Chuan-En Wang

Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity

Huntington disease (HD) is characterized by the preferential loss of striatal medium-sized spiny neurons (MSNs) in the brain. Because MSNs receive abundant glutamatergic input, their vulnerability to excitotoxicity may be largely influenced by the capacity of glial cells to remove extracellular glutamate. However, little is known about the role of glia in HD neuropathology. Here, we report that mutant huntingtin accumulates in glial nuclei in HD brains and decreases the expression of glutamate transporters. As a result, mutant huntingtin (htt) reduces glutamate uptake in cultured astrocytes and HD mouse brains. In a neuron–glia coculture system, wild-type glial cells protected neurons against mutant htt-mediated neurotoxicity, whereas glial cells expressing mutant htt increased neuronal vulnerability. Mutant htt in cultured astrocytes decreased their protection of neurons against glutamate excitotoxicity. These findings suggest that decreased glutamate uptake caused by glial mutant htt may critically contribute to neuronal excitotoxicity in HD.

N-Terminal Mutant Huntingtin Associates with Mitochondria and Impairs Mitochondrial Trafficking

Huntingtons disease (HD) is caused by polyglutamine (polyQ) expansion in huntingtin (htt), a large (350 kDa) protein that localizes predominantly to the cytoplasm. Proteolytic cleavage of mutant htt yields polyQ-containing N-terminal fragments that are prone to misfolding and aggregation. Disease progression in HD transgenic models correlates with age-related accumulation of soluble and aggregated forms of N-terminal mutant htt fragments, suggesting that multiple forms of mutant htt are involved in the selective neurodegeneration in HD. Although mitochondrial dysfunction is implicated in the pathogenesis of HD, it remains unclear which forms of cytoplasmic mutant htt associate with mitochondria to affect their function. Here we demonstrate that specific N-terminal mutant htt fragments associate with mitochondria in Hdh(CAG)150 knock-in mouse brain and that this association increases with age. The interaction between soluble N-terminal mutant htt and mitochondria interferes with the in vitro association of microtubule-based transport proteins with mitochondria. Mutant htt reduces the distribution and transport rate of mitochondria in the processes of cultured neuronal cells. Reduced ATP level was also found in the synaptosomal fraction isolated from Hdh(CAG)150 knock-in mouse brain. These findings suggest that specific N-terminal mutant htt fragments, before the formation of aggregates, can impair mitochondrial function directly and that this interaction may be a novel target for therapeutic strategies in HD.

Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice.

Huntingtons disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin–proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

Differential Activities of the Ubiquitin-Proteasome System in Neurons versus Glia May Account for the Preferential Accumulation of Misfolded Proteins in Neurons

A variety of neurological disorders and polyglutamine (polyQ) diseases are caused by misfolded proteins. The common feature of these diseases is late-onset cellular degeneration that selectively affects neurons in distinct brain regions. polyQ diseases, including Huntingtons disease (HD), present a clear case of selective neurodegeneration caused by polyQ expansion-induced protein misfolding, which also leads to predominant inclusions in neuronal nuclei. It remains unclear how these ubiquitously expressed disease proteins selectively kill neurons. In HD, mutant huntingtin accumulates in both neurons and glia, but more neuronal cells display huntingtin aggregates. These aggregates colocalize with components of the ubiquitin-proteasome system (UPS), which plays a critical role in clearing misfolded proteins. Using fluorescent reporters that reflect cellular UPS activity, we found that UPS activity in cultured neurons and glia decreases in a time-dependent manner. Importantly, UPS activity is lower in neurons than in glia and also lower in the nucleus than the cytoplasm. By expressing the UPS reporters in glia and neurons in the mouse brain, we also observed an age-dependent decrease in UPS activity, which is more pronounced in neurons than glial cells. Although brain UPS activities were similar between wild-type and HD 150Q knock-in mice, inhibiting the UPS markedly increases the accumulation of mutant htt in cultured glial cells. These findings suggest that the lower neuronal UPS activity may account for the preferential accumulation of misfolded proteins in neurons, as well as their selective vulnerability.

Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin

Mutant huntingtin accumulates in the neuronal nuclei and processes, which suggests that its subcellular localization is critical for the pathology of Huntingtons disease (HD). However, the contribution of cytoplasmic mutant huntingtin and its aggregates in neuronal processes (neuropil aggregates) has not been rigorously explored. We generated an intracellular antibody (intrabody) whose binding to a unique epitope of human huntingtin is enhanced by polyglutamine expansion. This intrabody decreases the cytotoxicity of mutant huntingtin and its distribution in neuronal processes. When expressed in the striatum of HD mice via adenoviral infection, the intrabody reduces neuropil aggregate formation and ameliorates neurological symptoms. Interaction of the intrabody with mutant huntingtin increases the ubiquitination of cytoplasmic huntingtin and its degradation. These findings suggest that the intrabody reduces the specific neurotoxicity of cytoplasmic mutant huntingtin and its associated neurological symptoms by preventing the accumulation of mutant huntingtin in neuronal processes and promoting its clearance in the cytoplasm.

Inhibiting the ubiquitin–proteasome system leads to preferential accumulation of toxic N-terminal mutant huntingtin fragments

An expanded polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt) causes misfolding and accumulation of htt in neuronal cells and the subsequent neurodegeneration of Huntingtons disease (HD). Clearing the misfolded htt is critical for preventing neuropathology, and this process is mediated primarily by both the ubiquitin-proteasome system (UPS) and autophagy. Although overexpression of mutant htt can inhibit UPS activity in cultured cells, mutant htt does not inhibit global UPS activity in the brains of HD transgenic mice. These findings underscore the importance of investigating the function of the UPS and autophagy in the brain when mutant proteins are not overexpressed. When cultured PC12 cells were treated with either UPS or autophagy inhibitors, more N-terminal mutant htt fragments accumulated via inhibition of the UPS. Furthermore, in HD CAG repeat knock-in mouse brain, inhibiting the UPS also resulted in a greater accumulation of N-terminal, but not full-length, mutant htt than inhibiting autophagy did. Our findings suggest that impairment of the UPS may be more important for the accumulation of N-terminal mutant htt and might therefore make an attractive therapeutic target.

Polyglutamine Expansion Reduces the Association of TATA-binding Protein with DNA and Induces DNA Binding-independent Neurotoxicity

TATA-binding protein (TBP) is essential for eukaryotic gene transcription. Human TBP contains a polymorphic polyglutamine (polyQ) domain in its N terminus and a DNA-binding domain in its highly conserved C terminus. Expansion of the polyQ domain to >42 glutamines typically results in spinocerebellar ataxia type 17 (SCA17), a neurodegenerative disorder that resembles Huntington disease. Our recent studies have demonstrated that polyQ expansion causes abnormal interaction of TBP with the general transcription factor TFIIB and induces neurodegeneration in transgenic SCA17 mice (Friedman, M. J., Shah, A. G., Fang, Z. H., Ward, E. G., Warren, S. T., Li, S., and Li, X. J. (2007) Nat. Neurosci. 10, 1519–1528). However, it remains unknown how polyQ expansion influences DNA binding by TBP. Here we report that polyQ expansion reduces in vitro binding of TBP to DNA and that mutant TBP fragments lacking an intact C-terminal DNA-binding domain are present in transgenic SCA17 mouse brains. polyQ-expanded TBP with a deletion spanning part of the DNA-binding domain does not bind DNA in vitro but forms nuclear aggregates and inhibits TATA-dependent transcription activity in cultured cells. When this TBP double mutant is expressed in transgenic mice, it forms nuclear inclusions in neurons and causes early death. These findings suggest that the polyQ tract affects the binding of TBP to promoter DNA and that polyQ-expanded TBP can induce neuronal toxicity independent of its interaction with DNA.

Differential ubiquitination and degradation of huntingtin fragments modulated by ubiquitin-protein ligase E3A

Significance Ubiquitination of misfolded proteins is an important process in a variety of age-dependent neurodegenerative diseases including Alzheimer’s disease, Parkinson disease, and Huntington disease (HD). However, we know little about how ubiquitination regulates the degradation and accumulation of misfolded proteins. By using HD knock-in mice and cultured cells, we found that the pathogenic form of the HD protein is differentially ubiquitinated via K48 and K63 in ubiquitin. K48-mediated ubiquitination promotes huntingtin (Htt) degradation while K63-mediated ubiquitination accelerates its aggregation. The K-48–mediated ubiquitination depends on Ube3a, whose expression declines in aged brain. Overexpression of Ube3a in old HD KI mouse brain can reduce mutant Htt accumulation and aggregation. The age-dependent decline in Ube3a may contribute to the late-onset neuropathology in HD. Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin and alters the levels of toxic proteins. In Huntington disease, polyglutamine expansion causes N-terminal huntingtin (Htt) to misfold, inducing neurodegeneration. Here we report that shorter N-terminal Htt fragments are more stable than longer fragments and find differential ubiquitination via K63 of ubiquitin. Aging decreases proteasome-mediated Htt degradation, at the same time increasing K63-mediated ubiquitination and subsequent Htt aggregation in HD knock-in mice. The association of Htt with the K48-specific E3 ligase, Ube3a, is decreased in aged mouse brain. Overexpression of Ube3a in HD mouse brain reduces K63-mediated ubiquitination and Htt aggregation, enhancing its degradation via the K48 ubiquitin–proteasome system. Our findings suggest that aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt.

Preferential accumulation of N-terminal mutant huntingtin in the nuclei of striatal neurons is regulated by phosphorylation

An expanded polyglutamine tract (>37 glutamines) in the N-terminal region of huntingtin (htt) causes htt to accumulate in the nucleus, leading to transcriptional dysregulation in Huntington disease (HD). In HD knock-in mice that express full-length mutant htt at the endogenous level, mutant htt preferentially accumulates in the nuclei of striatal neurons, which are affected most profoundly in HD. The mechanism underlying this preferential nuclear accumulation of mutant htt in striatal neurons remains unknown. Here, we report that serine 16 (S16) in htt is important for the generation of small N-terminal fragments that are able to accumulate in the nucleus and form aggregates. Phosphorylation of N-terminal S16 in htt promotes the nuclear accumulation of small N-terminal fragments and reduces the interaction of N-terminal htt with the nuclear pore complex protein Tpr. Mouse brain striatal tissues show increased S16 phosphorylation and a decreased association between mutant N-terminal htt and Tpr. These findings provide mechanistic insight into the nuclear accumulation of mutant htt and the selective neuropathology of HD, revealing potential therapeutic targets for treating this disease.

Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

Parkinsons disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2–3, 7–8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD.