Neeraj K. Tiwari

Cerebral hypoperfusion is associated with enhanced cognitive decline and increased risk of neuropsychiatric disorders. Erythropoietin (EPO) is a neurotrophic factor known to improve cognitive function in preclinical and clinical studies of neurodegenerative and psychiatric disorders. However, the clinical application of EPO is limited due to its erythropoietic activity that can adversely elevate hematocrit in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered non-erythropoietic derivative of EPO, does not alter hematocrit and maintains neurotrophic and behavioral effects comparable to EPO. Our study aimed to investigate the role of CEPO in cerebral hemodynamics. Magnetic resonance imaging (MRI) analysis indicated increased blood perfusion in the hippocampal and striatal region without altering tight junction integrity. In vitro and in vivo analyses indicated that hippocampal neurotransmission was unaltered and increased cerebral perfusion was likely due to EDRF, CGRP, and NOS-mediated vasodilation. In vitro analysis using human umbilical vein endothelial cells (HUVEC) and hippocampal vascular gene expression analysis showed CEPO to be a non-angiogenic agent which regulates the MEOX2 gene expression. The results from our study demonstrate a novel role of CEPO in modulating cerebral vasodilation and blood perfusion.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. This can be challenging when the researched protein lacks reliable antibodies. Here, we combine Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. This method is called Breasi-CRISPR (Brain Easi-CRISPR), and enables knock-in of both short and long epitope tag sequences in genes of interest with high efficiency. Using Breasi-CRISPR, we were able to visualize epitope tagged proteins known to have either high or low expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1, and RPL22. Detection was possible by immunohistochemistry as soon as one day after electroporation at embryonic day 13 (E13). Two and five days after electroporation, we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices two days after electroporation. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores in an efficient manner, allowing the visualization of endogenous proteins via live-imaging in organotypic brain slices two days after electroporation. Finally, we used Breasi-CRISPR to perform co-IP mass-spectrometry analyses of tagged autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data show Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

In recent years, erythropoietin (EPO) has emerged as a useful neuroprotective and neurotrophic molecule that produces antidepressant and cognitive-enhancing effects in psychiatric disorders. However, EPO robustly induces erythropoiesis and elevates red blood cell counts. Chronic administration is therefore likely to increase blood viscosity and produce adverse effects in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered modification of EPO, is non-erythropoietic but retains the neurotrophic and neurotrophic activity of EPO. Blood profile analysis after EPO and CEPO administration showed that CEPO has no effect on red blood cell or platelet counts. We conducted an unbiased, quantitative, mass spectrometry-based proteomics study to comparatively investigate EPO and CEPO-induced protein profiles in neuronal phenotype PC12 cells. Bioinformatics enrichment analysis of the protein expression profiles revealed the upregulation of protein functions related to memory formation such as synaptic plasticity, long term potentiation (LTP), neurotransmitter transport, synaptic vesicle priming, and dendritic spine development. The regulated proteins, with roles in LTP and synaptic plasticity, include calcium/calmodulin-dependent protein kinase type 1 (Camk1), Synaptosomal-Associated Protein, 25 kDa (SNAP-25), Sectretogranin-1 (Chgb), Cortactin (Cttn), Elongation initiation factor 3a (Eif3a) and 60S acidic ribosomal protein P2 (Rplp2). We examined the expression of a subset of regulated proteins, Cortactin, Grb2 and Pleiotrophin, by immunofluorescence analysis in the rat brain. Grb2 was increased in the dentate gyrus by EPO and CEPO. Cortactin was induced by CEPO in the molecular layer, and pleiotrophin was increased in the vasculature by EPO. The results of our study shed light on potential mechanisms whereby EPO and CEPO produce cognitive-enhancing effects in clinical and preclinical studies.

Radial neuron migration in the developing cerebral cortex is a complex journey, starting in the germinal zones and ending in the cortical plate. In mice, migratory distances can reach several hundreds of microns, or millimeters in humans. Along the migratory path, radially migrating neurons slither through cellularly dense and complex territories before they reach their final destination in the cortical plate. This task is facilitated by radial glia, the neural stem cells of the developing cortex. Indeed, radial glia have a unique bipolar morphology, enabling them to serve as guides for neuronal migration. The key guiding structure of radial glia is the basal process, which traverses the entire thickness of the developing cortex. Neurons recognize the basal process as their guide and maintain physical interactions with this structure until the end of migration. Thus, the radial glia basal process plays a key role during radial migration. In this review, we highlight the pathways enabling neuron-basal process interactions during migration, as well as the known mechanisms regulating the morphology of the radial glia basal process. Throughout, we describe how dysregulation of these interactions and of basal process morphology can have profound effects on cortical development, and therefore lead to neurodevelopmental diseases.