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 lab 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 lab 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 lab 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) (Wacker et al., Nat. Struct. Mol. Biol., 2004; Legleiter et al, J. Biol. Chem., 2010). 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 mm² scan of a protein aggregation reaction analyzed by AFM at 20 mm 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 (Wacker et al., Nat. Rev. Neurosci., 2005). How chaperones mediate protection against disease at the molecular and cellular levels, 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 lab. Our lab recently demonstrated by structural and biochemical methods that chaperones inhibit formation of spherical and annular oligomers formed by a mutant huntingtin fragment (Wacker et al., Nat. Struct. Mol. Biol., 2004; Lotz et al, J. Biol. Chem., 2010). More recently, we showed that the chaperones Hsp70 and Hsp40 can associate with a specific subset of soluble oligomers (Lotz et al, J. Biol. Chem., 2010). Studies from our lab also demonstrated that the disease process in HD mice lacking molecular chaperones is greatly exacerbated in comparison to mice that express these protective proteins (Wacker et al., J. Neurosci., 2009). 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 lab 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. 3) 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 (Willingham et al., Science, 2003). 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 (Giorgini et al., Nature Genetics, 2005). 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: Dissecting the role of the kynurenine pathway in health and disease

Metabolites in the kynurenine pathway (KP), generated by tryptophan degradation, are thought to play an important role in neurodegenerative disorders, including Alzheimer’s and Huntington’s diseases. In these disorders, glutamate receptor-mediated excitotoxicity and free radical formation have been correlated with increased levels of the toxic tryptophan metabolites 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN), and decreased levels of the neuroprotective metabolite kynurenic acid (KYNA). 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 (Giorgini et al., Nature Genetics, 2005). Among the most potent mutations identified in our screen was one in a gene that encodes kynurenine 3-monooxygenase (KMO), an enzyme that plays a critical role in the KP.

To test the hypothesis that KMO and the KP mediate neurodegeneration in mouse models, the Muchowski lab initiated a collaboration with Pauls’ father, a synthetic organic chemist with decades of experience in drug development, to develop novel small molecule inhibitors of KMO. We recently published a study that describes the synthesis and characterization of JM6, a small molecule prodrug inhibitor of KMO (Zwilling et al., Cell, 2011). Chronic oral administration of JM6 inhibits KMO in the blood, increasing KYNA levels and reducing extracellular glutamate in the brain (Fig. 4). In a transgenic mouse model of AD, JM6 prevents spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extends life span, prevents synaptic loss, and decreases microglial activation in a mouse model of HD. These findings support a critical link between tryptophan metabolism in the blood and neurodegeneration, and they provide a foundation for treatment of neurodegenerative diseases. The novel KMO inhibitors we generated are currently being tested in safety and toxicology assay in anticipation of clinical trials in humans.

Recent studies have now associated the KP as an important regulator of aging in model organisms, and our lab is actively pursuing studies to elucidate biological functions of the KP in health and diseases, and the molecular mechanisms that underlie the influence of the KP on neurodegeneration and aging.

Figure 4. A Model Illustrating the Mechanism by Which KMO Inhibition in Blood Cells Leads to Elevated Brain KYNA Levels and Neuroprotection. In neurodegenerative diseases like HD and AD, increased levels of the toxic kynurenine pathway metabolites 3-HK and QUIN and decreased levels of the neuroprotective pathway metabolite KYNA might contribute to increased glutamatergic neurotransmission, elevation of intracellular calcium levels, mitochondrial dysfunction, and ultimately neuronal dysfunction and cell death (inset labeled “Disease/Excitotoxicity”). We hypothesize that the biotransformation of JM6 to Ro 61-8048 in the gut (not shown) results in KMO inhibition in peripheral monocytes, causing the accumulation of both kynurenine (KYN) and KYNA in blood. Unlike KYNA, KYN is then actively transported into the brain, where it is rapidly converted by astrocytes to KYNA. KYNA released from astrocytes mediates neuroprotection, at least in part, by decreasing glutamate levels via antagonism of presynaptic α7 nicotinic acetylcholine receptors (inset labeled “Neuroprotection with JM6”). However, at high local concentrations, KYNA might also directly block glutamate receptors to reduce excitotoxicity. Neuroprotection by JM6 might also involve a decrease in inflammation and modulation of mitochondrial function (not shown). Reproduced from Zwilling et al., Cell (2011).

Project 5: Harnessing the immune system to suppress neurodegeneration.

All drugs that are currently used to treat brain diseases function by acting locally within the central nervous system (CNS). Yet many CNS penetrating drugs have serious undesirable side effects due to a lack of selectivity that have to be managed carefully. It has been known for many years that extensive communication occurs between the immune and nervous systems, and, based in part on our recent studies with JM6 (Zwilling et al., Cell, 2011), we hypothesize that manipulation of key signaling pathways in immune cells might be a unique approach to indirectly regulate CNS function with a high degree of specificity.

We are using cutting edge technologies, including in vivo 2-photon imaging and in vivo electrophysiology, to elucidate the molecular mechanisms that underlie how immune signaling systems modulate synaptic plasticity in the brain in healthy mice and disease models. Specifically, we are using genetic and pharmacological approaches using genetically engineered mice and novel small molecules we have recently generated, respectively, to manipulate signaling pathways in immune cells, and then are using the approaches described above to define how these manipulations are changing basic physiological properties of neurons. For example, after inhibition of KMO in blood cells we are examining electrophysiological correlates of learning and memory, such as long-term potentiation, in an effort to understand how the immune system is regulating the brain. Improving our understanding of the communication between the immune and nervous systems may ultimately lead to new approaches to treat brain diseases.