Gwendalyn DiAnn King, Ph.D. - US grants

DESCRIPTION (provided by applicant): The focus of our study is to examine how the age suppressor protein, Klotho, is regulated with aging in the brain. When Klotho expression is eliminated, mice develop normally, but age to death by 4 months of age. This rapid deterioration is accompanied with a phenotype not unlike what is observed in aged humans (cognitive impairment, atherosclerosis, ectopic calcification, emphysema, osteoporosis, skin atrophy and hair loss, thymic involution, infertility and decreased bone mineral density). Elimination of Klotho in mice causes cognitive impairment that is associated with increased oxidative stress. In contrast, Klotho overexpressing transgenic mice live longer by up to 30% and are resistant to oxidative stress. Our group found that Klotho is downregulated in the aging non-human primate, rat and mouse brains Together, these have lead us to hypothesize that Klotho is important in brain function and its downregulation with age may be the result of oxidative stress which, if prevented, could ameliorate decline into neurodegenerative disease. The work proposed, examines regulation of the Klotho promoter and 3'UTR with age and the effect of oxidative damage to the Klotho promoter with age. We will determine whether the high GC content of the Klotho promoter makes it a target for age-related downregulation because of damage that accumulates over time because of oxidative stress. This will be done by comparing the oxidation state of the Klotho promoter to that of other genes both in vitro and in post mortem samples from aged rhesus monkey brain. We will also work to characterize the transcription factors that bind and induce activation of the Klotho promoter. The Klotho promoter does not contain the classical elements for transcription initiation. Understanding what factors are important for Klotho transcription may shed light on signaling pathways leading to Klotho activation and the role of Klotho in the normal cell. Last, we will determine whether Klotho is regulated by microRNAs (miR) and how miR change in the brain with age. MiR bind and regulate translation of mRNA and nothing is known about whether and how miR affect Klotho processing. Again, understanding the processes that regulate Klotho will enable us to have a better understanding of the processes that affect Klotho and Klotho's wider role in cellular function. The results of this work will add new knowledge on both the anti-aging gene Klotho and elucidate a possible mechanism for how oxidative damage selectively downregulates specific genes. PUBLIC HEALTH RELEVANCE: With a rapidly aging population, increases in age related disorders are anticipated to rise to unprecedented levels in the next 50 years. Understanding the regulation of genes known to effect and be affected by the aging process is critical to developing novel therapies for a range of disorders. The aging suppressor protein, Klotho is decreased in the brain with age and may be a target for therapeutic development against neurodegenerative disorders.

? DESCRIPTION (provided by applicant): Changes in protein expression with age fundamentally alter the brain micro-environment. How changes in expression of specific proteins impact brain aging and development of disease remains poorly understood. Klotho, originally described as longevity protein, is age-downregulated in brain and promotes cognitive impairment or enhancement dependent on its level of protein expression. Our preliminary data show a role for klotho in both postnatal neurogenesis and synaptic plasticity of the hippocampus. Hippocampal function is critical in cognitive function and is particularly sensitive to the effects of advanced age. Klotho mediated effects on hippocampal function could underlie cognitive effects measured in knockout and overexpressing animals. The long-term goal of the proposed research is to understand the functional consequences on cognition caused by age-downregulation of klotho and thus how klotho affects hippocampal function over lifespan. Using mouse and viral vector models of klotho protein expression level differences, the central hypothesis that klotho expression is required for sustained hippocampal function with age will be interrogated. The hypothesis is based on preliminary data revealing differences in the neural stem cell development and synaptic plasticity concomitant with increased or decreased expression of klotho protein. To investigate the hypothesis three specific aims will be conducted: 1) Test the hypothesis that brain-derived klotho expression regulates hippocampal function by inhibition of neurotrophic signaling; 2) Determine whether long-term loss of brain- derived klotho causes hippocampal degeneration; 3) Test whether enhanced klotho expression is sufficient to prevent cognitive aging. In specific aim 1, we will utilize our novel mouse models to determine whether klotho in the brain mediates cognitive outcomes. Aim 2 will determine whether the rapid onset of cognitive impairment and markers of stress in the global klotho knockout are the precursors to neurodegeneration. Aim 3 will test whether klotho can be elevated in mid-life to support healthy brain aging. The work is significant in identifying a role for klotho in normal bran function and provides new information as to whether klotho's effects on cognition are mediated by its role in neurogenesis or synaptic plasticity. The application is innovative in its use of anial models to allow targeted manipulation of klotho expression level. By understanding how klotho functions in the brain, we will obtain greater insight into mechanisms underlying both normal brain aging and pathological disease development.

This REU site award to the University of Alabama at Birmingham (UAB), in Birmingham, AL, will support 10 students for 10 weeks during the summers of 2017-2019. The theme of the Summer Program in Neuroscience (SPIN) REU program, hosted by the Department of Neurobiology, is the development and plasticity of the nervous system. The program will allow students to conduct fundamental neuroscience research across a range of research questions and methodological approaches. Students will participate in activities and seminars to develop their graduate school application and prepare for graduate school interview. This REU site is devoted to supporting students seriously considering post-baccalaureate research careers following completion of their undergraduate degree program. While research experience is not a pre-requisite, students should be able to demonstrate a commitment to fundamental discovery science. Applications are accepted online through the program website (below). Students will be selected by the SPIN admissions committee based on their cumulative GPA in a STEM major, evidence of upper level coursework to prepare for research in a neuroscience lab, response to questions in the application, and recommendation letters.

It is anticipated that a total of 30 students primarily from schools with limited local research opportunity will be trained through the REU program. Students from under-represented groups are strongly encouraged to apply. Students will learn how research is conducted, will integrate into an active research team, will participate in professional development activities, and prepare for success in graduate level education. Students will build their technical skills, participate in journal clubs, present their research results, and attend the annual Neural Conference at UAB.

A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Students will be tracked after the program concludes to determine their career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://www.uab.edu/medicine/neurobiology/education/undergraduate-summer-research or by contacting the program directors: PI, Dr. Lucas Pozzo-Miller (lucaspm@uab.edu) or co-PI, Dr. Gwendalyn King (gdking@uab.edu).

PROJECT SUMMARY Age-related changes to protein and thus cellular function occur across the brain. Increased understanding of when age-related events become pathological is required both to support healthy brain aging and to develop early and efficacious neurodegenerative disease therapeutics. Among the cells of the brain to experience age- related impairment to structure and function, the choroid plexus epithelial cells stand out as a tissue with untapped potential to support the entire brain parenchyma. Choroid plexus epithelial cells express high levels of Klotho protein. Increasing expression of Klotho enhances but decreasing expression impairs neuronal memory function across species and in models of neurodegenerative disease. Recent work determined that a common human polymorphic variant of Klotho that increases protein is protective against Alzheimer?s disease development and pathology. While Klotho action in and upon cells of the brain parenchyma indicates its effects are brain-wide, little is known about the primary source of brain Klotho, the choroid plexus epithelial cells. Our pilot data indicate that cell-type specific klotho-deficiency impacts longevity, memory, and choroid plexus cell structure and function. These data suggest that Klotho may effects cells throughout the brain because it is critical to choroid plexus epithelial cell activities. Choroid plexus epithelial cells provide physical, nutrient, and signal transduction support to the entire brain. They also function as a physical protective barrier and communication center between body and brain. The long-term goal of the proposed research is to understand the health-related functional consequences of age-related klotho-deficiency from choroid plexus epithelial cells. We will use mouse models to interrogate the central hypothesis that Klotho-deficiency impairs brain parenchyma by disrupting the function of the choroid plexus epithelial cells. In specific aim one, we will use mouse models to determine the role of choroid plexus-specific Klotho expression on memory across lifespan. Using animal models and cellular and molecular techniques, aim two will determine whether choroid plexus epithelial cell structure or function are altered by Klotho-deficiency. The significance of the proposal lies in contributions to understanding the basic biology of Klotho as a protein critical for the normal choroid plexus epithelial cell functions required to sustain and support healthy brain parenchyma across lifespan. We are uniquely situated to expose undergraduate students interested in research and medically focused careers discovery neuroscience research using animal models. The proposal is innovative in its use of novel animal models to allow targeted manipulation of Klotho expression and, in studying the choroid plexus epithelia, studies a tissue-type with unrealized potential to support brain health across lifespan.