Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration—defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.     

Renal localization, expression, and developmental regulation of P450 4F cytochromes in three substrains of spontaneously hypertensive rats

Cytochrome P450 4F isoforms have been shown to metabolize arachidonic acid to generate 20-hydroxyeicosatetraenoic acid (20-HETE), a potent eicosanoid that modulates vascular tone and renal tubular function. 20-HETE production in the kidney is implicated in the development of essential hypertension in the spontaneously hypertensive rat (SHR). In this study, we determined CYP4F mRNA localization and distribution in rat liver and kidney by in situ hybridization and real time quantitative PCR. CYP4Fs are regionally distributed in the kidney with CYP4F1, 4F4, and 4F5 being expressed more in the renal cortex than medulla while CYP4F6 shows higher medullary expression. We investigated developmental CYP4F gene expression in three different substrains of SHR. Distinct age-dependent patterns of expression were seen for individual CYP4F isoforms in Wistar-Kyoto (WKY) and three SHR substrains (B2, C, and A3). A steady increase in CYP4F1 expression with age was seen in each of the three substrains which correlate well with increased 20-HETE levels and elevated blood pressure seen in these animals. CYP4F4 expression increased significantly at 8 weeks followed by a precipitous fall in WKY and A3 strains at 12 weeks of age. In strains B2 and C, CYP4F4 levels started declining as early as 8 weeks of age. CYP4F5 and 4F6 levels fluctuated with age in a biphasic manner with a different profile for each sub-strain. Based on the expression profile and catalytic activity, CYP4F1 seems to be the most critical 4F isoform involved in the production of 20-HETE in the SHR kidney.     

Expression of cytochromes P450 4F4 and 4F5 in infection and injury models of inflammation

Lipopolysaccharide (LPS) treatment of rats suppresses CYP 4F4 and 4F5 expression by 50 and 40%, respectively, in a direct fashion occurring in the liver. This contention is borne out by essentially parallel dose-dependent changes observed upon treatment of rat hepatocyte cultures with LPS. An alternate avenue of triggering the inflammatory cascade is traumatic brain injury by controlled cortical impact. Such injury brings about a dramatic change in the expression of CYP 4F4 and 4F5 mRNA which reaches its greatest effect 24 h after impact compared with sham-operated but uninjured controls. At time points after 24 h the expression of both isoforms increases dramatically reaching highest levels at 2 weeks post-injury. These changes in mRNA expression are mirrored by changes in protein expression. The results are consistent with the notion that immediately after injury concentrations of leukotriene and prostaglandin mediators are elevated by decreased CYP 4F concentrations. As time after injury increases those conditions reverse. Increased CYP 4F expression leads to diminished concentrations of leukotriene and prostaglandin mediators and then to recovery and repair.     

Inflammatory prompts produce isoform-specific changes in the expression of leukotriene B(4) omega-hydroxylases in rat liver and kidney

Cytochrome p450 (CYP) 4Fs metabolize leukotriene B(4) and other inflammatory mediators in the arachidonic acid cascade. Here we show that lipopolysaccharide (LPS) treatment suppresses CYP4F4 and up-regulates CYP4F5 mRNA expression in rat liver whereas renal CYP4Fs are essentially unchanged. BaSO(4) treatment, in contrast, increases both hepatic and renal CYP4F expression levels. Thus, distinct regulatory mechanisms in CYP4F expression might operate under different inflammatory prompts. To examine hepatic totipotency, primary hepatocytes were treated with varying doses of LPS resulting in decrease in all the CYP4F isoforms. Treatment of hepatocytes with 5 ng/ml of interleukin-1beta mimics the in vivo effects of LPS on CYP4F expression.