Increased Frataxin Levels Protect Retinal Ganglion Cells After Acute Ischemia/Reperfusion in the Mouse Retina In Vivo

Citation: Schultz R, Witte OW, Schmeer C. Increased frataxin levels protect retinal ganglion cells after acute ischemia/reperfusion in the mouse retina in vivo. Invest Ophthalmol Vis Sci. 2016;57:4115-4124. DOI:10.1167/iovs.16-19260 PURPOSE. The mitochondrial protein frataxin (FXN) is highly expressed in metabolically active tissues and has been shown to improve cell survival in response to oxidative stress after ischemia. Retinal ischemia/hypoxia is a complication of ocular diseases such as diabetic retinopathy and glaucoma. There are no effective therapeutic approaches currently available. This study was performed to evaluate the neuroprotective effects of FXN after acute retinal ischemia/reperfusion in vivo. METHODS. Retinal ischemia/reperfusion was induced in adult wild-type and FXN-overexpressing mice by transient elevation of intraocular pressure (IOP) for 45 minutes. Expression of FXN was evaluated by quantitative (q)RT-PCR and Western blot analysis between 6 and 48 hours after ischemia. Retinal ganglion cell (RGC) survival was determined with immunofluorescent staining and fluorescence microscopy 14 days after lesion. Expression of hypoxiainducible factors Hif-1a and Hif-2a and of oxidative stress markers heme oxygenase-1 (Hmox1), glutathione peroxidase 1 (Gpx1), superoxidase dismutase 1 and 2 (Sod1, Sod2), and catalase was evaluated by qRT-PCR. RESULTS. Endogenous FXN levels were upregulated for up to 24 hours after retinal ischemia in vivo. Retinal ganglion cell survival was significantly improved in FXN-overexpressing mice 14 days after ischemia. Expression of antioxidative enzymes Gpx1, Sod2, and catalase was significantly increased in FXN-overexpressing mice after lesion. CONCLUSIONS. Retinal FXN levels are increased in response to ischemia. Furthermore, elevated FXN levels had a clear neuroprotective effect as shown by increased ganglion cell survival after acute retinal ischemia/reperfusion. Frataxin's neuroprotective effect was associated with an upregulation of antioxidative enzymes. The data suggest that FXN induces neuroprotection by decreasing oxidative stress. Keywords: frataxin, retinal ischemia, retinal ganglion cell, antioxidants F rataxin (FXN) is a nuclear-encoded mitochondrial protein highly conserved among eukaryotes. The mouse gene (Frda) encodes a 207-amino acid protein showing 73% amino acid identity to its human counterpart. 1 It is abundantly expressed in metabolically active tissues such as liver, skeletal and cardiac muscle, and brain. 1 In the retina of normal mice, immunoreactivity to FXN is found in the external and internal plexiform layers, the ganglion cell layer, and the inner nuclear layer. 2 Frataxin deficiency is closely related to Friedreich ataxia (FRDA), a neurodegenerative disease associated with abnormal influx of iron into the mitochondria, which increases the susceptibility of the nervous system to oxidative stress. 14 There is evidence indicating that increasing FXN reduces the effects of oxidative stress and increases cell survival after oxidative stress-induced cell death. 17 A hallmark of the pathologic alteration in retinal ischemia is the generation of excessive reactive oxygen species (ROS) during reperfusion, which is involved in neuronal cell death. To date, the effect of ischemia on FXN levels and of FXN overexpression on oxidative stress levels and neuronal survival after acute retinal ischemia/reperfusion has not been evaluated. The aims of this study were to determine in vivo the effect of a transient ischemia/reperfusion on the expression of retinal FXN, whether increased FXN levels improve retinal ganglion cell (RGC) survival after an ischemic lesion, and the effect of increased FXN on the expression of antioxidative enzymes after ischemia. iovs.arvojournals.org

Optimized Protocol for Proportionate CNS Cell Retrieval as a Versatile Platform for Cellular and Molecular Phenomapping in Aging and Neurodegeneration

Efficient purification of viable neural cells from the mature CNS has been historically challenging due to the heterogeneity of the inherent cell populations. Moreover, changes in cellular interconnections, membrane lipid and cholesterol compositions, compartment-specific biophysical properties, and intercellular space constituents demand technical adjustments for cell isolation at different stages of maturation and aging. Though such obstacles are addressed and partially overcome for embryonic premature and mature CNS tissues, procedural adaptations to an aged, progeroid, and degenerative CNS environment are underrepresented. Here, we describe a practical workflow for the acquisition and phenomapping of CNS neural cells at states of health, physiological and precocious aging, and genetically provoked neurodegeneration. Following recent, unprecedented evidence of post-mitotic cellular senescence (PoMiCS), the protocol appears suitable for such de novo characterization and phenotypic opposition to classical senescence. Technically, the protocol is rapid, efficient as for cellular yield and well preserves physiological cell proportions. It is suitable for a variety of downstream applications aiming at cell type-specific interrogations, including cell culture systems, Flow-FISH, flow cytometry/FACS, senescence studies, and retrieval of omic-scale DNA, RNA, and protein profiles. We expect suitability for transfer to other CNS targets and to a broad spectrum of engineered systems addressing aging, neurodegeneration, progeria, and senescence

Memory-Like Responses of Brain Microglia Are Controlled by Developmental State and Pathogen Dose

Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1β, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p \u3c 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGF- β, MSR1, and IL-4 in newborn microglia (p \u3c 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation

Small Extracellular Vesicles from Peripheral Blood of Aged Mice Pass the Blood-Brain Barrier and Induce Glial Cell Activation

Extracellular vesicles (EVs), including small EVs (sEVs), are involved in neuroinflammation and neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Yet, increased neuroinflammation can also be detected in the aging brain, and it is associated with increased glial activation. Changes in EV concentration are reported in aging tissues and senescence cells, suggesting a role of EVs in the process of aging. Here, we investigated the effect of peripheral sEVs from aged animals on neuroinflammation, specifically on glial activation. sEVs were isolated from the peripheral blood of young (3 months) and aged (24 months) C57BL/6J wildtype mice and injected into the peripheral blood from young animals via vein tail injections. The localization of EVs and the expression of selected genes involved in glial cell activation, including Gfap , Tgf- β , Cd68 , and Iba1 , were assessed in brain tissue 30 min, 4 h, and 24 h after injection. We found that sEVs from peripheral blood of aged mice but not from young mice altered gene expression in the brains of young animals. In particular, the expression of the specific astrocyte marker, Gfap , was significantly increased, indicating a strong response of this glial cell type. Our study shows that sEVs from aged mice can pass the blood-brain barrier (BBB) and induce glial cell activation

In Situ Dividing and Phagocytosing Retinal Microglia Express Nestin, Vimentin, and NG2 In Vivo

BACKGROUND: Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin(+) cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo. METHODOLOGY/PRINCIPAL FINDINGS: For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin(+) microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin(+) microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU(+) and nestin(+), although no further local cell proliferation occurred. In addition, nestin(+) microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin(+)NG2(+) microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons. CONCLUSIONS/SIGNIFICANCE: The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed

Amitosenescence and Pseudomitosenescence: Putative New Players in the Aging Process

Replicative senescence has initially been defined as a stress reaction of replication-competent cultured cells in vitro, resulting in an ultimate cell cycle arrest at preserved growth and viability. Classically, it has been linked to critical telomere curtailment following repetitive cell divisions, and later described as a response to oncogenes and other stressors. Currently, there are compelling new directions indicating that a comparable state of cellular senescence might be adopted also by postmitotic cell entities, including terminally differentiated neurons. However, the cellular upstream inducers and molecular downstream cues mediating a senescence-like state in neurons (amitosenescence) are ill-defined. Here, we address the phenomenon of abortive atypical cell cycle activity in light of amitosenescence, and discuss why such replicative reprogramming might provide a yet unconsidered source to explain senescence in maturated neurons. We also hypothesize the existence of a G0 subphase as a priming factor for cell cycle re-entry, in analogy to discoveries in quiescent muscle stem cells. In conclusion, we propose a revision of our current view on the process and definition of senescence by encompassing a primarily replication-incompetent state (amitosenescence), which might be expanded by events of atypical cell cycle activity (pseudomitosenescence)

Statins modulate heat shock protein expression and enhance retinal ganglion cell survival after transient retinal ischemia/reperfusion in vivo. Invest Ophthalmol Vis Sci

PURPOSE. To evaluate putative mechanisms for the pleiotropic effects of statins, the expression of members of the heat shock family of proteins (HSPs) was compared between normal and ischemic rat retinas after transient retinal ischemia/reperfusion and statin treatment in vivo. METHODS. Retinal ischemia/reperfusion was induced by transient elevation of intraocular pressure (IOP). Retinal expression of HSPs was evaluated at different time points after drug and solvent injection and retinal ischemia/reperfusion by means of PCR and Western blot analysis. Immunofluorescent staining and confocal laser scanning microscopy were used to localize the expression of HSPs in normal and ischemic retinas. RESULTS. During the acute phase after retinal ischemia, ␣B-crystallin protein and mRNA expression were reduced after statin treatment. After 72 hours of reperfusion, statins increased the expression of ␣B-crystallin and reduced the expression of HSP27 in the retina. Increased expression of ␣B-crystallin early after lesion and statin delivery correlated with increased expression of the heat shock factors 1 and 2. Statins significantly enhanced retinal ganglion cell (RGC) survival 10 days after transient retinal ischemia in vivo. CONCLUSIONS. Systemic delivery of statins after a transient period of retinal ischemia significantly modulated HSP expression in the retina and enhanced RGC survival. Together, these results support the notion that statins constitute a feasible therapeutic approach to prevent some of the neuronal damage in the acute and possibly also the delayed phase and have beneficial effects in central nervous system (CNS) disorders directly affecting the visual system. (Invest Ophthalmol Vis Sci