Stroke is the leading cause of adult disability worldwide. The majority of stroke survivors are left with devastating functional impairments for which few treatment options exist. Recently, a number of studies have used ectopic expression of transcription factors that direct neuronal cell fate with the intention of converting astrocytes to neurons in various models of brain injury and disease. While there have been reports that question whether astrocyte-to-neuron conversion occurs in vivo, here, we have asked if ectopic expression of the transcription factor Neurod1 is sufficient to promote improved functional outcomes when delivered in the subacute phase following endothelin-1-induced sensory–motor cortex stroke. We used an adeno-associated virus to deliver Neurod1 from the short GFAP promoter and demonstrated improved functional outcomes as early as 28 days post-stroke and persisting to at least 63 days post-stroke. Using Cre-based cell fate tracking, we showed that functional recovery correlated with the expression of neuronal markers in transduced cells by 28 days post-stroke. By 63 days post-stroke, the reporter-expressing cells comprised ~20% of all the neurons in the perilesional cortex and expressed markers of cortical neuron subtypes. Overall, our findings indicate that ectopic expression of Neurod1 in the stroke-injured brain is sufficient to enhance neural repair.

Stroke is the leading cause of adult disability with few treatment options for stroke survivors. Astrocyte reprogramming to neurons enables the targetedin vivogeneration of new cells at the site of injury and represents a novel approach for brain repair. A number of studies have demonstrated successful conversion of astrocytes to neurons in various models of brain injury and disease; however, the impact of this strategy on tissue and functional outcome following stroke is not well established. Using AAV delivery of the transcription factor NeuroD1, we reprogrammed astrocytes 7 days after endothelin-1 induced cortical stroke, and studied the long-term cellular and functional outcomes. We found that by 63 days post-stroke, 20% of neurons in the perilesional cortex were reprogrammed. Furthermore, reprogrammed neurons had matured into regionally appropriate neuronal subtypes. Importantly, this treatment was associated with improved functional outcome using the foot fault test and gait analysis. Together, our findings indicate thatin vivoreprogramming is a promising regenerative approach for stroke repair.

SUMMARY: Stroke is the second leading cause of death and disability worldwide. Ischemic stroke mobilizes adult neural stem cells (NSCs) out of the quiescent state. The multifaceted responses of endogenous NSCs to ischemic stroke involve proliferation, migration, and differentiation. Hence one strategy which could be leveraged for recovery after ischemic stroke is the intrinsic mechanism of endogenous NSC mobilization. However, the survival rate of recruited endogenous NSCs is low. Moreover, the intercellular signals that activate NSCs after ischemic stroke are poorly understood. We hypothesized that after stroke, cells located in the cerebral lesion send signals to the NSC niche to initiate the regenerative response. To test this hypothesis, we used CellChat to computationally infer the cell-cell communication between the ischemic infarct region and ventricular-subventricular zone (V-SVZ) NSC niche from spatial gene expression profiles. We identified ligand-receptor pairs and signaling pathways involved in the signal transduction events at 2, 10, and 21 days after stroke. Out of several candidate genes of interest we identified, here we reported the regulatory function of galectin-9 on the proliferation of NSCs. Our present work portrays galectin-9 as a checkpoint signaling molecule that guards the responses of NSCs under physiological conditions and potentially during the recovery phase post-ischemic stroke. We suggest that TIM-3 mediates the inhibitory effect of galectin-9 on NSC proliferation and propose a working hypothesis that the stroke-induced proinflammatory factors stimulate the Toll-like receptor 4 (TLR4) on ependymal cells and result in the increased secretion of galectin-9, which in turn modulates neighboring NSCs. Our study paves the way for potential therapeutic approaches which leverage the TLR4 and galectin-9/TIM3 signaling pathways.