In this study, we investigated the role of hematopietic progenitor kinase 1 (HPK1) in delayed neuronal damage after cerebral ischemia and the possible regulatory mechanisms of this event. Our data show that tyrosine phosphorylation of HPK1 was significantly increased at 6 h of ischemic-reperfusion compared with sham control. The increase in p-HPK1, p-MLK3, p-MKK7, and p-JNK3 was attenuated by HPK1 antisense oligodeoxynucleotides intra-cerebroventricular infusion, but not MS-ODNs or vehicle. Intracerebroventricular infusion of antisense oligodeoxynucleotides also increased the number of surviving pyramidal neurons, whereas MS-ODNs or vehicle (TE) groups had no effects. These results indicate that knockdown of HPK1 expression provides neuroprotection through downregulation activation of the MLK3-MKK7-JNK3 pathway following cerebral ischemia in the rat hippocampus CA1 subfield.
Neural stem (NS) cells are a limitless resource, and thus superior to primary neurons for drug discovery provided they exhibit appropriate disease phenotypes. Here we established NS cells for cellular studies of Huntington’s disease (HD). HD is a heritable neurodegenerative disease caused by a mutation resulting in an increased number of glutamines (Q) within a polyglutamine tract in Huntingtin (Htt). NS cells were isolated from embryonic wild-type (Htt7Q/7Q) and “knock-in” HD (Htt140Q/140Q) mice expressing full-length endogenous normal or mutant Htt. NS cells were also developed from mouse embryonic stem cells that were devoid of Htt (Htt−/−), or knock-in cells containing human exon1 with an N-terminal FLAG epitope tag and with 7Q or 140Q inserted into one of the mouse alleles (HttF7Q/7Q and HttF140Q/7Q). Compared to Htt7Q/7Q NS cells, HD Htt140Q/140Q NS cells showed significantly reduced levels of cholesterol, increased levels of reactive oxygen species (ROS), and impaired motility. The heterozygous HttF140Q/7Q NS cells had increased ROS and decreased motility compared to HttF7Q/7Q. These phenotypes of HD NS cells replicate those seen in HD patients or in primary cell or in vivo models of HD. Huntingtin “knock-out” NS cells (Htt−/−) also had impaired motility, but in contrast to HD cells had increased cholesterol. In addition, Htt140Q/140Q NS cells had higher phospho-AKT/AKT ratios than Htt7Q/7Q NS cells in resting conditions and after BDNF stimulation, suggesting mutant htt affects AKT dependent growth factor signaling. Upon differentiation, the Htt7Q/7Q and Htt140Q/140Q generated numerous BetaIII-Tubulin- and GABA-positive neurons; however, after 15 days the cellular architecture of the differentiated Htt140Q/140Q cultures changed compared to Htt7Q/7Q cultures and included a marked increase of GFAP-positive cells. Our findings suggest that NS cells expressing endogenous mutant Htt will be useful for study of mechanisms of HD and drug discovery.
Deletion of the Saccharomyces gene, UTH1, a founding member of the SUN family of fungal genes, has pleiotropic effects. Several phenotypes of Δuth1 cells including their decreased levels of mitochondrial proteins, their impaired autophagic degradation of mitochondria, and their increased viability in the presence of mammalian BAX, a pro-apoptotic regulator localized to the mitochondria, have prompted others to propose that the Uth1p functions primarily at the mitochondria. In this report, we show that cells lacking UTH1 have more robust cell walls with higher levels of β-d-glucan that allows them to grow in the presence of calcofluor white (CFW) or sodium dodecyl sulfate (SDS), two reagents known to perturb the yeast cell wall. Moreover, these Δuth1 cells are also significantly more resistant to spheroplast formation induced by zymolyase treatment than their wildtype counterparts. Surprisingly, our data suggests that several of the enhanced growth phenotypes of Δuth1 cells, including their resistance to BAX-mediated toxicity, arise from a strengthened cell wall. Therefore, we propose that Uth1p's role at the cell wall and not at the mitochondria may better explain many of its effects on yeast physiology and programmed cell death.
Background: We previously demonstrated that chronic pulmonary infection with Cryptococcus neoformans results in enhanced allergic inflammation and airway hyperreactivity in a rat model. Because the cell wall of C. neoformans consists of chitin, and since acidic mammalian chitinase (AMCase) has recently been implicated as a novel mediator of asthma, we sought to determine whether such infection induces chitinase activity and expression of AMCase in the rat.
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