COX-2-deficient mice are less prone to MPTP-neurotoxicity than wild-type mice.

The primary lesion in Parkinson's disease is the death of dopaminergic neurons in the substantia nigra. The role of cyclooxygenase (COX)-2 in the etiology of Parkinson's disease was explored using COX-2 gene knockout mice. Mortality after injection of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP, a chemical known to cause parkinsonism in humans) in heterozygous COX-2-deficient mice was lower than that in wild-type mice. The number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta of MPTP-treated wild-type mice declined to a greater extent than in heterozygous mice. Inhibition of COX-2 protein expression decreased the lesion caused by MPTP and protected the dopaminergic neurons in substantia nigra pars compacta. This result suggested that inhibition of COX-2 has potential therapeutic implications.

Agents that stabilize mutated von Hippel-Lindau (VHL) protein: results of a high-throughput screen to identify compounds that modulate VHL proteostasis.

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.

Alternative polyadenylation produces two major transcripts of Alix.

The mammalian adaptor protein Alix participates in multiple cellular processes. Since mouse Alix cDNA detects two distinct transcripts of approximately 3.5 and approximately 7.0 kb in various mouse tissues, it is possible that there exist isoforms of Alix protein that perform varied biological functions. In this study, we first demonstrate that four different anti-Alix monoclonal antibodies immunoblot the single Alix protein in nine different mouse tissues. We then show that the two transcripts of 3.2 and 6.4 kb are widely expressed in various human tissues and cell lines. These two transcripts are generated from the same Alix gene localizing at 3p22.3 via alternative polyadenylation, thus containing an identical open reading frame. However, the 3.2-kb transcript is much more active in translation than the 6.4-kb transcript in a randomly selected cell line. These results eliminate the possibility that the two transcript variants encode different isoforms of Alix protein and suggest that alternative polyadenylation is one of the mechanisms controlling Alix protein expression.

[The effect of cyclooxygenase-2 on dopaminergic neurons in substantia nigra of Parkinson's disease mice induced by MPTP].

AIM AND METHODS: Using Strain C57/BL of COX-2 deficient mice, the effect of Cyclooxygenase-2 (COX-2) on dopaminergic neurons in substantia nigra of Parkinson's disease (PD) mice induced by intraperitoneal injections of MPTP-HCl were investigated by immunocytochemistry(ICC) . RESULTS: We found that the mortality in COX-2 heterozygous mice is much lower than that in wild type mice (P < 0.01) after injection of MPTP (30 mg/kg/day). The result of semiquantitative immunocytochemical staining showed that the number of tyrosine hydroxylase (TH) immunoreactive neurons in the substantia nigra pars compacta (SNc) declined more significantly in MPTP-treated wild type mice than that in COX-2 heterozygotes mice (P < 0.01). CONCLUSION: COX-2 may be related with lesion of dopaminergic neurons in the SNc of PD.