Tanja Diemer

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway.

Reduced dietary intake increases lifespan in a wide variety of organisms. It also retards disease progression. We tested whether dietary supplementation of citric acid cycle metabolites could mimic this lifespan effect. We report that oxaloacetate supplementation increased lifespan in Caenorhabditis elegans. The increase was dependent on the transcription factor, FOXO/DAF‐16, and the energy sensor, AMP‐activated protein kinase, indicating involvement of a pathway that is also required for lifespan extension through dietary restriction. These results demonstrate that supplementation of the citric acid cycle metabolite, oxaloacetate, influences a longevity pathway, and suggest a tractable means of introducing the health‐related benefits of dietary restriction.

Function of MYO7A in the human RPE and the validity of shaker1 mice as a model for Usher syndrome 1B.

PURPOSE To investigate the function of MYO7A in human RPE cells and to test the validity of using shaker1 RPE in preclinical studies on therapies for Usher syndrome 1B by comparing human and mouse cells. METHODS MYO7A was localized by immunofluorescence. Primary cultures of human and mouse RPE cells were used to measure melanosome motility and rod outer segment (ROS) phagocytosis and digestion. MYO7A was knocked down in the human RPE cells by RNAi to test for a mutant phenotype in melanosome motility. RESULTS The distribution of MYO7A in the RPE of human and mouse was found to be comparable, both in vivo and in primary cultures. Primary cultures of human RPE cells phagocytosed and digested ROSs with kinetics comparable to that of primary cultures of mouse RPE cells. Melanosome motility was also comparable, and, after RNAi knockdown, consisted of longer-range fast movements characteristic of melanosomes in shaker1 RPE. CONCLUSIONS The localization and function of MYO7A in human RPE cells is comparable to that in mouse RPE cells. Although shaker1 retinas do not undergo degeneration, correction of mutant phenotypes in the shaker1 RPE represents a valid preclinical test for potential therapeutic treatments.

Melanoregulin (MREG) modulates lysosome function in pigment epithelial cells.

Melanoregulin (MREG), the product of the Mregdsu gene, is a small highly charged protein, hypothesized to play a role in organelle biogenesis due to its effect on pigmentation in dilute, ashen, and leaden mutant mice. Here we provide evidence that MREG is required in lysosome-dependent phagosome degradation. In the Mreg-/- mouse, we show that loss of MREG function results in phagosome accumulation due to delayed degradation of engulfed material. Over time, the Mreg-/- mouse retinal pigment epithelial cells accumulate the lipofuscin component, A2E. MREG-deficient human and mouse retinal pigment epithelial cells exhibit diminished activity of the lysosomal hydrolase, cathepsin D, due to defective processing. Moreover, MREG localizes to small intracellular vesicles and associates with the endosomal phosphoinositide, phosphatidylinositol 3,5-biphosphate. Collectively, these studies suggest that MREG is required for lysosome maturation and support a role for MREG in intracellular trafficking.

Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis

Photoreceptor outer segment (POS) phagosomes associate with the kinesin light chain 1 (KLC1) and move bidirectionally along microtubules in retinal pigment epithelium cells; lack of KLC1 results in impaired POS phagosome motility and degradation and, in aged mice, pathogenesis resembling age-related macular degeneration.

NPAS2 Compensates for Loss of CLOCK in Peripheral Circadian Oscillators.

Heterodimers of CLOCK and BMAL1 are the major transcriptional activators of the mammalian circadian clock. Because the paralog NPAS2 can substitute for CLOCK in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, CLOCK-deficient mice maintain circadian rhythms in behavior and in tissues in vivo. However, when isolated from the SCN, CLOCK-deficient peripheral tissues are reportedly arrhythmic, suggesting a fundamental difference in circadian clock function between SCN and peripheral tissues. Surprisingly, however, using luminometry and single-cell bioluminescence imaging of PER2 expression, we now find that CLOCK-deficient dispersed SCN neurons and peripheral cells exhibit similarly stable, autonomous circadian rhythms in vitro. In CLOCK-deficient fibroblasts, knockdown of Npas2 leads to arrhythmicity, suggesting that NPAS2 can compensate for loss of CLOCK in peripheral cells as well as in SCN. Our data overturn the notion of an SCN-specific role for NPAS2 in the molecular circadian clock, and instead indicate that, at the cellular level, the core loops of SCN neuron and peripheral cell circadian clocks are fundamentally similar.

Lithium effects on circadian rhythms in fibroblasts and suprachiasmatic nucleus slices from Cry knockout mice.

Lithium is widely used as a treatment of bipolar disorder, a neuropsychiatric disorder associated with disrupted circadian rhythms. Lithium is known to lengthen period and increase amplitude of circadian rhythms. One possible pathway for these effects involves inhibition of glycogen synthase kinase-3β (GSK-3β), which regulates degradation of CRY2, a canonical clock protein determining circadian period. CRY1 is also known to play important roles in regulating circadian period and phase, although there is no evidence that it is similarly phosphorylated by GSK-3β. In this paper, we tested the hypothesis that lithium affects circadian rhythms through CRYs. We cultured fibroblasts and slices of the suprachiasmatic nucleus (SCN), the master circadian pacemaker of the brain, from Cry1-/-, Cry2-/-, or wild-type (WT) mice bearing the PER2:LUC circadian reporter. Lithium was applied in the culture medium, and circadian rhythms of PER2 expression were measured. In WT and Cry2-/- fibroblasts, 10mM lithium increased PER2 expression and rhythm amplitude but not period, and 1mM lithium did not affect either period or amplitude. In non-rhythmic Cry1-/- fibroblasts, 10mM lithium increased PER2 expression. In SCN slices, 1mM lithium lengthened period ∼1h in all genotypes, but did not affect amplitude except in Cry2-/- SCN. Thus, the amplitude-enhancing effect of lithium in WT fibroblasts was unaffected by Cry2 knockout and occurred in the absence of period-lengthening, whereas the period-lengthening effect of lithium in WT SCN was unaffected by Cry1 or Cry2 knockout and occurred in the absence of rhythm amplification, suggesting that these two effects of lithium on circadian rhythms are independent of CRYs and of each other.

Expression of the Mitochondrial Calcium Uniporter in Cardiac Myocytes Improves Impaired Mitochondrial Calcium Handling and Metabolism in Simulated Hyperglycemia

Diabetic cardiomyopathy is associated with metabolic changes, including decreased glucose oxidation (Gox) and increased fatty acid oxidation (FAox), which result in cardiac energetic deficiency. Diabetic hyperglycemia is a pathophysiological mechanism that triggers multiple maladaptive phenomena. The mitochondrial Ca2+ uniporter (MCU) is the channel responsible for Ca2+ uptake in mitochondria, and free mitochondrial Ca2+ concentration ([Ca2+]m) regulates mitochondrial metabolism. Experiments with cardiac myocytes (CM) exposed to simulated hyperglycemia revealed reduced [Ca2+]m and MCU protein levels. Therefore, we investigated whether returning [Ca2+]m to normal levels in CM by MCU expression could lead to normalization of Gox and FAox with no detrimental effects. Mouse neonatal CM were exposed for 72 h to normal glucose [5.5 mM glucose + 19.5 mM mannitol (NG)], high glucose [25 mM glucose (HG)], or HG + adenoviral MCU expression. Gox and FAox, [Ca2+]m, MCU levels, pyruvate dehydrogenase (PDH) activity, oxidative stress, mitochondrial membrane potential, and apoptosis were assessed. [Ca2+]m and MCU protein levels were reduced after 72 h of HG. Gox was decreased and FAox was increased in HG, PDH activity was decreased, phosphorylated PDH levels were increased, and mitochondrial membrane potential was reduced. MCU expression returned these parameters toward NG levels. Moreover, increased oxidative stress and apoptosis were reduced in HG by MCU expression. We also observed reduced MCU protein levels and [Ca2+]m in hearts from type 1 diabetic mice. Thus we conclude that HG-induced metabolic alterations can be reversed by restoration of MCU levels, resulting in return of [Ca2+]m to normal levels.

O-GlcNAcylation of 8-Oxoguanine DNA Glycosylase (Ogg1) Impairs Oxidative Mitochondrial DNA Lesion Repair in Diabetic Hearts.

mtDNA damage in cardiac myocytes resulting from increased oxidative stress is emerging as an important factor in the pathogenesis of diabetic cardiomyopathy. A prevalent lesion that occurs in mtDNA damage is the formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), which can cause mutations when not repaired properly by 8-oxoguanine DNA glycosylase (Ogg1). Although the mtDNA repair machinery has been described in cardiac myocytes, the regulation of this repair has been incompletely investigated. Here we report that the hearts of type 1 diabetic mice, despite having increased Ogg1 protein levels, had significantly lower Ogg1 activity than the hearts of control, non-type 1 diabetic mice. In diabetic hearts, we further observed increased levels of 8-OHdG and an increased amount of mtDNA damage. Interestingly, Ogg1 was found to be highly O-GlcNAcylated in diabetic mice compared with controls. In vitro experiments demonstrated that O-GlcNAcylation inhibits Ogg1 activity, which could explain the mtDNA lesion accumulation observed in vivo. Reducing Ogg1 O-GlcNAcylation in vivo by introducing a dominant negative O-GlcNAc transferase mutant (F460A) restored Ogg1 enzymatic activity and, consequently, reduced 8-OHdG and mtDNA damage despite the adverse hyperglycemic milieu. Taken together, our results implicate hyperglycemia-induced O-GlcNAcylation of Ogg1 in increased mtDNA damage and, therefore, provide a new plausible biochemical mechanism for diabetic cardiomyopathy.

Cellular circadian oscillators in the suprachiasmatic nucleus remain coupled in the absence of connexin-36

In mammals, the master circadian clock resides in the suprachiasmatic nucleus (SCN). The SCN is characterized by robust circadian oscillations of clock gene expression and neuronal firing. The synchronization of circadian oscillations among individual cells in the SCN is attributed to intercellular coupling. Previous studies have shown that gap junctions, specifically those composed of connexin-36 (Cx36) subunits, are required for coupling of electrical firing among SCN neurons at a time scale of milliseconds. However, it remains unknown whether Cx36 gap junctions also contribute to coupling of circadian (∼24h) rhythms of clock gene expression. Here, we investigated circadian expression patterns of the clock gene Period 2 (Per2) in the SCN of Cx36-deficient mice using luminometry and single-cell bioluminescence imaging. Surprisingly, we found that synchronization of circadian PER2 expression rhythms is maintained in SCN explants from Cx36-deficient mice. Since Cx36 expression levels change with age, we also tested circadian running-wheel behavior of juvenile (3-4weeks old) and adult (9-30weeks old) Cx36-deficient mice. We found that impact of connexin-36 expression on circadian behavior changes greatly during postnatal development. However, consistent with the intact synchrony among SCN cells in cultured explants, Cx36-deficient mice had intact locomotor circadian rhythms, although adults displayed a lengthened period in constant darkness. Our data indicate that even though Cx36 may be required for electrical coupling of SCN cells, it does not affect coupling of molecular clock gene rhythms. Thus, electrical coupling of neurons and coupling of circadian clock gene oscillations can be regulated independently in the SCN.