Triptolide increases transcript and protein levels of survival motor neurons in human SMA fibroblasts and improves survival in SMA-like mice.

BACKGROUND AND PURPOSE: Spinal muscular atrophy (SMA) is a progressive neuromuscular disease. Since disease severity is related to the amount of survival motor neuron (SMN) protein, up-regulated functional SMN protein levels from the SMN2 gene are considered a major SMA drug-discovery strategy. In this study, we investigated the possible effects of triptolide, a diterpene triepoxide purified from Tripterygium wilfordii Hook. F., as a new compound for increasing SMN protein. EXPERIMENTAL APPROACH: The effects and mechanisms of triptolide on the production of SMA protein were determined by cell-based assays using the motor neuronal cell line NSC34 and skin fibroblasts from SMA patients. Wild-type (Smn(+/+) SMN2(-/-) , C57BL/6) and SMA-like (Smn(-/-) SMN2) mice were injected with triptolide (0.01 or 0.1 mg·kg(-1) ·day(-1) , i.p.) and their survival rate and level of change in SMN protein in neurons and muscle tissue measured. KEY RESULTS: In NSC34 cells and human SMA fibroblasts, pM concentrations of triptolide significantly increased SMN protein expression and the levels of SMN complex component (Gemin2 and Gemin3). In human SMA fibroblasts, triptolide increased SMN-containing nuclear gems and the ratio of full-length transcripts (FL-SMN2) to SMN2 transcripts lacking exon 7 (SMN2Δ7). Furthermore, in SMA-like mice, triptolide significantly increased SMN protein levels in the brain, spinal cord and gastrocnemius muscle. Furthermore, triptolide treatment increased survival and reduced weight loss in SMA-like mice. CONCLUSION AND IMPLICATIONS: Triptolide enhanced SMN protein production by promoting SMN2 activation, exon 7 inclusion and increasing nuclear gems, and increased survival in SMA mice, which suggests triptolide might be a potential candidate for SMA therapy.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) regulates the level of SMN expression through ubiquitination in primary spinal muscular atrophy fibroblasts.

BACKGROUND: Spinal muscular atrophy (SMA), a lethal hereditary disease caused by mutations of the survival of motor neuron 1 (SMN1) gene, is the leading genetic cause of infant mortality. Its severity directly correlates to the expression level of SMN protein in patients with SMA, but the regulatory mechanisms of SMN protein expression remain incompletely defined. In the present study, we aimed to identify candidate proteins to distinguish SMA fibroblasts from normal fibroblasts. METHODS: To identify cellular targets regulating the expression of SMN, we initially utilized a proteomics approach combining 2D electrophoresis and LC-MS/MS, wherein the total proteins extracted from type I SMA patients and normal skin fibroblast cells were compared. RESULTS: Our initial proteomics analysis discovered significant increase of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in type I SMA fibroblasts when compared to normal fibroblasts. Significantly, UCHL1 proteins directly interacted with SMN protein, as determined by immunoprecipitation and immunofluorescence assays in P19 and NSC34 cells. Over-expression of UCHL1 in P19 and NSC34 cells significantly reduced the level of SMN proteins in vivo, and, in fact, purified UCHL1 was shown to be able to enhance, in a dose-dependent manner, the level of ubiquitinated SMN in vitro. Further, inhibition of UCHL1 activity by UCHL1 inhibitor (LDN-57444) increased cellular SMN protein and gems number in the nucleus in NSC34 and SMA skin fibroblasts. The same results were observed in cells with UCHL1-specific knockdown. CONCLUSIONS: These results suggested that UCHL1 may be a critical regulator in controlling cellular SMN protein turnover, and that it may serve as an attractive therapeutic target for SMA.

KMUP-1 attenuates serum deprivation-induced neurotoxicity in SH-SY5Y cells: roles of PKG, PI3K/Akt and Bcl-2/Bax pathways.

Aging populations with neurodegenerative disorders will gradually become a greater problem for society. Serum deprivation-induced cell death is recognized as one of the standard models for the study of neurotoxicity. Increasing evidence indicates that cGMP/PKG pathway may play a rescue role in serum deprivation-induced toxicity. The aim of this study was to investigate protective effects of KMUP-1, an enhancer of cGMP/PKG signaling on serum deprivation-induced neurotoxicity in SH-SY5Y neuroblastoma cells. Under normal serum condition, KMUP-1 enhanced protein expression of nNOS, PKG and sGCalpha1, increased intracellular cyclic GMP level, and attenuated PDE5 expression. KMUP-1 also increased expression of BDNF and Bcl-2, but it did not affect Bax expression. The phosphorylation of Akt and CREB induced by KMUP-1 was inhibited by tyrosine kinase (TrK) inhibitor K252a and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, respectively. Under serum deprivation condition, flow cytometric analysis using Annexin V showed KMUP-1 increased cell viability, but lacked protective effects in the presence of nitric oxide synthase inhibitor l-NAME, PKG inhibitor Rp-8-pCPT-cGMPS or LY294002. KMUP-1 not only enhanced expression of nNOS, sGCalpha1, PKG, p-CREB, p-Akt and Bcl-2, but also attenuated Bax expression in serum deprivation-treated cultures. In conclusion, cGMP/PKG, PI3K/Akt/CREB and Bcl-2/Bax signals play critical roles in the neuroprotective effects of KMUP-1 on serum deprivation-induced toxicity.

Effects of San-Huang-Xie-Xin-Tang on U46619-induced increase in pulmonary arterial blood pressure.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Huang-Xie-Xin-Tang (SHXT), composed of Coptidis rhizoma, Scutellariae radix and Rhei rhizoma, is traditionally used to treat hypertension. AIM OF THE STUDY: Our aim was to investigate the pharmacology effect of SHXT on a thromboxane A(2) analogue U46619-induced increase in pulmonary hypertension and protein expression in primary pulmonary smooth muscle cells (PASMCs). MATERIALS AND METHODS: Arterial blood pressure and isometric tension in the aorta and pulmonary artery of rats were measured by pressure and force transducers, respectively. Protein expressions on PASMCs were detected by Western blotting. RESULTS: SHXT significantly attenuated U46619-induced increase in arterial blood pressure. The inhibitory effect of SHXT on pulmonary arterial pressure was greater than systemic arterial pressure in U46619 treated rats. Similarly, the inhibitory effect of SHXT on U46619-induced vasoconstriction in rat pulmonary arterial rings was greater than that in aortic rings. In U46619 treated PASMCs, SHXT down-regulated expression of phosphodiesterase type 5 (PDE5), Rho-kinase (ROCK) II, cyclooxygenase-2 (COX-2) and up-regulated expression of soluble guanylyl cyclase (sGC) alpha(1) and sGCbeta(1). CONCLUSIONS: SHXT attenuated U46619-induced increase in systemic and pulmonary arterial blood pressure. Inhibition of PDE5, ROCK-II, COX-2 and stimulation of sGC may play important roles in the cardiovascular effects of SHXT.

The unusual electrochemical and photophysical behavior of 2,2'-bis(1,3,4-oxadiazol-2-yl)biphenyls, effective electron transport hosts for phosphorescent organic light emitting diodes.

The fluorescence and phosphorescence of 2,2'-bis(5-phenyl-1,3,4-oxadiazol-2-yl)biphenyl shows good spectral matching with the absorption spectra of the MLCT1 and MLCT3 transitions of Ir(ppy)3. The red-shift of the 0-0 band in the phosphorescence at 77 K is due to the intramolecular pi-pi interactions between the oxadiazole side chains. Maximum brightness of 43,000 cd/m2 with an efficiency of 26 cd/A at 200 cd/m2 was achieved when BOBP was used as the host material for Ir(ppy)3 in the PHOLED study. [structure: see text].