The Catabolism Of Huntingtin-552 And Its Effect On The Production And Secretion Of BDNF In Astrocytes

Aim: To establish a glia Huntington's disease (HD) model expressing wild-type and mutant N-terminal huntingtin fragment 1–552 amino acids (htt552), to study the contribution of autophagy/lysosomal pathway on the metabolism of the wild-type and mutant htt552 and to study the effects of wild-type and mutant htt552 on the production and secretion of brain-derived neurotrophic factor (BDNF) in astrocytes.Method: In this study, we developed an in vitro model of HD by infecting primary cortical astrocytes of new-born rat with adenoviral vectors encoding the first 552 amino acids of wild-type (18Q) and mutant (100Q) htt. Western Blot analysis and double immunofluorescence (IF) were used to measure the levels and duration of htt552 expression in astrocytes. MTT assay was used to detect the viability of astrocytes after viral infection. The effects of autophagy specific inhibitor 3-methyadenine (3-MA), autophagy activator rapamycin, and the proteasome inhibitor lactacystin on htt552 accumulation were assessed in astrocytes expressing htt552 with Western Blot analysis and double immunofluorescence (IF). Proteins related to autophagy -- LC3 and Beclin1 were determined with Western Blot analysis and Real-time PCR. Autophagosomes were detected by monodansylcardeverine (MDC) immuofluorescence. Protein levels of BDNF in cell lysates and medium were detected by enzyme-linked immunosorbent assay (ELISA) and Western Blot analysis. The levels of BDNF mRNA and its four transcripts were detected with Real-time PCR to estimate the influence of htt552 on the transcription of BDNF. The co-location of htt552 with cAMP-response element binding protein (CBP) or Golgi complex was detected by double IF to estimate the mechanisms by which htt552 affects the transcription and secretion of BDNF.Results: Wild-type and mutant htt552 transfected by adenovirus successfully expressed in primary astrocytes. They distributed predominantly in the cytoplasm with relatively low levels in the nucleus. Meanwhile, htt552-100Q produced the characteristic HD pathology by the appearance of cytoplasmic aggregates and intranuclear inclusions. To obtain high infection rate and low toxicity, the viral dose with an m.o.i. of 40 was optimal to our cell model, the duration of expression of htt552 lasted for about 7 days with a relatively high levels from 1 to 4 days post-infection. Treatments with the autophagy inhibitor (3-MA) or proteasome inhibitor (lactacystin) resulted in the augments of htt552 levels in cells, especially in those expressing mutant htt552. The activator of autophagy, rapamycin, significantly decreased the levels of mutant htt552 and the number of aggregates, however, had little effect on htt552-18Q accumulation. Western Blot analysis and Real time PCR revealed that the expression of LC3 and Beclin1 were up-regulated, and autophagosomes detected by MDC increased in cells expressing wild-type htt552 but it was more robust in the cells expressing mutant htt552. The result of IF assay shown that neurons cultured with astrocyte conditioned medium (ACM) from the cell expressing htt552-100Q exhibited shorter processes and fewer branchings than those with either ACM from the cell cultured normally or ACM from the cell expressing htt552-18Q. ELISA analysis revealed that total BDNF in the cell expressing htt552-100Q was increased compared with the cells expressing htt552-18Q. Western blot and ELISA analysis revealed that BDNF in ACM was down-regulated by htt552-100Q, and was not significantly changed by wild-type htt552. Western Blot assay further showed that proBDNF in astrocytes expressing with htt552-100Q was increased, but mature BDNF was decreased compared with the astrocytes expressing with htt552-18Q. Real time PCR revealed that BDNF mRNA levels were decreased in the astrocytes expressing htt552-100Q, as a result of the inhibition of promoter II,III,IV in BDNF gene. The result also showed that the levels of BDNF and its four transcripts were not significant change in the astrocytes expressing htt552-18Q. The result of double IF showed that htt552-100Q co-localized with CBP in the nucleus, which may have influence on the function of CBP. The result of double IF showed that htt552-100Q and its aggregates colocalized with BDNF puncta or Golgi complex, moreover, the morphology of Golgi complex and size of Golgi complex were changed by mutant htt552. Conclusion: In vitro astrocytes HD model expressing wild-type and mutant N-terminal huntingtin fragment htt552 had been successfully established, which provided an advantageous system for research of HD pathogenesis in primary cortical astrocyte cultures. The ubiquitin-proteasome pathway (UPP) and autophagy/lysosomal pathway participated in the catabolism of htt552, and autophagy/lysosomal pathway was the major pathway in the catabolism of mutant htt552 and its aggregates. Wild-type and mutant htt552 fragments can both activate autophagy in cells and mutant fragments activated autophagy more robustly. Htt552-100Q can inhibit the transcription and secretion of BDNF in astrocytes, and then have influence on the development and function of neurons. Htt552-100Q and its aggregates may sequester CBP in the nucleus and BDNF vesicles in the cytoplasm. In addition, Htt552-100Q may have an aberrant effect on Golgi complex, thus disrupt the processing and secretion of BDNF.