Although huntingtin (htt) can be cleaved at many sites by caspases, calpains, and aspartyl proteases, amino acid (aa) 552 was defined as a preferred site for cleavage in human Huntington disease (HD) brains in vivo. To date, the normal function of wild-type N-terminal htt fragment 1-552 aa (htt552) and its pathological roles of mutant htt552 are still unknown. Although mutant htt (mhtt) is also expressed in astrocytes, whether and how mhtt contributes to the neurodegeneration through astrocytes in HD remains largely unknown. In this study, a glia HD model, using an adenoviral vector to express wild-type htt552 (htt552-18Q) and its mutation (htt552-100Q) in rat primary cortical astrocytes, was generated to investigate the influence of htt552 on the transcription of brain-derived neurotrophic factor (BDNF). Results from enzyme linked immunosorbent assay showed that the level of BDNF in astrocyte-conditioned medium was decreased in the astrocytes expressing htt552-100Q. Quantitative real-time polymerase chain reaction demonstrated that htt552-100Q reduced the transcripts of the BDNF III and IV, hence, repressed the transcription of BDNF. Furthermore, immunofluorescence showed that aggregates formed by htt552-100Q entrapped transcription factors cAMP-response element-binding protein and stimulatory protein 1, which might account for the reduction of BDNF transcription. These findings suggest that mhtt552 reduces BDNF transcription in astrocytes, which might contribute to the neuronal dysfunction in HD.

Huntington Disease (HD) is caused by an abnormal expansion of polyQ tract in the protein named huntingtin (Htt). HD pathology is featured by accumulation and aggregation of mutant Htt in striatal and cortical neurons. Aberrant Htt degradation is implicated in HD pathogenesis. The aim of this study was to investigate the regulatory role of chaperone-mediated autophagy (CMA) components, heat shock protein cognate 70 (Hsc70) and lysosome-associated protein 2A (LAMP-2A) in degradation of Htt fragment 1-552aa (Htt-552). A cell model of HD was produced by overexpression of Htt-552 with adenovirus. The involvement of CMA components in degradation of Htt-552 was determined with over-expression or silencing of Hsc70 and LAMP-2A. The results confirmed previous reports that both macroautophagy and CMA were involved in degradation of Htt-552. Changing the levels of CMA-related proteins affected the accumulation of Htt-552. The lysosomal binding and luminal transport of Htt-552 was demonstrated by incubation of Htt-552 with isolated lysosomes. Expansion of the polyQ tract in Htt-552 impaired its uptake and degradation by lysosomes. Mutation of putative KFERQ motif in wild-type Htt-552 interfered with interactions between Htt-552 and Hsc70. Endogenous Hsc70 and LAMP-2A interacted with exogenously expressed Htt-552. Modulating the levels of CMA related proteins degraded endogenous full-length Htt. These studies suggest that Hsc70 and LAMP-2A through CMA play a role in the clearance of Htt and suggest a novel strategy to target the degradation of mutant Htt.

Huntington's disease (HD) is an inheritable neurological disorder caused by an abnormal expansion of the polyglutamine tract in the N-terminus of the protein huntingtin (htt). Mutant htt (mhtt) leads to selective neurodegeneration that preferentially affects striatal medium spiny neurons. Although mhtt is also expressed in astrocytes, whether and how astrocyte derived mhtt contributes to the neurodegeneration in HD remains largely unknown. In this study, a glia HD model, using an adenoviral vector to express wild-type and mutant N-terminal huntingtin fragment 1–552 aa (htt552) in rat primary cortical astrocytes, was generated. The influence of htt552 on the protein level of brain-derived neurotrophic factor (BDNF) in astrocytes was evaluated. Immunofluorescence showed that htt552-100Q formed aggregates in some astrocytes. These mhtt aggregates sequestered clathrin immunoreactivities and dispersed the Golgi complex. ELISA and immunofluorescence demonstrated an increase in BDNF levels in the astrocytes expressing htt552-100Q. Western blot analysis showed that there was an increase in pro-BDNF, but a decrease in mature BDNF in the astrocytes expressing htt552-100Q. Furthermore, medium collected from astrocytes expressing htt552-100Q showed a lower level of mature BDNF and less activity in supporting neurite development of primary cortical neurons. These results suggest that aggregates formed by mutant htt552 affect processing and secretion of the BDNF in astrocytes, which might contribute to the neuronal dysfunction and degeneration in HD.

To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552). Methods: Primary astrocyte cultures were prepared from the cortex of postnatal rat pups. An astrocytes model of Huntington's disease was established using the astrocytes infected with adenovirus carrying coden gene of N-terminal 552 residues of Huntingtin. The protein levels of glutamate transporters GLT-1 and GLAST, the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the autophagy substrate p62 in the astrocytes were examined using Western blotting. The mRNA expression levels of GLT-1 and GLAST in the astrocytes were determined using Real-time PCR. 3H]glutamate uptake by the astrocytes was measured with liquid scintillation counting. The expression of mutant Htt-552 in the astrocytes significantly decreased both the mRNA and protein levels of GLT-1 but not those of GLAST. Furthermore, Htt-552 significantly reduced 3H]glutamate uptake by the astrocytes. Treatment with the autophagy inhibitor 3-MA (10 mmol/L) significantly increased the accumulation of mutant Htt-552, and reduced the expression of GLT-1 and 3H]glutamate uptake in the astrocytes. Treatment with the autophagy stimulator rapamycin (0.2 mg/mL) significantly reduced the accumulation of mutant Htt-552, and reversed the changes in GLT-1 expression and 3H]glutamate uptake in the astrocytes. Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.

The development of a reliable PCR assay for genetic testing of spinocerebellar ataxia type 3. Touchdown PCR conditions were tested and different primer sets were evaluated with genomic DNA from blood sample of patients suffering from spinocerebellar ataxia type 3 (SCA3). An improved PCR assay was developed with a new set of primers and using the optimized touchdown PCR protocol. This new assay had been successfully employed in the screening of one identificated SCA3 family. Results from the present study document a simple and reliable PCR assay for genetic testing of SCA3. Strategies used in the present study may find applications in the optimization of PCR assay for triplet expansion with GC rich in the sequence context.

Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the Hdh Q111/Q111 knock-in mouse.

Huntington's disease (HD) is an autosomal dominant neurological disease. It is a fatal neurological disorder affecting 5–10 out of 10 000 people. While there are intensive research efforts focusing on uncovering molecular mechanisms of the pathogenesis of HD, a number of studies have begun to look for effective therapies for HD. There is a large body of encouraging news on novel therapeutic developments. The present paper reviews drugs used for symptomatic treatment of HD and experimental therapies targeting HD molecular pathology.

Huntington's disease (HD) is caused by an increase in the length of the poly(Q) tract in the huntingtin (Htt) protein, which changes its solubility and induces aggregation. Aggregation occurs in two general phases, nucleation and elongation, and agents designed to block either phase are being considered as potential therapeutics. We demonstrate that inclusion formation can be retarded by introducing modified, single-stranded oligonucleotides into a model neuronal cell line. This cell-based assay is used in conjunction with a standardized biochemical assay to identify molecules that can disrupt the process of aggregate formation. Active oligonucleotides include a 6-mer containing a single phosphorothioate linkage on each terminus, a 53-mer and a 9-mer containing three phosphorothioate linkages at each end, and a 25-mer consisting of all modified RNA residues. The disruption process directed by the active oligonucleotides appears to be independent of sequence specificity and complementarity. In contrast, the activity is more dependent on the type of chemical modifications contained within the oligonucleotide. Some oligonucleotides that demonstrated inhibition activity were also found to extend the life span of PC12 cells after the toxic Htt aggregation process was induced. Our data provide the first evidence that short synthetic oligonucleotides inhibit a fundamental pathological pathway of HD and may provide the basis for a novel therapeutic approach.