Early alterations of autophagy in Huntington disease-like mice

In a recent study, we reported in vivo evidence of early and sustained alterations of autophagy markers in a novel knock-in mouse model of Huntington disease (HD). The novel model is derived from selective breeding of HdhQ150 knock-in mice to generate mice with ~200 CAG/polyglutamine repeats (HdhQ200). HdhQ200 knockin mice exhibit an accelerated and more robust motor phenotype than the parent line with detectable abnormalities at 50 weeks and substantial impairments at 80 weeks. Heterozygous HdhQ200 knock-in mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age followed by striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Increased LC3-II protein expression is noted at 9 weeks and sustained throughout the disease course, and is paralleled by increased expression of p62. Early and sustained expression of: autophagy-related proteins in this genetically precise mouse model of HD suggests that alteration of autophagic flux is an important and early component of neuronal response to polyglutamine expanded huntingtin.     

Hsp70 and Hsp40 chaperones do not modulate retinal phenotype in SCA7 mice

Nine neurodegenerative diseases, including spinocerebellar ataxia type 7 (SCA7), are caused by the expansion of polyglutamine stretches in the respective disease-causing proteins. A hallmark of these diseases is the aggregation of expanded polyglutamine-containing proteins in nuclear inclusions that also accumulate molecular chaperones and components of the ubiquitin-proteasome system. Manipulation of HSP70 and HSP40 chaperone levels has been shown to suppress aggregates in cellular models, prevent neuronal death in Drosophila, and improve to some extent neurological symptoms in mouse models. An important issue in mammals is the relative expression levels of toxic and putative rescuing proteins. Furthermore, overexpression of both HSP70 and its co-factor HSP40/HDJ2 has never been investigated in mice. We decided to address this question in a SCA7 transgenic mouse model that progressively develops retinopathy, similar to SCA7 patients. To co-express HSP70 and HDJ2 with the polyglutamine protein, in the same cell type, at comparable levels and with the same time course, we generated transgenic mice that express the heat shock proteins specifically in rod photoreceptors. While co-expression of HSP70 with its co-factor HDJ2 efficiently suppressed mutant ataxin-7 aggregation in transfected cells, they did not prevent either neuronal toxicity or aggregate formation in SCA7 mice. Furthermore, nuclear inclusions in SCA7 mice were composed of a cleaved mutant ataxin-7 fragment, whereas they contained the full-length protein in transfected cells. We propose that differences in the aggregation process might account for the different effects of chaperone overexpression in cellular and animal models of polyglutamine diseases.     

RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.     

Transgenic models of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion. A mouse model of this disease has been generated by the introduction of exon 1 of the human HD gene carrying highly expanded CAG repeats into the mouse germ line (R6 lines). Transgenic mice develop a progressive neurological phenotype with a movement disorder and weight loss similar to that in HD. We have previously identified neuronal inclusions in the brains of these mice that have subsequently been established as the pathological hallmark of polyglutamine disease. Inclusions are present before symptoms, which in turn occur long before any selective neuronal cell death can be identified. We have extended the search for inclusions to skeletal muscle, which, like brain, contains terminally differentiated cells. We have conducted an investigation into the skeletal muscle atrophy that occurs in the R6 lines, (i) to provide possible insights into the muscle bulk loss observed in HD patients, and (ii) to conduct a parallel analysis into the consequence of inclusion formation to that being performed in brain. The identification of inclusions in skeletal muscle might be additionally useful in monitoring the ability of drugs to prevent inclusion formation in vivo.     

E46K human alpha-synuclein transgenic mice develop Lewy-like and tau pathology associated with age-dependent, detrimental motor impairment

Synucleinopathies are a group of neurodegenerative disorders associated with the formation of aberrant amyloid inclusions composed of the normally soluble presynaptic protein α-synuclein (α-syn). Parkinson disease is the most well known of these disorders because it bears α-syn pathological inclusions known as Lewy bodies (LBs). Mutations in the gene for α-syn, including the E46K missense mutation, are sufficient to cause Parkinson disease as well as other synucleinopathies like dementia with LBs. Herein, we describe transgenic mice expressing E46K human α-syn in CNS neurons that develop detrimental age-dependent motor impairments. These animals accumulate age-dependent intracytoplasmic neuronal α-syn inclusions that parallel disease and recapitulate the biochemical, histological, and morphological properties of LBs. Surprisingly, the morphology of α-syn inclusions in E46K human α-syn transgenic mice more closely resemble LBs than the previously described transgenic mice (line M83) that express neuronal A53T human α-syn. E46K human α-syn mice also develop abundant neuronal tau inclusions that resemble neurofibrillary tangles. Subsequent studies on the ability of E46K α-syn to induce tau inclusions in cellular models suggest that both direct and indirect mechanisms of protein aggregation are probably involved in the formation of the tau inclusions observed here in vivo. Re-evaluation of presymptomatic transgenic mice expressing A53T human α-syn reveals that the formation of α-syn inclusions in mice must be synchronized; however, inclusion formation is diffuse within affected areas of the neuroaxis such that there was no clustering of inclusions. Collectively, these findings provide insights in the mechanisms of formation of these aberrant proteinaceous inclusions and support the notion that α-syn aggregates are involved in the pathogenesis of human diseases.     

Influence of species differences on the neuropathology of transgenic Huntington's disease animal models

Transgenic animal models have revealed much about the pathogenesis of age-dependent neurodegenerative diseases and proved to be a useful tool for uncovering therapeutic targets.Huntington's disease is ...     

Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease

Neurodegenerative disorders like Huntington's disease (HD) are characterized by progressive and putative irreversible clinical and neuropathological symptoms, including neuronal protein aggregates. Conditional transgenic models of neurodegenerative diseases therefore could be a powerful means to explore the relationship between mutant protein expression and progression of the disease. We have created a conditional model of HD by using the tet-regulatable system. Mice expressing a mutated huntingtin fragment demonstrate neuronal inclusions, characteristic neuropathology, and progressive motor dysfunction. Blockade of expression in symptomatic mice leads to a disappearance of inclusions and an amelioration of the behavioral phenotype. We thus demonstrate that a continuous influx of the mutant protein is required to maintain inclusions and symptoms, raising the possibility that HD may be reversible.     

Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The N-terminal fragment of AR containing the expanded polyglutamine tract aggregates in cytoplasm and/or in nucleus and induces cell death. Some chaperones such as Hsp40 and Hsp70 have been identified as important regulators of polyglutamine aggregation and/or cell death in neuronal cells. Recently, Hsp105alpha, expressed at especially high levels in mammalian brain, has been shown to suppress apoptosis in neuronal cells and prevent the aggregation of protein caused by heat shock in vitro. However, its role in polyglutamine-mediated cell death and toxicity has not been studied. In the present study, we examined the effects of Hsp105alpha on the aggregation and cell toxicity caused by expansion of the polyglutamine tract using a cellular model of SBMA. The transient expression of truncated ARs (tARs) containing an expanded polyglutamine tract caused aggregates to form in COS-7 and SK-N-SH cells and concomitantly apoptosis in the cells with the nuclear aggregates. When Hsp105alpha was overexpressed with tAR97 in the cells, Hsp105alpha was colocalized to aggregates of tAR97, and the aggregation and cell toxicity caused by expansion of the polyglutamine tract were markedly reduced. Both beta-sheet and alpha-helix domains, but not the ATPase domain, of Hsp105alpha were necessary to suppress the formation of aggregates in vivo and in vitro. Furthermore, Hsp105alpha was found to localize in nuclear inclusions formed by ARs containing an expanded polyglutamine tract in tissues of patients and transgenic mice with SBMA. These findings suggest that overexpression of Hsp105alpha suppresses cell death caused by expansion of the polyglutamine tract without chaperone activity, and the enhanced expression of the essential domains of Hsp105alpha in brain may provide an effective therapeutic approach for CAG repeat diseases.     

Polyglutamine-mediated neurodegeneration: Use of chaperones as prevention strategy

Polyglutamine diseases are a class of inherited neurodegenerative disorders caused by the expansion of a polyglutamine tract within the respective proteins. Clinical studies have revealed that the forming of neuronal intranuclear inclusions by the disease protein is a common pathological feature of polyglutamine diseases. Although there has been considerable progress in understanding polyglutamine diseases, many questions regarding their mechanism are still unanswered. The finding that molecular chaperones are associated with ubiquitinated intranuclear inclusions clearly indicates a crucial role of molecular chaperones in the generation of these fatal diseases. Molecular and chemical chaperones have been found to be a good agent for suppressing many polyglutamine diseases in several animal models. In this review, I discuss the roles of the ubiquitin-proteasome pathway and molecular chaperones in the development of polyglutamine diseases and probable approach for the prevention of many of these fatal disorders using molecular chaperones as a therapeutic agent. Newly found chemical chaperones have been demonstrated to be potentially useful and could be used as a therapeutic strategy in preventing many versions of polyglutamine diseases.     

Increased expression of p62 in expanded polyglutamine-expressing cells and its association with polyglutamine inclusions

Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60-kDa protein was increased in Neuro2a cells expressing the N-terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin-associated domain-containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine-expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine-expressing cells. We also found that p62 formed aggresome-like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock-down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine-induced stress rather than as a cross-linker between ubiquitylated proteins.     

HSP70 interacting protein prevents the accumulation of inclusions in polyglutamine disease1

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over‐expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co‐expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over‐expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.     

Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation

The inherited neurodegenerative diseases caused by an expanded glutamine repeat share the pathologic feature of intranuclear aggregates or inclusions (NI). Here in cell-based studies of the spinocerebellar ataxia type-3 disease protein, ataxin-3, we address two issues central to aggregation: the role of polyglutamine in recruiting proteins into NI and the role of nuclear localization in promoting aggregation. We demonstrate that full-length ataxin-3 is readily recruited from the cytoplasm into NI seeded either by a pathologic ataxin-3 fragment or by a second unrelated glutamine-repeat disease protein, ataxin-1. Experiments with green fluorescence protein/polyglutamine fusion proteins show that a glutamine repeat is sufficient to recruit an otherwise irrelevant protein into NI, and studies of human disease tissue and a Drosophila transgenic model provide evidence that specific glutamine-repeat–containing proteins, including TATA-binding protein and Eyes Absent protein, are recruited into NI in vivo. Finally, we show that nuclear localization promotes aggregation: an ataxin-3 fragment containing a nonpathologic repeat of 27 glutamines forms inclusions only when targeted to the nucleus. Our findings establish the importance of the polyglutamine domain in mediating recruitment and suggest that pathogenesis may be linked in part to the sequestering of glutamine-containing cellular proteins. In addition, we demonstrate that the nuclear environment may be critical for seeding polyglutamine aggregates.     

Curcumin enhances the polyglutamine-expanded truncated N-terminal huntingtin-induced cell death by promoting proteasomal malfunction

Formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various proteasome components is the major hallmark of the polyglutamine diseases. Curcumin is a polyphenolic compound having anti-inflammatory, anti-tumor, and anti-oxidative properties. Recently, curcumin has been reported to suppress the amyloid-beta accumulation, oxidative damage, and inflammation in the transgenic mice model of Alzheimer's disease. Here, we found that the treatment of curcumin increases the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-dependent cell death. Curcumin also causes rapid proteasomal malfunction in the mutant huntingtin expressing cells in comparison with normal glutamine repeat expressing cells. Finally, we show that N-acetyl cysteine (NAC), a potent antioxidant, reverted the curcumin-induced mutant huntingtin aggregation and proteasomal malfunction in the mutant huntingtin expressing cells. NAC also protects curcumin-induced cell death. Our result suggests that curcumin promotes mutant huntingtin-induced cell death by mimicking proteasomal dysfunction.     

Inhibition of proteasomal activity causes inclusion formation in neuronal and non-neuronal cells overexpressing Parkin

Association between protein inclusions and neurodegenerative diseases, including Parkinson's and Alzheimer's diseases, and polyglutamine disorders, has been widely documented. Although ubiquitin is conjugated to many of these aggregated proteins, the 26S proteasome does not efficiently degrade them. Mutations in the ubiquitin-protein ligase Parkin are associated with autosomal recessive juvenile Parkinsonism. Although Parkin-positive inclusions are not detected in brains of autosomal recessive juvenile Parkinsonism patients, Parkin is found in Lewy bodies in sporadic disease. This suggests that loss of Parkin ligase activity via mutation, or sequestration to Lewy bodies, is a contributory factor to sporadic disease onset. We now demonstrate that decreased proteasomal activity causes formation of large, noncytotoxic inclusions within the cytoplasm of both neuronal and nonneuronal cells overexpressing Parkin. This is not a general phenomenon as there is an absence of similar inclusions when HHARI, a structural homolog of Parkin, is overexpressed. The inclusions colocalize with ubiquitin and with proteasomes. Furthermore, Parkin inclusions colocalize with gamma-tubulin, acetylated alpha-tubulin, and cause redistribution of vimentin, suggesting aggresome-like properties. Our data imply that lower proteasomal activity, previously observed in brain tissue of Parkinson's disease patients, leads to Parkin accumulation and a concomitant reduction in ligase activity, thereby promoting Lewy body formation.     

Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such as Huntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survival in a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreased polyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse model of Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutamines and stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacy upon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutamine diseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutamine diseases.     

Expression of Expanded Polyglutamine Protein Induces Behavioral Changes in Drosophila (Polyglutamine-Induced Changes in Drosophila)

Spinocerebellar ataxia type-3 or Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disease caused by triplet nucleotide expansion. The expansion of the polyglutamine tract near the C terminus of the MJD1 gene product, ataxin-3, above a threshold of 40 glutamine repeats causes neuronal loss and degeneration. The expanded ataxin-3 forms aggregates, and nuclear inclusions, within neurons, possibly due to the misfolding of mutant proteins. Here we report upon the behavioral test changes related to truncated and expanded forms of MJD protein (MJDtr) in Drosophila, and show that expanded MJDtr, when expressed in the nervous system, causes characteristic locomotor dysfunction and anosmia. This phenomenon has not been previously reported in humans or in transgenic Drosophila models. In addition, the in vivo expression of the antiapoptotic gene bcl-2 showed no evidence of ameliorating the deleterious effect of MJDtr-Q78s, either in the eye or in the nervous system. The study shows that such Drosophila transgenic models express olfactory dysfunction and ataxic behavior as observed in human patients.