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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.
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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).
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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)
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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.
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