Marc Diamond, M.D. - US grants

As our population ages, neurodegenerative diseases are a growing problem, and trinucleotide repeat diseases are increasingly important. This is a proposal to determine the mechanism of motor neuron degeneration in Spinal and Bulbar Muscular Atrophy (SBMA) or Kennedy's disease, in which repeat expansion in the androgen receptor (AR) gene causes polyglutamine tract expansion in AR protein (termed ARK henceforth). First AR and ARK expression and localization will be compared in transfected cells through RNA and protein quantification, and immunohistochemical methods. Receptor binding to HSP9O, hormone response and regulation of transcription at known hormone response elements will be assayed. A yeast two hybrid screen and interaction cloning will be select proteins that bind ARK in vivo and vitro. ARK expressed in cultured cells will be used to study early cell death. and transgenic mice will be constructed with human ARK to establish an animal model. This research will be conducted with Keith R. Yamamoto, PhD, who cloned the glucocorticoid receptor (GR) and has described many aspects of steroid receptor function in molecular detail, including HSP9O association, hormone binding, transcriptional regulation, transcription factor interactions, and three dimentional protein structure. His laboratory has developed broad expertise in mammalian cell culture, yeast genetics, and protein biochemistry. Keith is a leader in fundamental research and scientific education. This proposal stemmed from my experience as a neurology resident, a fascination with trinucleotide repeat disease, and previous research experience with Keith on GR regulation through interactions with other factors. Initially I hope to to clarify molecular mechanisms of neurodegeneration in SBMA while broadening my knowledge of molecular and cellular biology, neuroscience and neurodegenerative diseases, While I conduct research I will attend basic science courses and journal clubs. Neurology departmental lectures, and outside courses on neurodegenerative diseases and transgenics. Ultimately I hope to become an academic investigator focused on mechanisms of neurodegenerative disease. By combining an important clinical problem with a strong basic science environment I will use a well defined protein as a tool to understand the molecular basis of the progressive motor neuron degeneration of SBMA.

DESCRIPTION (provided by applicant): The long-term objectives of this application are to design cell-based systems for high throughput screening of compounds that will modify the toxicity and abnormal processing associated with polyglutamine proteins. The specific aims of the project are: 1. Establish neural precursor cell lines with regulated gene expression of polyglutamine proteins suitable for high throughput applications. a. Express of polyglutamine-flourescent proteins for FRET-based studies of aggregation. b. Express of native polyglutamine proteins for cell viability studies. 2. Establish a fluorescent plate reader-based assay to monitor expanded polyglutamine protein aggregation. a. Establish FPR-based assays of protein aggregation based on FRET. b. Establish FPR-based assays of cell toxicity. The project is directly related to drug development for treatment and prevention of human neurodegenerative disease due to polyglutamine expansion proteins. The research design has two basic elements: development of appropriate cell models using inducible promoter systems in neural precursor cells; development of systems to monitor cell viability and abnormal processing using a fluorescent plate reader. Methods for cell development include culture and transfection of neural cell lines, establishment of inducible cell expression systems using fluroscence activated cell sorting to isolate cells with regulated expression, and characterization of responses to induced expression of polyglutamine proteins. Methods for FPR development are based on detection of FRET signal from polyglutamine proteins with abnormal aggregation, and detection of fluorescent signals in viable and dead cells, using expression of fluorescent proteins and detection dyes. To corroborate FPR results, standard methods of cell imaging and protein biochemistry shall be employed.

Polyglutamine neurodegenerative diseases are a devastating family of inherited disorders that include Huntington Disease and spinobulbar muscular atrophy (SBMA). Long-term goals of this project are to identify molecular mechanisms of polyglutamine neurodegenerative disease, to determine specific therapeutic targets, and to develop mechanism-based therapies. Previous work demonstrated that Y-27632, an inhibitor of the rho-associated kinase p160ROCK, reduced polyglutamine aggregation and toxicity in cell and Drosophila models. Aim 1: Identify and characterize novel regulatory pathways and target molecules. We will complete a screen of a library of biologically active small molecules. We will also analyze hits from two prior screens of biologically active compounds. Results will be analyzed in a systematic fashion using a combination of genetic and pharmacologic approaches to determine new pathways of potential significance. Aim 2: Determine the molecular mechanism by which p160ROCK signaling influences polyglutamine aggregation and toxicity. The role of specific components of the p160ROCK signaling pathway will be tested in Drosophila. The molecular basis of polyglutamine protein association with actin will be tested, and its role in modulating polyglutamine aggregation determined. Aim 3: Test the activity of Y-27632 in preventing neurodegeneration in vivo. Bioactivity of Y-27632 in brain, and systemic toxicity shall be determined in order to plan an appropriate dosing regimen. Y-27632 inhibition of polyglutamine-dependent pathology in vivo shall be tested using a variety of behavior, rotarod, pathological and biochemical analyses in the R6/2 mouse model of HD.

DESCRIPTION (provided by applicant): The androgen receptor (AR) is the single best molecular target for prostate cancer (PCa). Novel anti-androgens will likely be useful in treating both primary and recurrent PCa. Such anti-androgens need not be competitive antagonists, and could conceivably block downstream events in AR signaling. We have used cellular screens to identify two novel, potent anti-androgens: one is an FDA-approved drug, and the other a natural product. These compounds function as non-competitive antagonists, and synergize with classical anti-androgens such as hydroxy-flutamide or bicalutamide. Preliminary evidence suggests one compound is effective in vivo, especially in combination with BiC. The long term goals of this grant are to develop a better therapy for PCa. Aim 1: Efficacy studies in mice. We will treat animals for via IP injection, testing the candidate compound with bicalutamide alone or in combination, with castration as a positive control. We will measure effects on prostate weight and morphology, and will use RT-PCR to determine the degree to which it can down-regulate AR-dependent gene expression in the prostate gland. If successful, we will then establish an optimal ratio of the compound with bicalutamide to achieve maximal responses. These studies will underlie translation of this compound to the clinic for the treatment of hormone-refractory PCa in humans. Aim 2: Determine the mechanism of action of lead compounds. Both compounds identified have marked potency in cellular systems. Determination of their molecular mechanism of action will help elucidate biological mechanisms that control AR activity, and may lead to new therapeutic targets. We will use the extensive knowledge of steroid receptor biology to characterize at which step compounds inhibit AR activity. We will use FRET-based assays to determine effects on intramolecular and intermolecular conformational change. We will test for effects on AR nuclear localization via cell fractionation and western blot. Since the compounds specifically inhibit AR, but not the closely related glucocorticoid receptor, we will use domain transposition to test which regions of AR mediate its sensitivity to inhibition. This will facilitate identification of factors that interact with these regions, and which may be modulated by the compounds. We will measure effects of test compounds on gene regulation and promoter occupancy using quantitative RT-PCR and chromatin immunoprecipitation (ChIP) to determine whether they affect AR transcription globally, or only at specific subsets of promoters. These approaches will pinpoint the precise mechanisms of AR inhibition, will facilitate identification of intracellular targets, and will expand our knowledge of AR biology, facilitating new treatment strategies for PCa.

DESCRIPTION (provided by applicant): Cell-cell transfer and propagation of tau aggregates. Tauopathies are devastating neurodegenerative diseases. All are linked pathologically to misfolding and aggregation of the microtubule-associated protein tau, and include common disorders such as Alzheimer disease and frontotemporal dementia. Both wild-type tau protein and mutant forms associated with dominantly inherited diseases have the propensity to misfold and aggregate. In virtually all cases, disease begins in one brain region before spreading to involve other regions, and there is emerging evidence that this could be based on movement of protein aggregates between cells. This project seeks to understand the cellular mechanisms that govern tau aggregate uptake and release from cells, and how a tau aggregate taken into a cell manages to corrupt the endogenous, normally folded protein. The answers to these questions will provide immediate new opportunities for therapeutic strategies. The goals of this work are as follows: (1) Determine molecular mechanisms of tau uptake. We will use cell-based assays we have developed to study tau aggregate uptake, and to evaluate the molecular mechanisms that underlie this phenomenon. We will use mouse models to test predictions about pathways derived from our experiments in cell models. (2) Determine mechanisms of tau aggregate degradation and release. We believe that cellular pathways linked to protein degradation may play a role in processing tau protein aggregates, and might also be involved in allowing these aggregates to transfer between cells. We will test these ideas using a cellular model of aggregate transfer between cells. (3) Determine mechanisms of tau aggregate propagation. It is unclear how tau protein aggregates that move from one cell to another might lead to misfolding of protein in the recipient cell, and whether this movement can occur across synapses, as is suggested by new clinical studies. We will test whether tau protein aggregates "corrupt" protein on the cell interior through templated conformation change, whereby normally folded protein directly contacts aggregated forms. We will additionally test whether aggregates can move across synapses using a novel mouse model. PUBLIC HEALTH RELEVANCE: Tauopathies afflict millions of Americans, and hundreds of millions of individuals worldwide. As our population ages, the incidence of these diseases will rise acutely. There is no cure, and we lack sufficient mechanistic understanding of pathogenesis to design well-targeted therapies. This research will help solve these problems, as it is focused on understanding the cellular mechanisms that influence pathogenesis.

DESCRIPTION (provided by applicant): This is a proposal to develop a retinal model of neurodegeneration that will ultimately enable much more rapid and efficient evaluation of chemical modifiers of disease than is currently possible. We are carrying out proof-of-concept experiments using the huntingtin (Htt) protein, which causes Huntington disease when it contains an elongated tract of glutamines. The significance of this work is high, because if we are successful we will show that it is possible to isolate a discrete portion of the CNS for chemical and genetic studies, using anatomy, physiology, and function. This could help bridge cellular studies of neurodegeneration, which can rapidly generate much data, with whole animal studies, which are expensive and time consuming. The retinal model allows independent measurement of function in each eye, enabling internal controls within an animal that vastly improve statistical power. In preliminary work, we have shown that it is possible to detect retinal degeneration in the R6/2 mouse model of HD. We have further demonstrated that this is associated with progressive loss of function in tests of retinal physiology (electroretinography). We have shown that it is possible to deliver a disease-modifying compound in tiny amounts using liposome-mediated drug delivery, and to observe a beneficial effect on retinal physiology. We have also developed a behavior assay that lets us independently measure vision in the right vs. left eye of an unrestrained mouse. Finally, we have shown that it is possible to transduce large numbers of retinal neurons after a single injection of a modified adeno-associated virus. This work puts us in an excellent position to test whether we can build a model of retinal degeneration based on expression of various Htt proteins with mutations of broad interest to the HD field. Aim 1: Characterize viral transduction of Htt proteins within the retina. We will use AAV2 to express various forms of Htt and determine their effects on retinal neurons over time. Aim 2: Use ERG to measure effects on retinal physiology and compare mutants. We will use electroretinography to measure effects of various Htt proteins on retinal physiology. Aim 3. Use optomotry to measure effects on retinal neuron function and compare mutants. We will use a specialized behavior assay to measure the effects of various Htt proteins on visual function. The work is based on the use of recombinant adeno-associated virus, micro-injection of this virus into the intraocular space of wild-type mice, and the determination of the consequences of virus-mediated Htt gene expression within the eye. We will also use state-of-the-art techniques to monitor retinal pathology, physiology, and function. If successful, this work will set the stage for much more rapid in vivo analyses of chemical and genetic modifiers of neurodegeneration. This could have an enormous impact on human health, since neurodegenerative disease is one of the single biggest health problems faced by the United States. PUBLIC HEALTH RELEVANCE: It is very difficult to translate cellular studies of neurodegenerative disease into animal models. Cell models are very useful for identifying early drug leads that could be translated into patient use, as well as for the identification of genes that modify the neurodegenerative disease process. However, it is very difficult, time consuming, and expensive to evaluate experimental compounds in whole animals, and to test genetic modifiers. Evaluation of small molecules is difficult because it requires large amounts of compound, and any early compound must be safe, and easily administered, with good bioavailability. Evaluation of genetic modifiers is difficult because this requires creating hybrid mice that express multiple genes of interest. This is a proposal to develop a mouse retinal model of neurodegenerative disease that could be useful for evaluating experimental compounds and modifier genes. It is based on the idea that experimental compounds can be safely administered locally to each individual eye via injection, and the genetic modifications can be delivered by a single injection of a specialized virus. If we are successful, we will show that it is possible to direct expression of a disease-associated gene to the eye, and then use highly quantitative measures of visual function to determine the relative toxicity of various mutants. We will be studying huntingtin, the protein that causes Huntington disease, a devastating neurodegenerative condition. In Aim 1 we will create specialized viruses that allow us to efficiently express various forms of the huntingtin protein within the retina. We will determine that we are getting good expression, and will monitor animals over long periods to determine any effects of gene expression on the integrity of retinal neurons. In Aim 2 we will use a technique called electroretinography to measure the effects of the various forms of the huntingtin protein on the physiology of neurons in the mouse retina. We will correlate any changes with pathology observed in Aim 1. We will independently measure the physiologic changes in the left vs. right eyes of the animals. This will enable much more effective studies of relative effects of the various huntingtin mutations. In Aim 3 we will use a state-of-the-art behavior technique to measure the effects of huntingtin protein expression on visual acuity in the injected mice. This allows us to independently measure the function of the left vs. right retina, which enables much more effective studies of the various huntingtin mutations. Relevance to public health Neurodegenerative diseases represent an enormous cost to society, and afflict millions of people. There are no effective therapies, and the methods to develop new treatments and to study these diseases are relatively slow and cumbersome. If successful, this work will develop a new method to study neurodegenerative disease that could vastly improve our ability to develop new drugs and to study the basic mechanisms of disease. This will help speed the development of more effective treatments.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is a devastating neurodegenerative disease caused by CAG codon expansion in exon 1 of the huntingtin (htt) gene. Exon 1 spanning protein products, referred to as Httex1 are the major components of neuronal intranuclear inclusions that are the hallmarks of HD. Y-27632, a small molecule inhibitor of the rho-associated kinase (ROCK), was shown by the Diamond lab to reduce Httex1 aggregation in cells and ameliorate Httex1-mediated toxicity in Drosophila and mouse models. Serine-137 of profilin was established as the direct target of ROCK. Phospho-profilin does not reduce Httex1 aggregation whereas unphosphorylated profilin modulates Httex1 aggregation through direct interactions thus explaining the effect of Y-27632. We envisage a direct therapeutic approach that involves the design of molecules to mimic the effects of profilin. Such an approach requires a comprehensive understanding of the mechanisms by which profilin suppresses Httex1 aggregation, and this is the focus of our proposal. Our goal is to understand how profilin modulates the aggregation of exon 1 of huntingtin through interactions with its polyproline regions. Our approaches will include intracellular assays of aggregation and in vitro biophysical studies that combine fluorescence spectroscopies, electron paramagnetic resonance spectroscopy, and electron microscopy. The relevant entity for modulation of Httex1 aggregation by profilin is the 38-residue proline-rich stretch (C38) that is C-terminal to polyglutamine in Httex1. This stretch encompasses two polyproline modules that are connected via a 17-residue flexible linker. Profilin binds to the polyproline modules in C38. Our preliminary data show that in the presence of profilin, a higher total concentration of Htt-NTFs is required to form large spherical and fibrillar aggregates because profilin binds preferentially to smaller oligomeric species. Our preliminary data also establish that the apparent affinity of profilin for Httex1 constructs is higher when compared to C38 alone. This appears to be due to increased avidity that derives from oligomerization of Htt-NTFs in the M-phase. Avidity refers to the increased local concentration of C38 modules within oligomers. We will build on our preliminary data to uncover the mechanisms by which profilin binding impacts the phase behavior of disease- relevant N-terminal fragments of Httex1.