Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 M epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.

Members of the FE65 family of adaptor proteins, FE65, FE65L1, and FE65L2, bind the C-terminal region of the amyloid precursor protein (APP). Overexpression of FE65 and FE65L1 was previously reported to increase the levels of ␣-secretase-derived APP (APPs␣). Increased ␤-amyloid (A␤) generation was also observed in cells showing the FE65-dependent increase in APPs␣. To understand the mechanism for the observed increase in both A␤ and APPs␣ given that ␣-secretase cleavage of a single APP molecule precludes A␤ generation, we examined the effects of FE65L1 overexpression on APP Cterminal fragments (APP CTFs). Our data show that FE65L1 potentiates ␥-secretase processing of APP CTFs, including the amyloidogenic CTF C99, accounting for the ability of FE65L1 to increase generation of APP Cterminal domain and A␤40. The FE65L1 modulation of these processing events requires binding of FE65L1 to APP and APP CTFs and is not because of a direct effect on ␥-secretase activity, because Notch intracellular domain generation is not altered by FE65L1. Furthermore, enhanced APP CTF processing can be detected in early endosome vesicles but not in endoplasmic reticulum or Golgi membranes, suggesting that the effects of FE65L1 occur at or near the plasma membrane. Finally, although FE65L1 increases APP C-terminal domain production, it does not mediate the APP-dependent transcriptional activation observed with FE65.Processing of the amyloid precursor protein (APP) 1 results in the generation of the amyloidogenic peptide, A␤, which plays a central role in the pathogenesis of Alzheimer's disease. Cleavage of C99, the APP C-terminal fragment derived from ␤-secretase processing of APP, by ␥-secretase generates the A␤ peptide. Furthermore, ␥-secretase cleavage of C99 and C83, the ␣-secretase derived APP C-terminal fragment (APP CTF), releases the APP C-terminal domain (AICD), a 6-kDa peptide also called CTF␥ or AID, that regulates transcription after translocation to the nucleus (1-4).The majority of proteins reported to bind the 47-amino acid intracellular region of APP (5-7), including the FE65 protein family members FE65, FE65L1, and FE65L2, bind the YENPTY sorting motif of APP via a phosphotyrosine interaction domain (PID/PTB). YENP is a clathrin-coated pit internalization domain required for trafficking of APP into the endocytic pathway (8, 9). Previous studies have shown that FE65 protein family members can alter the processing of APP by influencing APP trafficking. Increased maturation of APP and increased ␣-secretase-cleaved APP (APPs␣) secretion was observed in H4 neuroglioma cells induced for FE65L1 overexpression (10). Furthermore, enhanced secretion of APPs␣ and A␤ was reported in Madin-Darby canine kidney APP695 cells stably overexpressing FE65 (11). Cell surface APP levels were elevated in these cells, and increased routing of APP into the endocytic pathway from the plasma membrane was suggested to account for the observed increase in A␤ (11).The FE65 proteins are adaptor proteins that have three protein-protein interaction dom...

Traumatic brain injury (TBI) induces microglial activation, which can contribute to secondary tissue loss. Activation of mGluR5 reduces microglial activation and inhibits microglial-mediated neurodegeneration in vitro, and is neuroprotective in experimental models of CNS injury. In vitro studies also suggest that the beneficial effects of mGluR5 activation involve nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibition in activated microglia. We hypothesized that activation of mGluR5 by the selective agonist CHPG after TBI in mice is neuroprotective and that its therapeutic actions are mediated by NADPH oxidase inhibition. Vehicle, CHPG, or CHPG plus the mGluR5 antagonist (MPEP), were administered centrally, 30 minutes post-TBI, and functional recovery and lesion volume was assessed. CHPG significantly attenuated post-traumatic sensorimotor and cognitive deficits, and reduced lesion volumes; these effects were blocked by MPEP, thereby indicating neuroprotection involved selective activation of mGluR5. CHPG treatment also reduced NFjB activity and nitrite production in lipopolysaccharide-stimulated microglia and the protective effects of CHPG treatment were abrogated in NADPH oxidase deficient microglial cultures (gp91 phox-/-). To address whether the neuroprotective effects of CHPG are mediated via the inhibition of NADPH oxidase, we administered the NADPH oxidase inhibitor apocynin with or without CHPG treatment after TBI. Both apocynin or CHPG treatment alone improved sensorimotor deficits and reduced lesion volumes when compared with vehicle-treated mice; however, the combined CHPG + apocynin treatment was not superior to CHPG alone. These data suggest that the neuroprotective effects of activating mGluR5 receptors after TBI are mediated, in part, via the inhibition of NADPH oxidase.