Delayed Neuregulin-1 Treatment Initiates a Late Neuroprotective Early Regenerative and Autophagic Spatial Profiles in Mice following Ischemic Stroke

Abstract

Abstract Neuregulin-1 (NRG-1) has been shown to be neuroprotective and anti-inflammatory in rodent models following focal brain ischemia. However, the spatial transcriptional mechanisms involved in the effects of NRG-1 have not been investigated. In this study, we examined the spatial cellular and neuroinflammatory mechanisms employed by NRG-1 following stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO). Animals were treated with NRG-1 (5ug/kg/day NRG-1 β) or vehicle beginning 24 hours after surgery, and then sacrificed at 3 days post-ischemia (dpi). Using GeoMX DSP technology, we examined the relative expression of 73 proteins in selected regions of interest (ROI) within the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in controls and NRG-1 treated mice. FJB staining showed that delayed NRG-1 treatment did not significantly alter overall neuronal death, but it had profound effects on the neuroregenerative outgrowth environment. The ipsilateral core ROI demonstrated the most altered proteomic profile with 37 differentially regulated proteins related to autophagy and mitophagy, immune cell phagocytosis, and synaptic activity. NRG-1 upregulated synaptophysin by 2.10-fold while marginally rescuing neuronal proteins NeuN and MAP2. NRG-1 treatment induced the upregulation of ULK1, LC3B, ATG5, PINK1, and Park7 within the core region, suggesting successful autophagic flux restoration. NRG-1 treatment also increased Mertk, CD9, CSF1R/CD115, MHCII and Myelin Basic Protein (MBP). Phospho-Tau (S199) was increased 57-fold following stroke and further increased to 90-fold after NRG-1 treatment. The peri-infarct region showed increased astrocyte proteins along with BAG3, CD204/MSR1, phospho-Tau (S199) and Ki67, a marker of cell proliferation. Together, our findings showed that mechanisms of neuroprotection by NRG-1 following stroke occur in distinct spatial domains of the injured brain following ischemia. The data suggest that NRG-1 provides protection against further neuronal apoptosis and priming of an anti-inflammatory, neuroregenerative niche in a spatial manner, which could lead to novel treatment strategies for stroke.