RNA Binding Protein HuD Regulates Proliferation and Differentiation of Adult Neural Stem Cells

1. RNA Binding Protein HuD Regulates Proliferation and Differentiation of Adult Neural Stem Cells HuD Nestin-GFP Merge GFAP/Tuj1/DAPI WT Weixiang Guo1, Eric Polich1, Amy Gardiner2, Nora I. Perrone-Bizzozero2and Xinyu Zhao1 Waisman Centerand Department of Neuroscience University of Wisconsin-Madison, Madison, Wisconsin, United States of America 2. Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America Merge DCX HuD KO HuD Abstract Adult neurogenesis in the adult brain has been shown to exist primarily in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. These two distinct pools of neural stem cells (NSCs) generate different subtypes of neurons and respond independently to environmental cues. However, the mechanisms that differentially controls neurogenesis in these two brain regions are still unclear. The RNA binding protein HuD is a regulator of neuronal differentiation and involved in paraneoplastic encephalomyelitis disorders with learning deficits. Here, we found HuD has distinct expression patterns in these two neurogenic regions. HuD is expressed throughout the process of SVZ neurogenesis from NSCs to mature neurons, whereas HuD is only expressed in NSCs and doublecortin-positive immature neurons in the DG. We found that HuD exhibits differential regulatory roles in NSCs derived from these two neurogenic regions. Furthermore, we found HuD may regulate SVZ-NSCs and DG-NSCs differentiation through different molecular mechanisms. Taken together, investigation of the differential regulation in the SVZ and DG NSCs by HuD will not only shed light on a mechanism that governs the different neurogenesis in these two neurogenic zones, but also provide mechanistic insights into paraneoplastic encephalomyelitis. Results • HuD expression is different in the Subventricular Zone • HuD expression is different in the Dentate Gyrus a,b a Merge HuD Nestin-GFP Merge b DCX HuD Dapi Figure 4. HuD expression pattern in DG in vivo as analyzed using HuD immunostaining of Nestin-GFP transgenic mouse brain. a. HuD is not expressed in radial glia cells (arrow), but HuD is expressed in the Nestin positive type 2A cells (NPCs) (arrow head). b. HuD is expressed in the Doublecortin (DCX) positive immature neuron but not mature neurons of the granule cell layer. Background • HuDInhibits Neuronal Differentiation in the Dentate Gyrus HuD Knock-down DG NSCs HuD Overexpression DG NSCs a b Astocyte differentiation Neuronal differentiation Astocyte differentiation Neuronal differentiation a SVZ • HuD Promotes Neuronal Differentiation in the Subventricular Zone c DAPI a b HuD exp. HuD expression * c b a b RMS Figure 5. HuD inhibits neuronal differentiation in the DG NSCs. HuD knock down using shRNAin DG NSCs has no effect on astrocyte differentiation but increases neuronal differentiation compared to the control. b. HuD overexpression in DG NSCs slightly decreases astrocyte differentiation and inhibits neuronal differentiation compared to the control. Data were obtained using luciferase assays as described in Fig 3. Summary Olfactory Bulb neurogenesis Hippocampal neurogenesis c OB • HuD demonstrates distinct expression patterns in the SVZ and the DG NSCs. • Adult NSCs lacking or deficient in HuD showed increased neuronal differentiation in the DG • Adult NSCs lacking or deficient in HuD showed decreasedneuronal differentiation in the SVZ. • Understanding this unique relationship could be critical for understanding of the unique regulation of separate NSC populations and and provide insight into the mechanisms of of neurodegenerative diseases, injury, and memory. • Future Direction: • In vivo NSC differentiation assay for DG neurogenesis • NSC proliferation assay for SVZ and DG. • Identification of HuD targets in SVZ and DG. • Adult Neurogenesis – Implications • Neurodevelopmental Diseases • e.g. Fragile X syndrome, Rett, Asperger’s (Autism spectrum disorders) • Brain Injury and Repair • e.g. Stroke, Trauma • Learning and Memory • e.g. Special, Episodic • Neurodegenerative Diseases • e.g. Alzheimer’s, Huntington’s, Parkinson’s Figure 3. HuD is required for neuronal differentiation in SVZ NSCs. a. Quantitative immunocytochemistry using cell lineage markers indicate that SVZ-NSCs isolated from HuD mutant mice exhibit reduced neuronal differentiation, with no change in astrocyte differentiation. b. HuD knock down using shRNAin SVZ NSCs has no effect on astrocyte differentiation but decreases neuronal differentiation compared to the control, as assessed by neuronal (NeuoD1) and astrocyte (GFAP) promoter driven luciferase assays. c. HuD overexpression in SVZ NSCs has no effect on astrocyte differentiation but increased neuronal differentiation compared to the control as assessed by luciferase assays. Figure 2. HuD expression pattern in SVZ, RMS, and OB in vivo using immunostaining in nesting GFP mouse brain. a. HuD is co-localized in Nestin positive NSCs. b. HuD is co-localized with DCX positive neuroblasts in RMS. c. HuD is expressed in the GCs of the olfactory bulb (OB) References • HuD • [1]E Gould.Nature Reviews Neuroscience 8, 481-488 (June 2007) | doi:10.1038/nrn2147 • [2] Fuentealba LC, Obernier K, Alvarez-Buylla A: Adult neural stem cells bridge their niche. • Cell Stem Cell 2012, 10(6):698-708. • [3] C. Colombrita, V. Silani, A. Ratti. ELAV proteins along evolution: back to the nucleus? Mol. Cell. Neurosci., 56 (2013), pp. 446–454 • ELAV or Hu family of genes • RNA binding protein that binds to AU rich element and regulates mRNA stability • elavproteins are proposed to function by binding to specific mRNAs and regulating their expression to control developmental programs [3] • involved in paraneoplastic encephalomyelitis disorders with learning deficits • Precise mechanism behind this regulation is still unknown Figure 1. Adult neurogenesis is known to predominantly occur in two distinct regions in the adult mammalian brain. These regions are the subventricular zone (SVZ) of the lateral ventricles, and the dentate gyrus (DG) of the hippocampus. Many of these stem cells generated in the SVZ will migrate via the rostral migratory stream to the olfactory bulb (OB) where they will undergo terminal differentiation to form GABAergic inhibitory neurons. In contrast the NSC in the subgranular layer of the DG differentiate into neurons that migrate a short distance into the granule cell layer to form glutamatergic excitatory neurons and incorporate into existing neural networks. [1] These two regions of active neural stem (NSC) and progenitor cell (NPC) proliferation have created two distinct pools of stem cells, each with a unique environment. [2] Acknowledgements Technical support from Yina Xing, IsmatBhuiyan, Dan Magyar, and JanessaMladucky This work was funded by NIH grants R01MH080434 and R01MH078972 to XZ; funds from Waisman Center, UW Graduate School, UW School of Medicine and Public Health, and WARF to XZ, R01NS30255 to NPB, a center grant from the NIH to the Waisman Center (P30HD03352); a postdoctoral fellowship from University of Wisconsin Center for Stem Cells and Regenerative Medicine to W.G. and an undergraduate Hilldale fellowship from University of Wisconsin to E.P.