Muchowski Lab

Research

A growing number of neurodegenerative disorders are caused by mutations that result in altered protein conformations, often leading to a loss or gain of function for the mutant protein. For many of these diseases, structural alterations in the mutant proteins lead to the accumulation of insoluble, ubiquitinated protein aggregates in cellular structures called inclusion bodies that are found inside and sometimes outside of the affected cells. In many cases the aggregates adopt an ordered fibrillar structure rich in b sheet called amyloid, which has been implicated in the pathology of many human diseases, including Alzheimer’s disease (AD), Huntington’s Disease (HD) and Parkinson’s Disease (PD). The molecular basis for why accumulation of misfolded protein in the brain causes neurodegeneration is unresolved, and is actively being pursued in our laboratory using multiple independent approaches that span from molecular and cellular studies, to molecular genetic approaches in mouse models of disease. Several ongoing projects from our laboratory are described below.

Project 1: Elucidation of the structures and biological activities of huntingtin oligomers

Despite being a prominent feature of neurodegenerative disorders, surprisingly little is known about how and when altered protein conformations give rise to aberrant protein interactions that trigger pathological cascades. Recent studies have shown that the assembly of misfolded proteins into amyloid is a complex, multi-step process that is characterized by the population of transient or metastable non-fibrillar structures that include spherical and annular species. These oligomeric structures have been hypothesized to be potent neurotoxins in AD and other neurological diseases. Our goal is to use biochemical and biophysical techniques to elucidate the structures and biological activities of huntingtin oligomers, the protein that is mutated in HD. My laboratory has recently shown by atomic force microscopy and biochemical analyses that a mutant huntingtin fragment assembles into spherical and annular oligomeric species in addition to fibrillar structures (Fig. 1). We are now developing tools and reagents that will allow us to test in vivo the hypothesis that non-fibrillar oligomers are causal for triggering neurodegeneration in animal models of HD.

Figure 1. Atomic force microscopy (AFM) of polyglutamine aggegation reactions. Shown is a 3 m m 2 scan of a protein aggregation reaction analyzed by AFM at 20 m m huntingtin-53Q. Mature amyloid fibrils, as well as different types of spherical pre-fibrillar intermediates are observed.

Project 2: Mechanisms of neuroprotection by molecular chaperones

All cells and organelles possess a machinery of molecular chaperones whose function is to mediate the proper folding of other proteins and to insure that these proteins maintain their native conformations during conditions of stress. In addition to their functions in co- and post-translational folding, chaperones are required for the translocation of many proteins across cellular membranes, are involved in macromolecular assembly and disassembly, and facilitate the transfer of misfolded proteins to the proteasome for degradation (Fig. 2). Many chaperones are also components of signal transduction cascades that mediate transcriptional responses to stress, including those that trigger apoptosis. At a molecular level, chaperones are thought to function by binding transiently to exposed hydrophobic surfaces in a manner that is regulated by ATP-induced conformational changes. Chaperones were originally identified as proteins that are induced in response to heat shock (i.e., heat shock proteins). However, it is now appreciated that their elevated expression is observed during numerous types of stress (e.g. oxidative damage or treatment with heavy metals) and are therefore also commonly referred to as ‘stress’ proteins.

Chaperones are up-regulated and re-distribute to sites of aggregation in human neurodegenerative diseases associated with protein misfolding. Exciting recent studies suggest that over-expression of chaperones protects against neurodegeneration in fly and mouse models of HD and PD. How chaperones mediate protection against disease at the molecular and cellular levels (Fig. 3), and whether over-expression of chaperones can induce a protective effect in other protein misfolding diseases such as AD remain unresolved questions which are currently being examined in our laboratory. Our laboratory recently demonstrated by structural and biochemical methods that chaperones inhibit formation of spherical and annular oligomers formed by a mutant huntingtin fragment. Studies from our laboratory also demonstrated that the disease process in HD mice lacking molecular chaperones is greatly exacerbated in comparison to mice that express these protective proteins. Furthermore, similar to our test tube studies, we observed that molecular chaperones suppress formation of oligomeric protein aggregates in vivo. Thus, our studies confirm that the endogenous machinery of molecular chaperones can critically regulate neurodegeneration in vivo. As oligomers are proposed to mediate toxicity in animal models of HD and other disorders, these provocative results suggest that the identification of small molecules that can increase chaperone levels in vivo may have therapeutic benefit. Indeed, small molecules that turn on expression of molecular chaperones have shown much promise in mouse models of neurological diseases, and are currently being evaluated in clinical trials.

Figure 2. Molecular chaperones regulate several important cellular processes. Molecular chaperones facilitate protein folding and prevent protein aggregation. However, they also regulate several other cellular processes, such as autophagy,
vesicle fusion, signal transduction, apoptosis and proteasomal degradation. AIF, apoptosis-inducing factor; ER, endoplasmic
reticulum; ERAD, endoplasmic reticulum–associated degradation; HSF1, heat shock transcription factor 1; HSP, heat shock protein; LAMP, lysosomal-associated membrane protein; ROS, reactive oxygen species. Reproduced from Muchowski and Wacker, Nat. Rev. Neurosci., 6:11-22 (2005).

Project 3: Genetic analysis of toxicity due to protein misfolding in yeast and mice

The yeast Saccharomyces cerevisiae, which has been used to brew beer and wine for millennia, is a commonly used laboratory organism because numerous intracellular processes are well conserved between yeast and higher organisms, including humans. Studies in yeast have led to many significant, and often unexpected, advances in our knowledge of human diseases.

We recently used yeast as a model eukaryotic organism (Fig. 4) to test the hypothesis that the downstream targets and molecular mechanisms by which a mutant huntingtin fragment and a-synuclein mediate toxicity are unique. Using a genome-wide screening approach with 4,850 haploid yeast mutants containing deletions of nonessential genes, we identified 52 that are sensitive to a mutant huntingtin fragment, 86 that are sensitive to a-synuclein, and only one mutant that is sensitive to both. Genes that enhance huntingtin toxicity are enriched in the functionally related cellular processes of protein folding, response to stress and the ubiquitin degradation pathway, while genes that modify a-synuclein toxicity are enriched in vesicular transport and lipid metabolism pathways. In addition, we completed genome-wide screens to identify loss-of-function (LOF) mutations that suppress huntingtin or a-synuclein toxicity in yeast. The results from these screens are particularly appealing from a therapeutic standpoint, as pharmacological inhibition of the products of genes identified in these screens would presumably be beneficial in animal models of HD and PD.

We are now translating our studies from yeast into mouse models of HD and PD. More specifically, we have generated conditional mutations in mouse homologs of yeast genes identified in our genomic screens, and are evaluating the effects of these mutations on pathogenesis. Some of the LOF suppressor mutants identified in our screens encode proteins for which small molecule inhibitors exist, and we are now testing these molecules in cell and animal models of HD and PD. The long-term goal of these studies is to elucidate the genes, pathways and molecular mechanisms that are responsible for neurodegeneration in AD, HD and PD.

Figure 3. Yeast cells expressing huntingtin (green) and labeled with a vacuole marker (red)

Project 4: Analysis of the roles of microglia and the kynurenine pathway in neurodegeneration

In 2005 our lab reported the results from a large-scale genetic screen in yeast that identified mutations in 28 genes that suppress toxicity of mutant huntingtin. Among the most potent mutations identified in our screen was one in a gene that encodes kynurenine 3-monooxygenase (KMO). KMO functions in the kynurenine pathway (KP) of tryptophan degradation, and intrastriatal injection in rodents of a metabolite in this pathway called quinolinic acid (QUIN) reproduces behavioral and pathological features of HD, raising the possibility that alterations in QUIN metabolism may be central to HD pathophysiology. Indeed, metabolites in the KP are activated in early stage HD patients and in animal models of HD.

One of the most intriguing outcomes of our yeast study is that they implicate a cell type called microglia (the resident macrophages of the brain) in HD pathogenesis. Since KMO is expressed predominantly in microglia, and microglia are activated in HD patients and animal models, our results suggest that some of the toxic effects of mutant huntingtin may result from its expression in non-neuronal cell types. However, little is known about the role of microglia in HD pathophysiology. We hypothesize that mutant huntingtin induces a transcriptional defect that activates the KP in microglia, and that inhibiting the KP via pharmacological and genetic approaches will improve behavioral and pathological outcome measures in HD mouse models. Ongoing experiments in our lab will establish whether genetic or pharmacological inhibition of the KP is protective in mouse models of HD and other neurological disorders.

Figure 4. Co-culture studies are used in our lab to study the role of microglia on neuronal functions. Neurons are labeled red and activated microglia green in this image.

Figure 5. A model depicting the non-cell autonomous contribution by microglia to neuronal dysfunction in Huntington’s disease (HD). In this model, cell-autonomous expression of mutant huntingtin (Htt) in microglia causes dysfunction, perhaps by interactions with mitochondria or the mitochondrial membrane protein KMO, leading to upregulation of 3HK and QUIN synthesis, and thereby increased levels of ROS. The combined effects of ROS and NMDA receptormediated excitotoxicity by QUIN contribute to the dysfunction of neurons expressing mutant Htt. Other functional categories represented in our genomic screen, and previously implicated in HD, are highlighted in the dysfunctional neuron.