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Dissertation, RWTH Aachen University, 2020; Aachen 1 Online-Ressource (XIII, 93 Seiten) : Illustrationen, Diagramme (2020). = Dissertation, RWTH Aachen University, 2020, Cerebral ischemia is the most common type of stroke and one of the leading causes of disability and death worldwide. Ischemic stroke occurs due to an insufficient blood perfusion to the brain leading to rapid neuronal cell death in the infarct core. The adjacent penumbra region is meta-stable and heterogeneously susceptible to ischemia making it amenable to therapeutic interventions. The currently approved reperfusion modalities are systemic thrombolysis by recombinant tissue plasminogen activator (rtPA) and endo-vascular treatments (e.g. mechanical thrombectomy). Due to their time-dependency they are applicable for only 15% of stroke patients. Neuroprotective approaches have not been successful so far. Modulation of apoptosis and endogenous cytoprotective pathways may provide a promising therapeutic strategy after ischemic stroke. The evolutionary highly conserved and ubiquitously expressed transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) protein family is attributed a significant role in the maintenance of intracellular calcium homeostasis and in the inhibition of apoptosis. TMBIM2/FAIM2 and TMBIM3/GRINA share a similar secondary structure and are found in membranes of the endoplasmic reticulum (ER) and/or in plasma membranes of the central nervous system (CNS). FAIM2 showed neuroprotective and regenerative effects in a murine transient middle cerebral artery occlusion (tMCAo) model and is regulated by erythropoietin (EPO). The role of GRINA in transient brain ischemia, its potential compensatory or synergistic effects with FAIM2 and its regulation by EPO treatment were not elucidated yet. To address this, tMCAo or Sham surgery for 30 minutes followed by a subsequent reperfusion time of 6 h and/or 72 h were performed in GRINA-deficient (Grina-/-), FAIM2-deficient (Faim2-/-), double-deficient (Grina-/-Faim2-/-) and wildtype littermate (WT) mice. EPO or saline were administered 0, 24 and 48 hours after tMCAo or Sham surgery. Neurological out- come was evaluated at various time points. Primary murine cortical neurons (pMCN) of all mouse strains were subjected to oxygen-glucose deprivation (OGD) after Grina and/or Faim2 gene transfection. High expression levels of both TMBIM family members were found in the murine brain. Grina-/- led to a similar increase in infarct volumes as Faim2-/- mice. The double-deficient mice revealed the highest neurological deficits and largest infarct sizes after stroke. EPO administration upregulated Grina and Faim2 mRNA levels in wildtype littermates. EPO significantly decreased infarct sizes and abrogated neurological impairments in WT mice only. While the absence of FAIM2 potentiated the activation of caspase 8, a strong activation of caspase 9 was observed in Grina-/- mice. Likewise, pMCN of FAIM2- and/or GRINA-deficient mice showed a pronounced impaired ischemia tolerance after OGD compared to WT. Over- expression of Grina and Faim2 in WT neurons and the reintroduction of both TMBIM genes alone and in combination in double-deficient neurons significantly decreased the OGD-induced cell death rates. Cerebral ischemia is known to cause the accumulation of misfolded proteins and loss of calcium homeostasis leading to endoplasmic reticulum (ER) stress. This activates an ER- located and cytoprotective pathway, the unfolded protein response (UPR). Since GRINA is mostly located at the ER membrane suppressing ER calcium release by inositol-1,4,5- trisphosphate receptors, we hypothesized that GRINA might play a crucial role in the UPR. Thus, we investigated the influence of GRINA and EPO on the post-ischemic UPR in a second study. We subjected Grina-/- and WT mice to 30 minutes of tMCAo or Sham surgery followed by 6 h or 72 h of reperfusion. We administered EPO or saline 0, 24 and 48 h after tMCAo/sham surgery. OGD and pharmacological stimulation of the UPR using Tunicamycin and Thapsigargin were carried out in primary murine cortical mixed cell cultures. Treatment with the PERK-inhibitor GSK-2606414, IRE1a-RNase-inhibitor STF-083010 and EPO was performed 1 h prior to 1 h, 2 h or 3 h of OGD. This study provided evidence that both the IREα and the PERK branch of the UPR are activated in the early reperfusion phase (6 h) after cerebral ischemia. Moreover, Grina-/- increased apoptosis and the activation of the corresponding PERK arm of the UPR after stroke compared to WT mice. EPO was able to enhance the post-ischemic activation of the protective IREα pathway and attenuated the pro-apoptotic PERK cascade. In addition to EPO, the PERK inhibitor GSK-2606414 also reduced post-ischemic cell death and regulated Grina transcription after ODG. In conclusion, both TMBIM family members play an important role in EPO-mediated neuro- protection after cerebral ischemia. FAIM2 appears to be involved in the extrinsic apoptosis pathway (caspase 8) and GRINA in the intrinsic apoptosis pathway (caspase 9). Further- more, GRINA seems to be implicated in the post-ischemic modulation of the UPR, particularly of the PERK arm. Besides the findings on the mechanism of EPO after cerebral ischemia, our studies also indicate a potential therapeutic relevance of PERK inhibitors after stroke., Published by Aachen

Delayed cell death in the penumbra region of acute ischemic stroke occurs through apoptotic mechanisms, making it amenable to therapeutic interventions. Fas/CD95 mediates apoptotic cell death in response to external stimuli. In mature neurons, Fas/CD95 signaling is modulated by Fas-apoptotic inhibitory molecule 2 (Faim2), which reduces cell death in animal models of stroke, meningitis, and Parkinson disease. Erythropoietin (EPO) has been studied as a therapeutic strategy in ischemic stroke. Erythropoietin stimulates the phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway, which regulates Faim2 expression. Therefore, up-regulation of Faim2 may contribute to neuroprotection by EPO. Male Faim2-deficient mice (Faim2(-/-)) and wild-type littermates (WT) were subjected to 30 min of middle cerebral artery occlusion (MCAo) followed by 72 h of reperfusion. EPO was applied before (30 min) and after (24 and 48 h) MCAo. In WT mice application of EPO at a low dose (5000 U/kg) significantly reduced stroke volume, whereas treatment with high dose (90 000 U/kg) did not. In Faim2(-/-) animals administration of low-dose EPO did not result in a significant reduction in stroke volume. Faim2 expression as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) increased after low-dose EPO but not with high dose. An extensive phenotyping including analysis of cerebral vessel architecture did not reveal confounding differences between the genotypes. In human post-mortem brain Faim2 displayed a differential expression in areas of penumbral ischemia. Faim2 up-regulation may contribute to the neuroprotective effects of low-dose erythropoietin in transient brain ischemia. The dose-dependency may explain mixed effects of erythropoietin observed in clinical stroke trials.