Effect Of Mutant Huntingtin On Expression Of Free Zinc And Zinc Transporter 3 And The Underlying Mechanism

Huntington's disease (HD) is an autonomic, dominantly inherited neurological disorder caused by a CAG expansion within the coding sequence of HD gene, which results in a long stretch of polyglutamine of huntingtin. Clinically, Huntington's disease is characterized by irrepressible motor dysfunction, psychiatric disturbances and cognitive deterioration to dementia. The pathological characteristic feature of HD is the dramatic regional neurodegeneration in the brain, which occur most prominently in the striatumand deep layers of the cerebral cortex. Studies have suggested that mutant huntingtin (mHtt) can cause synaptic dysfunction by altering the availability of various synaptic proteins and formation aggregates in axonal terminals. Mutant huntingtin has the obvious toxicity to synaptic function.Zinc is one of the essential trace elements in the biological functions of the mammals. Most of which zinc, about 85% of total content, is tightly bound to metalloenzymes where zinc is involved as a combination form and as the deposition of free zinc. A considerable amount of zinc, approximately 15% of total zinc is concentrated in synaptic vesicles as a pool of free or loosely bound zinc ions. Synaptic vesicles zinc, over 95% of free zinc exists in the synaptic vesicles, involved in synaptic transportation and synaptic activity. In synaptic activity, the zinc released into the synaptic cleft with other neurotransmitters and regulated receptors on the postsynaptic neurons. So the free zinc homeostasis has an important roal in the synaptic transmission. The free zinc couldn't pass the membrane through the passive diffusion and the free zinc trafficking is mediated by zinc transporters (ZnTs). Previously results demonstrated that ZnT3 is required for transport of zinc into synaptic vesicles and suggest that vesicular zinc concentration is determined by the abundance of ZnT3.Recent data have shown that vesicular Zinc, transported by ZnT3, was all changes in transgenic mice brainin of the neurodegenerative disease.However, free zinc and ZnT3 expression in HD have no reported. For this study, we characterized the association of mHtt with free zinc and its transporter ZnT3 and explored the mechanism of ZnT3 on the transcription dysfunction caused by mHtt. The results show that Mutant huntingtin reduces vesicular zinc level by inhibiting the binding of Spl to ZnT 3 promoter.Decrease in level of free zinc in the brain of HD transgenic mice. To investigate the levels of total zinc element in the TG mice, metal content was measured by flame atomic absorption spectrometry. To compare with the WT mice, in the hippocampus cortex and striatum regions the zinc element was reduced significantly in the HD mice. To determine whether the vesicular zinc is affected in the TG mice, autometallography (AMG) staining method (sodium sulfide solution perfusion) was performed to stain the free zinc. Importantly, zinc density in the hippocampus, striatum, lateral globus pallidus and substantia nigra was reduced marked in the HD mice compared with WT mice. These results indicated that zinc homeostasis was damaged by the mHtt and then caused synaptic dysfunction.Mutant Htt decreases expression of ZnT3. The free zinc couldn't pass the membrane through the passive diffusion. Studies have shown that vesicular zinc concentration is determined by the abundance of ZnT3. To investigate the ZnT3 mRNA and protein expression on the vesicle membranes, we performed the RT-PCR assay, immunohistochemical method and Western blotting analysis in the brain of TG mice and in the BHK cells transfected with pJRed-20 Q-Htt and pJRed-160 Q-Htt. Western blots analysis showed that the ZnT3 protein expression of cortex, hippocampus and striatum tissues from the 14W,18W and 20W HD mice was significantly reduced gradually compared with respectively WT mice. Moreover, the ZnT3 protein expression of the BHK cells transfected with pJRed-160 Q-Htt was decrease at the 24 h after the transfection and the 48 h and 72 h reduced seriously. Consistent with the ZnT3 protein expression, the mRNA level of ZnT3 in the HD mice and 160 Q/BHK cells was reduced marked. Immunohistochemical analysis observed that ZnT3 expression decreased in the hippocampus, striatum, medial and lateral globus pallidus and substantia nigra of TG mice at light-microscopic levels. Above results showed that the the mutant Htt suppressed the transcription of ZnT3 so that the free zinc and ZnT3 expression reduced significantly. Above results suggest that the free zinc levels reduced in the TG mice caused by the inhibition of the ZnT3 gene transcription disorders and expression reduced.Sp1 activates transcription of ZnT3 gene. The mHtt has been shown to suppress the activities of many transcription factors and-subsequently causes transcriptional dysfunction. To further explore the mechanism of ZnT3 mRNA suppression on the above results, we have to find out which ones were the transcription factors of ZnT3 gene. Bioinformatics analysis showed that the ZnT3 gene promoter region contains Spl, WT1+ KTS, WT1-KTS, NF-κB in p50, NF-κB of p65 and other transcription factors may be binding with the ZnT3 promoter sequence, in which the Sp1 has a higher score than the others. Western blot detection showed that over-expression of Sp1 to upregulate the expression of ZnT3 in the BHK cell lines and the other ranscription factors have no this effection on the expression of ZnT3. Furthermore, we found that ZnT3 expression increased after the BHK cells transfected with pEBGN-Sp1 and decreased after the Sp1 RNA inteference applied in BHK cells. According to the ZnT3 promoter sequence, there were two conserved Sp1 binding site (-269bp～-261bp,-184bp～-174bp) were named Spla and Splb. Dual Luciferase Reporter Gene Assay showed that pEBGN-Spl over-expression enhanced the pGL3-ZnT3(-283-+10) and pGL3-ZnT3(-193～+10) promoter activity. The ChIP results revealed that Spl is one of the transcriptional factors and Sp1 can combine with the ZnT3 promoter sequences at Sp1a and Sp1b sites directly.Mutant huntingtin reduces binding of Sp1 to ZnT3 promoter sequence. To investigate whether the mHtt suppress the ZnT3 expression by reducing the activity of ZnT3 promoter,we construct the plasmid of pGL3-ZnT3(-283～+10). And then,we inspected the dual luciferase activity after the co-transfected pJRed-160 Q-Htt and PGL3-ZnT3(-283～+10bp) in the BHK cells for 48 h. When compared the dual luciferase activity of cells transfected pJRed-160 Q-Htt at the different does with the cells transfected with pJRed-20 Q-Htt, the value was less gradually and the decrease tendency was does depended. The mHtt appeared to inhibit the expression of ZnT3 at the transcription level in BHK cells. We also co-transfected the Spl-pEBGN and ZnT3 promoter construct with different does of pJRed-20 Q-Htt or pJRed-160 Q-Htt in the BHK cells. Thus the results showed that the ZnT3 promoter sequence dual luciferase activity of co-transfected with pJRed-160 Q-Htt and Spl-pEBGN was corrected compared with co-transfected with pJRed-160 Q-Htt and pEBGN vector. Furthermore, we have inspected transcriptional inhibition of the ZnT3 gene by mHtt and the rectification by 160 Q and Spl co-transfected in BHK cells. To explore the paradox of Spl-driven gene ZnT3 transcription suppression and the Spl expression increased in the TG mcie and BHK/160 Q cells, we performed the ChIP assay to it. Having revealed specificity of the ChIP technique, we sought to compare the binding of Spl to the ZnT3 promoter in BHK/160 Q cells to BHK/20 Q control cells. Sp1a and Sp1b sites of Spl combined with in the ZnT3 promoter sequence, were amplified 106bp and 141bp. RT-PCR results showed that the both of ZnT3 promoter sequence bands in BHK/160 Q cells were weaker than in the BHK/20 Q control cells.The difference indicates that there was less Spl associated with the ZnT3 promoter in BHK/160 Q cells as compared to the BHK/20 Q control cells. The decreases of Spl binding to target gene ZnT3 explained the previous results of decreased overall level of ZnT3 and increased overall of Spl.The results of this study showed that the mHtt inhibit the combination of transcription factor Spl and ZnT3 gene promoter sequence, resulting in the ZnT3 gene transcription and expression decreased and then free zinc level in the synaptic vesicles suppression and synaptic dysfunction. This study provides a new experimental basis to reveal the mechanism of HD synaptic transmission and synaptic dysfunction.