MicroRNAs (miRNAs) are highly conserved non-coding RNAs modulating gene expression via mRNA binding. Recent work suggests an involvement of miRNAs in cardiovascular diseases including stroke. As such, the brain-abundant miR-124 and its transcriptional repressor RE1-silencing transcription factor (REST) do not only have elementary roles in the developing and the adult brain, but also alter expression upon cerebral ischemia. However, the therapeutic potential of miR-124 against stroke and the mechanisms involved remain elusive. Here, we analyzed the therapeutic potential of ectopic miR-124 against stroke and its underlying mechanisms with regard to the interaction between miR-124 and REST. Our results show that viral vector-mediated miR-124 delivery increased the resistance of cultured oxygen-glucose-deprived cortical neurons in vitro and reduced brain injury as well as functional impairment in mice submitted to middle cerebral artery occlusion. Likewise, miR-124 induced enhanced neurovascular remodeling leading to increased angioneurogenesis 8 weeks post-stroke. While REST abundance increased upon stroke, the increase was prevented by miR-124 despite a so far unknown negative feedback loop between miR-124 and REST. Rather, miR-124 decreased the expression of the deubiquitinating enzyme Usp14, which has two conserved miR-124-binding sites in the 3'UTR of its mRNA, and thereby mediated reduced REST levels. The down-regulation of REST by miR-124 was also mimicked by the Usp14 inhibitor IU-1, suggesting that miR-124 promotes neuronal survival under ischemic conditions via Usp14-dependent REST degradation. Ectopic miR-124 expression, therefore, appears as an attractive and novel tool in stroke treatment, mediating neuroprotection via a hitherto unknown mechanism that involves Usp14-dependent REST degradation.

Intravenous transplantation of neural progenitor cells (NPCs) induces functional recovery after stroke, albeit grafted cells are not integrated into residing neural networks. However, a systematic analysis of intravenous NPC delivery at acute and post-acute time points and their long-term consequences does not exist. Male C57BL6 mice were exposed to cerebral ischemia, and NPCs were intravenously grafted on day 0, on day 1 or on day 28. Animals were allowed to survive for up to 84 days. Mice and tissues were used for immunohistochemical analysis, flow cytometry, ELISA and behavioral tests. Density of grafted NPCs within the ischemic hemisphere was increased when cells were transplanted on day 28 as compared with transplantation on days 0 or 1. Likewise, transplantation on day 28 yielded enhanced neuronal differentiation rates of grafted cells. Post-ischemic brain injury, however, was only reduced when NPCs were grafted at acute time points. On the contrary, reduced post-ischemic functional deficits due to NPC delivery were independent of transplantation paradigms. NPC-induced neuroprotection after acute cell delivery was due to stabilization of the blood–brain barrier (BBB), reduction in microglial activation and modulation of both peripheral and central immune responses. On the other hand, post-acute NPC transplantation stimulated post-ischemic regeneration via enhanced angioneurogenesis and increased axonal plasticity. Acute NPC delivery yields long-term neuroprotection via enhanced BBB integrity and modulation of post-ischemic immune responses, whereas post-acute NPC delivery increases post-ischemic angioneurogenesis and axonal plasticity. Post-ischemic functional recovery, however, is independent of NPC delivery timing, which offers a broad therapeutic time window for stroke treatment.

In view of its profound effect on cell survival and function, the modulation of the ubiquitin-proteasome-system has recently been shown to promote neurological recovery and brain remodeling after focal cerebral ischemia. Hitherto, local intracerebral delivery strategies were used, which can hardly be translated to human patients. We herein analyzed effects of systemic intraperitoneal delivery of the proteasome inhibitor BSc2118 on neurological recovery, brain injury, peripheral and cerebral immune responses, neurovascular integrity, as well as cerebral neurogenesis and angiogenesis in a mouse model of transient intraluminal middle cerebral artery occlusion. Systemic delivery of BSc2118 induced acute neuroprotection reflected by reduced infarct volume when delivered up to 9 h post-stroke. The latter was associated with reduced brain edema and stabilization of blood-brain-barrier integrity, albeit cerebral proteasome activity was only mildly reduced. Neuronal survival persisted in the post-acute stroke phase up to 28 days post-stroke and was associated with improved neurological recovery when the proteasome inhibitor was continuously delivered over 7 days. Systemic proteasome inhibition prevented stroke-induced acute leukocytosis in peripheral blood and reversed the subsequent immunosuppression, namely, the reduction of blood lymphocyte and granulocyte counts. On the contrary, post-ischemic brain inflammation, cerebral HIF-1α abundance, cell proliferation, neurogenesis, and angiogenesis were not influenced by the proteasome inhibitor. The modulation of peripheral immune responses might thus represent an attractive target for the clinical translation of proteasome inhibitors.

Cerebral ischemia stimulates endogenous neurogenesis. However, the functional relevance of this phenomenon remains unclear because of poor survival and low neuronal differentiation rates of newborn cells. Therefore, further studies on mechanisms regulating neurogenesis under ischemic conditions are required, among which ephrin-ligands and ephrin-receptors (Eph) are an interesting target. Although Eph/ephrin proteins like ephrin-B3 are known to negatively regulate neurogenesis under physiological conditions, their role in cerebral ischemia is largely unknown. We therefore studied neurogenesis, brain injury and functional outcome in ephrin-B3−/− (knockout) and ephrin-B3+/+ (wild-type) mice submitted to cerebral ischemia. Induction of stroke resulted in enhanced cell proliferation and neuronal differentiation around the lesion site of ephrin-B3−/− compared to ephrin-B3+/+ mice. However, prominent post-ischemic neurogenesis in ephrin-B3−/− mice was accompanied by significantly increased ischemic injury and motor coordination deficits that persisted up to 4 weeks. Ischemic injury in ephrin-B3−/− mice was associated with a caspase-3-dependent activation of the signal transducer and activator of transcription 1 (STAT1). Whereas inhibition of caspase-3 had no effect on brain injury in ephrin-B3+/+ animals, infarct size in ephrin-B3−/− mice was strongly reduced, suggesting that aggravated brain injury in these animals might involve a caspase-3-dependent activation of STAT1. In conclusion, post-ischemic neurogenesis in ephrin-B3−/− mice is strongly enhanced, but fails to contribute to functional recovery because of caspase-3-mediated aggravation of ischemic injury in these animals. Our results suggest that ephrin-B3 might be an interesting target for overcoming some of the limitations of further cell-based therapies in stroke.