Ischemic Post-Conditioning Induces Post-Stroke Neuroprotection via Hsp70-Mediated Proteasome Inhibition and Facilitates Neural Progenitor Cell Transplantation.

In view of the failure of pharmacological therapies, alternative strategies promoting post-stroke brain repair are needed. Post-conditioning is a potentially promising therapeutic strategy, which induces acute neuroprotection against ischemic injury. To elucidate longer lasting actions of ischemic post-conditioning, mice were exposed to a 60-min stroke and post-conditioning by an additional 10-min stroke that was induced 10 min after reperfusion onset. Animals were sacrificed 24 h or 28 days post-stroke. Post-conditioning reduced infarct volume and neurological deficits 24 h post-stroke, enhancing blood-brain barrier integrity, reducing brain leukocyte infiltration, and reducing oxidative stress. On the molecular level, post-conditioning yielded increased Hsp70 expression, whereas nuclear factor (NF)-κB and proteasome activities were decreased. Reduced infarct volume and proteasome inhibition were reversed by Hsp70 knockdown, suggesting a critical role of the Hsp70 proteasome pathway in ischemic post-conditioning. The survival-promoting effects of ischemic post-conditioning, however, were not sustainable as neuroprotection and neurological recovery were lost 28 days post-stroke. Although angioneurogenesis was not increased by post-conditioning, the favorable extracellular milieu facilitated intracerebral transplantation of neural progenitor cells 6 h post-stroke, resulting in persisted neuroprotection and neurological recovery. Thus, post-conditioning might support brain repair processes, but in view of its transient, neuroprotection is unlikely useful as stroke therapy in its current form.

MicroRNA-124 protects against focal cerebral ischemia via mechanisms involving Usp14-dependent REST degradation.

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.

Enhancement of endogenous neurogenesis in ephrin-B3 deficient mice after transient focal cerebral ischemia.

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.