[Effect of p65 gene inhibited by siRNA on differention of rat marrow mesenchymal stem cells into neurons].

OBJECTIVE: To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs). METHODS: The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTT at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The expressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method after transfection for 6 h. RESULTS: The fluorescence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile, the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased (P < 0.05); MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group (P < 0.05). CONCLUSION: The p65 gene inhibited by siRNA can promote the marrow mesenchymal stem cells to differentiate into neurons.

Transglutaminase 6 interacts with polyQ proteins and promotes the formation of polyQ aggregates.

A common feature of polyglutamine (polyQ) diseases is the presence of aggregates in neuronal cells caused by expanded polyglutamine tracts. PolyQ proteins are the substrates of transglutaminase 2, and the increased activity of transglutaminase in polyQ diseases suggests that transglutaminase may be directly involved in the formation of the aggregates. We previously identified the transglutaminase 6 gene to be causative of spinocerebellar ataxia type 35 (SCA35), and we found that SCA35-associated mutants exhibited reduced transglutaminase activity. Here we report that transglutaminase 6 interacts and co-localizes with both normal and expanded polyQ proteins in HEK293 cells. Moreover, the overexpression of transglutaminase 6 promotes the formation of polyQ aggregates and the conversion of soluble polyQ into insoluble polyQ aggregates. However, SCA35-associated mutants do not affect their interactions with polyQ proteins. These data suggest that transglutaminase 6 could be involved in polyQ diseases and there may exist a common pathological link between polyQ associated SCA and SCA35.

Spinocerebellar ataxia type 35 (SCA35)-associated transglutaminase 6 mutants sensitize cells to apoptosis.

Spinocerebellar ataxia type 35 (SCA35) is an autosomal dominant neurodegenerative disorder. In our previous study, using exome sequencing and linkage analysis, two missense mutations of the transglutaminase 6 (TGM6) gene were identified as causative for SCA35. TGM6 encodes transglutaminase 6 (TG6), a member of the transglutaminase family of enzymes that catalyze the formation of a covalent bond between a free amine group and the γ-carboxamide group of protein- or peptide-bound glutamine. However, the precise role of TG6 in contributing to SCA35 remains unclear. In this study, we analyzed the subcellular distribution, expression and in vitro activity of two missense mutations of TG6 (D327G, L517W) and found that both mutants exhibited decreased transglutaminase activity and stability. Furthermore, overexpressing the TG6 mutants sensitized cells to staurosporine-induced apoptosis by increasing the activity of caspases. We propose that the pro-apoptotic role of these mutants might underlie the pathogenesis of SCA35.

[Effect of notch signaling on differentiation of rat marrow mesenchymal stem cells into neurons induced by fasudil hydrochloride].

OBJECTIVE: To investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons induced by fasudil hydrochloride. METHODS: The experiments were divided into non-transfected group, transfected group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (transfected with negative control siRNA). Fasudil hydrochloride induced MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notch1 mRNA, Hes1 mRNA and MAPK1 mRNA in MSCs was detected by RT-PCR. The expression of Notch1 protein, NSE, neurofilament M (NF-M) and glial fibrillary acidic protein(GFAP)was detected by immunocytochemical method. The viability of MSCs was detected by MTT. RESULTS: (1) The fluorescence of MSCs was mostly displayed after transfection for 72 h and the efficiency of transfection was up to 91.3% +/- 4.2%. Meanwhile, the notch1 mRNA and Hes1 mRNA expressed by MSCs of transfected group were significantly decreased (P < 0.05) and MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). (2) Fasudil hydrochloride could induce MSCs differentiate into neurons and the best efficiency of induction was observed in the transfected group. There was higher expression of NSE and neurofilament-M (NF-M) than the other groups (P < 0.05). CONCLUSION: There may be notch1 signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promote the differentiation of MSCs into neurons.

[Effects of DSCAM on differentiation of rat marrow mesenchymal stem cells into neurons in vitro].

OBJECTIVE: To study the effects of Down syndrome cellular adhesion molecule (DSCAM) on differentiation of rat marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: MSCs from Sprague-Dawley rats were induced into neurons by baicalin. The expression of DSCAM before and after induction was evaluated by immunocytochemical staining and Western blot assay. After knockdown of DSCAM by siRNA transfection, the differentiation rate of neurons derived from MSCs was measured. RESULTS: Before induction, the expression of DSCAM was not detectable in MSCs. After bFGF preinduction for 24 hrs, DSCAM was slightly expressed in MSCs (1.71+/- 0.67%). The DSCAM expression increased 6 hrs after baicalin induction (15.79+/- 4.24%), reached a peak at 3 days (53.16+/- 5.94%) and then decreased gradually. The DSCAM expression 6 days after baicalin induction (28.99+/- 6.72%) was significantly lower than that at 3 days (P<0.01). However, after DSCAM-siRNA transfection, the DSCAM expression in MSCs was significantly reduced. MSCs did not express neuron-specific beta-III-tubulin before induction. After baicalin induction for 6 hrs, 3 days and 6 days, the expression of beta-III-tubulin was 1.40+/- 0.79%, 41.59+/- 3.17% and 59.11+/- 4.76% respectively. But the beta-III-tubulin expression significantly decreased 3 and 6 days after DSCAM-siRNA transfection (28.57+/- 2.91% and 43.90+/- 12.31% respectively). CONCLUSIONS: DSCAM may play an important role in MSCs differentiation into neural cells.