Peter Baciu

Mouse mtDNA mutant model of Leber hereditary optic neuropathy

An animal model of Leber hereditary optic neuropathy (LHON) was produced by introducing the human optic atrophy mtDNA ND6 P25L mutation into the mouse. Mice with this mutation exhibited reduction in retinal function by elecroretinogram (ERG), age-related decline in central smaller caliber optic nerve fibers with sparing of larger peripheral fibers, neuronal accumulation of abnormal mitochondria, axonal swelling, and demyelination. Mitochondrial analysis revealed partial complex I and respiration defects and increased reactive oxygen species (ROS) production, whereas synaptosome analysis revealed decreased complex I activity and increased ROS but no diminution of ATP production. Thus, LHON pathophysiology may result from oxidative stress.

Retinal pigment epithelial cell death by the alternative complement cascade: role of membrane regulatory proteins, calcium, PKC, and oxidative stress.

PURPOSE Retinal pigment epithelial (RPE) cell death is an important feature of the advanced forms of AMD. Complement alternative pathway (AP) activation is associated with RPE cell death in AMD. In this study, we developed a new model to initiate AP activation on RPE cells and investigated the cellular mechanisms modulating AP activation-mediated RPE cell death. METHODS An anti-RPE antibody was developed. A spontaneously arising human RPE cell line (ARPE-19) and donor RPE cells were primed with this antibody followed by stimulation with 6% C1q-depleted human serum (C1q-Dep) to activate AP. Complement activation was evaluated by flow cytometry and immunofluorescent staining. Cellular response to complement activation was examined by measurement of intracellular calcium and adenosine triphosphate (ATP) release. Cell viability was assessed by Sytox orange, tetrazolium salt, and lactate dehydrogenase release assays. RESULTS Alternative pathway complement-mediated RPE cell death was associated with membrane attack complex formation and a rapid rise in intracellular calcium followed by release of ATP. Downregulation of membrane complement regulatory proteins and protein kinase C (PKC) inhibition increased cell susceptibility to complement attack. Pretreatment of RPE cells with either hydrogen peroxide or hydroquinone enhanced cell death. Chronic repetitive treatment of RPE cells with low levels of oxidants also enhanced complement-mediated cell death. CONCLUSIONS Activation of complement through the alternative pathway induces sublytic and lytic phases of complement attack on RPE cells, leading to cell death modulated by extracellular calcium, membrane complement regulatory proteins, and intracellular signaling mechanisms. Single-dose oxidant exposure and low-dose repetitive oxidant exposure rendered RPE cells more susceptible to complement-mediated death.

All-trans-Retinal Sensitizes Human RPE Cells to Alternative Complement Pathway–Induced Cell Death

PURPOSE Retinal pigment epithelial (RPE) cell death occurs early in the pathogenesis of age-related macular degeneration (AMD) and Stargardts disease. Emerging evidence suggests that all-trans-retinal (atRal) and alternative complement pathway (AP) activation contribute to RPE cell death in both of these retinal disorders. The aim of this study was to investigate the combined effect of atRal and AP activation on RPE cell viability. METHODS RPE cells were treated with atRal and then incubated with a complement-fixing antibody followed by stimulation with C1q-depleted serum to activate AP. Cell viability was assessed by tetrazolium salt and lactate dehydrogenase release assays. Changes in cell surface CD46 and CD59 expression were assessed by flow cytometry. Cells were pretreated with the antioxidant resveratrol, and C1q-depleted serum was incubated with an anti-C5 antibody prior to initiating AP attack to determine the protective effects of antioxidant therapy and complement inhibition, respectively. RESULTS Both atRal and AP activation independently caused RPE cell death. When AP attack was initiated following atRal treatment, a synergistic increase in cell death was observed. Following 24-hour atRal treatment, CD46 and CD59 expression decreased, corresponding temporally to increased susceptibility to AP attack. Resveratrol and the anti-C5 antibody both protected against AP-induced cell death following atRal exposure and were most effective when used in combination. CONCLUSIONS atRal sensitizes RPE cells to AP attack, which may be mediated in part by atRal-induced downregulation of CD46 and CD59. Despite increased susceptibility to AP attack following exposure to atRal, resveratrol and anti-C5 antibody effectively prevent AP-mediated cell death.

Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

Purpose. Complement activation is implicated in the pathogenesis of age-related macular degeneration (AMD). Apolipoprotein E (ApoE) and complement activation products such as membrane attack complex (MAC) are present in eyes of individuals with AMD. Herein, we investigated the effect of complement activation on induction of ApoE accumulation in human retinal pigment epithelial (RPE) cells. Methods. Cultured human RPE cells were primed with a complement-fixing antibody followed by treatment with C1q-depleted (C1q-Dep) human serum to elicit alternative pathway complement activation. Controls included anti-C5 antibody-treated serum and heat-inactivated C1q-Dep. Total protein was determined on RPE cell extracts, conditioned media, and extracellular matrix (ECM) by Western blot. ApoE and MAC colocalization was assessed on cultured RPE cells and human eyes by immunofluorescent stain. ApoE mRNA expression was evaluated by quantitative PCR (qPCR). Results. Complement challenge upregulated cell-associated ApoE, but not apolipoprotein A1. ApoE accumulation was blocked by anti-C5 antibody and enhanced by repetitive complement challenge. ApoE mRNA levels were not affected by complement challenge. ApoE was frequently colocalized with MAC in complement-treated cells and drusen from human eyes. ApoE was released into complement-treated conditioned media after a single complement challenge and accumulated on ECM after repetitive complement challenge. Conclusions. Complement challenge induces time-dependent ApoE accumulation in RPE cells. An understanding of the mechanisms by which complement affects RPE ApoE accumulation may help to better explain drusen composition, and provide insights into potential therapeutic targets.