Research Description: Therapeutic Mechanisms in Human Disease

Effective treatment of many human diseases will derive from understanding and modulating protein interactions, modifications, and conformation changes. We are using biochemical and biophysical methods in conjunction with cell and animal models to study these processes, and to elucidate basic biological mechanisms of disease. We thus hope to develop highly specific therapies. We work in two broad areas: neurodegenerative disease (polyglutamine diseases and tauopathy), and the regulation of the androgen receptor (AR). AR is linked to development and progression of prostate cancer, in addition to its role in neurodegeneration in X-linked spinobulbar muscular atrophy (SBMA). We have made advances that are leading towards new mechanism-based therapies.

A. Polyglutamine Diseases

Polyglutamine diseases are devastating, dominantly inherited neurodegenerative conditions that are caused by CAG triplet repeat expansions within unrelated genes. CAG encodes the amino acid glutamine, and the affected proteins have enlarged tracts of this amino acid. An expanded glutamine tract is subject to a critical conformational shift that leads to a misfolded protein, and these diseases are characterized by insoluble intracellular inclusions comprised of the mutant proteins, particularly within vulnerable neurons. Huntington disease (HD) is caused by repeat expansion in the huntingtin (Htt) protein. SBMA derives from repeat expansion in AR, and causes progressive motor neuron degeneration and muscle weakness.

Several years ago we suggested that the transition from a normal to a toxic conformation is subject to cellular regulation. We used intracellular polyglutamine protein aggregation as a marker for this conformational shift, and designed a cellular assay based on fluorescence resonance energy transfer (FRET) to find genes and small molecules that influence this process. We identified a specific kinase inhibitor (Y-27632) that inhibits AR and Htt aggregation. Y-27632 inhibits the rho-associated kinase ROCK, which participates in the rearrangement of the actin cytoskeleton via phosphorylation of multiple downstream targets. It reduces polyglutamine toxicity in cell, Drosophila and mouse models of HD, suggesting that a ROCK inhibitor might ultimately be an effective therapy. We have determined that the actin binding protein profilin is the critical downstream ROCK target that controls Htt misfolding. We have further identified two phosphorylation sites on this protein that regulate its activity.

Current Projects in Polyglutamine Disease Pathogenesis

1. Profilin and Htt: We have identified actin and profilin as factors that might control the misfolding and aggregation of Htt. In collaboration with a computational biologist (Paolo Carloni, Ph.D.), we have developed a model for profilin, Htt and actin interactions. We are now testing predictions of this model using molecular biology and biochemistry to determine how the interactions of these proteins, and profilin phosphorylation, directly control Htt misfolding and aggregation in vitro. Finally, we are using behavioral, pathological, and biochemical studies in mouse models of HD to test the efficacy of ROCK inhibition, and the protective role of profilin.

2. Genetic Modifiers of HD Pathogenesis. The FRET-based cellular model of Htt aggregation has now been well validated as a predictor of genes and small molecules that modify Htt misfolding and toxicity. We are very interested in extending the use of this system in genetic screens (siRNA) to identify modifiers of misfolding. Further, our development of phospho-specific antibodies to profilin has enabled high-throughput methods to identify the spectrum of factors that regulate its phosphorylation.

3. Novel Mouse Models. Standard modeling of disease in transgenic mice is time consuming and expensive. We have developed a mouse retinal model of HD that allows independent measurement of neuronal physiology (via ERG) and function (via visual acuity) in each eye. This has created unique opportunities to study chemical and genetic modifiers of pathogenesis in vivo much more efficiently than standard approaches, since a modifier can be delivered unilaterally, using the contralateral eye as a control. We are currently evaluating profilin in this system, which will also be amenable to evaluating other factors identified by cellular studies and genetic screens.

B. Propagation of Protein Misfolding in Tauopathy.

Tauopathies are a large family of neurodegenerative diseases that feature misfolding and intracellular aggregation of the microtubule-associated protein tau, such as Alzheimer disease and frontotemporal dementia. In tauopathies a single protein accounts for a diversity of clinical phenotypes. In addition to other degenerative conditions (e.g. amyotrophic lateral sclerosis), tauopathies exhibit inexorable spread of pathology from one involved brain region to another, apparently based on neuronal connectivity and/or proximity. These features are quite reminiscent of prionopathies. We have observed that tau protein has key "prion-like" characteristics. It exhibits conformational diversity in vitro specified by templated conformational change; it propagates a misfolded state from the outside to the inside of a cell; and tau aggregates within a cell can transfer between cells in vitro. This work may establish a new, unifying paradigm to understand the propagation of protein misfolding in tauopathies and other neurodegenerative diseases associated with fibrillar proteins.

Current Projects in Tauopathy

1. The role for Intracellular Tau: Prions cannot cause toxicity unless a normal form of the protein is expressed. We are testing whether this is also true for tau by using cell and animal approaches. We are determining whether the presence of intracellular tau renders a cell vulnerable to toxicity from tau aggregates, and we are testing whether extracellular tau aggregates transmit a specific conformation to the intracellular protein.

2. Molecular Mechanisms of Cell Entry and Cell-Cell Propagation: Tau aggregates are rapidly taken up by cells, and transfer between co-cultured cells. The mechanism is not clear, however. We are using a cell-based assay of tau uptake to dissect the molecular mechanisms of endocytosis of tau fibrils, whether the process is specific for tau, and whether it also accounts for uptake of other types of fibrillar protein. The specific molecular mechanisms of this process this could provide novel drug targets. We are evaluating propagation of tau misfolding in vivo using directed expression of aggregation-prone or aggregation-resistant tau mutants within subsets of neurons in the brain, against the background of wild-type, tau knockout, or tau over-expression. We are interested in whether propagation of pathology can occur across synapses, as is predicted by human studies which indicate that degeneration occurs along neural networks.

C. The Androgen Receptor and Prostate Cancer.

In addition to its role in neurodegeneration in SBMA, AR is also the primary therapeutic target for prostate cancer, a leading cause of cancer death in males. We have created FRET-based systems to measure the conformation changes (both intra and intermolecular) that occur in AR after it binds its ligand, DHT. This has allowed us to study AR function using biophysical measures of conformation, as opposed to more indirect downstream events such as gene activation. We are identifying genes and small molecules that control AR ligand-induced conformational change, and which may be relevant for treating prostate cancer.

Current Projects for AR

1. Novel Therapeutics for Prostate Cancer: Our high-throughput approaches have allowed us to identify several FDA-approved drugs and natural products that block ligand-induced conformational change in AR. These compounds function at low nanomolar concentrations and synergize with each other and with bicalutamide, the best available competitive antagonist. We have taken an early lead and modified it to create a new compound that is effective in vivo. We are now organizing toxicity studies that we hope will lead to early clinical trials in patients.

2. High-Throughput Screening: The development of a high-throughput system to detect AR conformation changes has allowed us to initiate chemical and genetic screens to identify small molecules that directly (via interaction with AR) or indirectly (via modulation of cell signaling pathways) modulate AR conformation. We are pursuing current chemical hits, and will analyze genes identified in these efforts to extend our understanding of the biology of AR activation, and the potential for new therapeutic approaches.