Altered PPARgamma expression and activation after transient focal ischemia in rats.

Stroke is a devastating disease with limited treatment options. Recently, we found that the peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists troglitazone and pioglitazone reduce injury and inflammation in a rat model of transient cerebral ischemia. The mechanism of this protection is unclear, as these agents can act through PPAR-gamma activation or through PPAR-gamma-independent mechanisms. Therefore, we examined PPAR-gamma expression, DNA binding and transcriptional activity following stroke. In addition, we used a PPAR-gamma antagonist, T0070907, to determine the role of PPAR-gamma during ischemia. Using immunohistochemical techniques and real-time PCR, we found low levels of PPAR-gamma mRNA and PPAR-gamma immunoreactivity in nonischemic brain; however, PPAR-gamma expression dramatically increased in ischemic neurons, peaking 24 h following middle cerebral artery occlusion. Interestingly, we found that in both vehicle- and agonist-treated brains, DNA binding was reduced in the ischemic hemisphere relative to the contralateral hemisphere. Expression of a PPAR-gamma target gene, lipoprotein lipase, was also reduced in ischemic relative to nonischemic brain. Both DNA binding and lipoprotein lipase expression were increased by the addition of the PPAR-gamma agonist rosiglitazone. Finally, we found that rosiglitazone-mediated protection after stroke was reversed by the PPAR-gamma antagonist T0070907. Interestingly, infarction size was also increased by T0070907 in the absence of PPAR-gamma agonist, suggesting that endogenous PPAR-gamma ligands may mitigate the effects of cerebral ischemia.

The WW domain of dystrophin requires EF-hands region to interact with beta-dystroglycan.

Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with beta-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of beta-dystroglycan. This region of dystrophin implicated in binding beta-dystroglycan contains four modular protein domains: a WW domain, two putative Ca2+-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38-40 amino acids with two highly conserved tryptophan residues spaced 20-22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding beta-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of beta-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxy-terminus of beta-dystroglycan occurs via a beta-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/beta-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.