Construction And Functional Analysis Of Eukaryotic Expression Vector Of IT15 Gene Fragment In Huntington's Disease

[Background and objective] Huntington disease is an autosomal dominant neurodegenerative disorder that is characterized by motor dysfunction, cognitive decline and psychiatric disturbance. It is caused by an unstable expansion in the number of CAG trinucleotide repeat in exon 1 of the IT15 gene on chromosome 4, which encodes huntingtin. In the nomal population, the number of CAG repeats varies from 6 to 35, but length of 37 and over invariably cause HD. There is currently no treatment to prevent or palliate the progress of HD. The striatum is the primary area of neuronal degeneration in HD. The exact cause of the selective neuronal death in HD remains unknown. A crucial step in the pathogenesis of this disorder is the progressive truncation of mutant huntingtin resulting in nuclear location and aggregate formation. Mutant huntingtin is cleaved to form N-terminal fragments containing the polyglutamine repeats, which are believed to be the toxic species found in aggregates. Accordingly, the pathogenesis of HD is frequently modeled with exon 1 fragments, which cause toxicity and aggregate in cell models and in vivo. The mechanism by which mutant huntingtin accumulates intranuclearly is no known yet. Cornett found that the first 17 amino acids of Htt can interact withnuclear pore protein TPR (translocated promoter region), which involved in nuclear export. PolyQ expansion and aggregation decrease this interaction and increase the nuclear accumulation of huntingtin. Transcriptional dysregulation and loss of function of transcriptional coactivator proteins have been implicated in the pathogenesis of the disease. The N-terminal fragments of mutant huntingtin directly bind the acetyltransferase domains of CREB-bingding protein (CBP) and inhibit the acetyltransferase activity of CBP. Acetylation and deacetylation of histone proteins regulate transcription by modifying the nucleosomal assembly. HDAC inhibitor sodium butyrate reduced neuronal death in drosophila and transgenic mice HD models.We had genetically confirmed 5 HD patients from a Chinese family. A 18-year-old patient had an expanded CAG allele of 68 repeats in the FT 15 gene. To study the pathogenesis of Huntington disease, we transfected SH-SY5Y neurons and Hela cells with plasmid constructs containing exonl of IT 15 gene of the HD patient with reporter protein EGFP. We investigated the effects of sodium butyrate on the transiently transfected cells and the mechanism in N-terminal huntingtin nuclear-endoplasmic trafficking.[Method] Genomic DNA from the HD patient who had 68 CAG repeats and a nomal family member who had 17 CAG repeats was used to amplify codons 1 to 53 inclusive of IT 15 exonl ( numbering applies to huntingtin with 19Q residues -equivalent to 17 uninterrupted CAG repeats ). We used primers HD1 5'- gcg cag ate tat ggc gac cct gga aaa gc-3' and HD2 5'- gcg cga att egg egg ctg agg aag ctg agg a -3', which have Bglll and EcoR I restriction sites incorporated to allow cloning into pEGFP-Cl. The human neuroblastoma SH-SY5Y and Hela cells were transiently transfected with GFP-tagged exonl of human Huntingtin (GFP-Htt-19Q and GFP-mHtt-70Q). The cells were observed with fluorescence microscopy. Western blot was used to detect the expression of the fusion protein. The HDAC inhibitorsodium butyrate was added to the culture medium of SH-SY5Y cells after transfection. MTT assay and flow cytometric analysis were used to measure SH-SY5Y cell viability and cell death ratio. The transfected Hela cells were treatedby Leptomycin B for 2 hours then these cells were observed by inverted fluorescence1 microscope. A plasmid encoding N-terminal huntingtins with aa M deletion(GFP-Htt-19Q-Sl) was constructed from gDNA of the normal family member by a PCR using the primers 5'- gcg cag ate tgc gac cct gga aaa gc -3' and 5'- gcg cga art egg egg ctg agg aag ctg agg a -3'. A plasmid encoding N-terminal huntingtins with1 3with aa MAT deletion (GFP-Htt-19Q-S3) was constructed from gDNA of the normal family member by a PCR using the primers 5'- gcg cag ate tct gga aaa get gat g -3'and 5'- gcg cga att egg egg ctg agg aag ctg agg a -3'. A plasmid encoding1 5N-terminal huntingtins with MATLE deletion (GFP-Htt-19Q-S5) was constructed from gDNA of the normal family member by a PCR using the primers 5'- gcg cag ate taa get gat gaa ggc c -3' and 5'- gcg cga att egg egg ctg agg aag ctg agg a -3'. A1 7plasmid encoding N-terminal huntingtins with MATLEKL deletion (GFP-Htt-19Q-S7) was constructed from gDNA of the normal family member by a PCR using the primers 5'- gcg cag ate tat gaa ggc ctt cga gtc cct caa gtc ctt c -3' and 5'- gcg cga att egg egg ctg agg aag ctg agg a -3'. Hela cells were transiently transfected with GFP-Htt-19Q-Sl and GFP-Htt-19Q-S3 and GFP-Htt-19Q-S5 and GFP-Htt-19Q-S7. The subcellular localization of these N-terminal huntingtin fragments was observed by fluorescence microscope.[Results] The recombinant eukaryotic expression vectors of IT 15 gene fragment were successfully constructed. Both GFP-Htt-19Q and GFP-mHtt-70Q were primarily localized in the cytoplasm of transfected SH-SY5Y cells or Hela cells initially. Some cells transfected with GFP-mHtt-70Q had nuclear or perinuclearaggregates that changed with time. Western blotting of SH-SY5Y cells transfected with GFP-Htt-19Q and GFP-mHtt-70Q demonstrated that GFP-Htt-19Q had an apparent molecular mass of 34 KDa as expected and GFP-mHtt-70Q had an apparent molecular mass of 40 KDa as expected. Expression of N-terminal mutant huntingtin caused cell death in SH-SY5Y cells. Sodium butyrate mitigated SH-SY5Y cells death induced by N-terminal mutant huntingtin with a maximum effect at 0.5mmol/l. Sodium butyrate did not attenuate mutant huntingtin aggregates formation. Addition of leptomycin B to Hela cells expressing N-terminal nomal huntingtin caused the fluorescence to shift from the cytoplasm to the nucleus. Addition of leptomycin B to Hela cells expressing N-terminal mutant huntingtin increased nuclear mutant huntingtin accumulation. Fluorescence was localized predominantly within the cytoplasm in the GFP-Htt-19Q-Sl transfected Hela cells and GFP-Htt-19Q-S3 transfected Hela cells. In contrast, fluorescence in GFP-Htt-19Q-S5 transfected Hela cells and in GFP-Htt-19Q-S7 transfected Hela cells were observed throughout both the cytoplasm and the nucleus.[Conclusions] We constructed eukaryotic expression vectors of IT 15 gene fragment. We developed a cell culture model of HD. This model will be useful for future experiments to test mechanism of aggregation and toxicity and potentially for testing experimental therapeutic interventions. Sodium butyrate can directly mitigate SH-SY5Y cells death induced by N-terminal mutant huntingtin. Our finding strengthen the hypothesis that transcriptional dysfunction plays a role in the pathogenesis of HD and suggest that therapies aimed at modulating transcription may target early pathological events. Sodium butyrate did not attenuate mutant huntingtin aggregates formation. Neurons death does not correlate with the formation of aggregates. The N-terminal huntingtin can shuttle between the cytoplasm and nucleus. The export of N-terminal huntingtin from nucleus to cytoplasm may utilize1 5the CRM-1/exportin pathway. N-terminal huntingtins with aa MATLE deletion abrogated the nuclear export property. We presume that N-terminal amino acid4 17sequence of huntingtin, LEKLMKAFESLKSF , involved nuclear export. Defectivenuclear export, rather than nuclear import, of N-terminal huntingtin may beimportant for its nuclear pathology.