Scholars program, 10 undergraduate research students will be paired with faculty mentors (PhD and/or MDs) within the Center for Transformative Research on Health Behaviors at VT. Currently, TOUR Scholar mentors have $14M in past and current NIDDK funding, as well as $27.5M in current research funding from other sources. The program has a spring semester independent study component to prepare students for their 10-week research intensive immersion. Within the 10-week program, students will work an average of 38 hours per week within a research lab as well as attend TOUR Scholar specific curriculum weekly (e.g., grantsmanship, diversity and inclusion, communication skills, research ethics). The program culminates with research site visits, including the NIDDK campus, as well as presentation of their work at the VT Undergraduate Research Symposium. We aim to increase the proportion and representativeness of students who pursue NIDDK-related jobs, improve scholars' competencies with research skills (specific to each lab), and explore factors that lead to mentor retention. These outcomes will be measured through a mixed- methods approach, including a third-party evaluator as well as scholar and program advisory committee feedback. The impact of this 5-year NIDDK grant at Virginia Tech will be to produce 50 NIDDK-trained researchers, and at least 20 NIDDK-oriented mentors in a learner-centered, experiential undergraduate research program.