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WNT3A Promotes Neuronal Regeneration Upon Traumatic Brain Injury

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International Journal of Molecular Sciences Article WNT3A Promotes Neuronal Regeneration upon Traumatic Brain Injury 1, 1, 2 1 1 Chu-Yuan Chang y, Min-Zong Liang y, Ching-Chih Wu , Pei-Yuan Huang , Hong-I Chen , Shaw-Fang Yet 3, Jin-Wu Tsai 4, Cheng-Fu Kao 5,* and Linyi Chen 1,6,* 1 Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan; [email protected] (C.-Y.C.); [email protected] (M.-Z.L.); [email protected] (P.-Y.H.); [email protected] (H.-I.C.) 2 Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan; [email protected] 3 Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan 35053, Taiwan; [email protected] 4 Institute of Brain Science, National Yang-Ming University, Taipei 11221, Taiwan; [email protected] 5 Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 11574, Taiwan 6 Department of Medical Science, National Tsing Hua University, Hsinchu 30013, Taiwan * Correspondence: [email protected] (C.-F.K.); [email protected] (L.C.); Tel.: +886-3-574-2775 (L.C.); Fax: +886-3-571-5934 (L.C.) These authors contributed equally to this work. y Received: 20 January 2020; Accepted: 19 February 2020; Published: 21 February 2020 Abstract: The treatment of traumatic brain injury (TBI) remains a challenge due to limited knowledge about the mechanisms underlying neuronal regeneration. This current study compared the expression of WNT genes during regeneration of injured cortical neurons. Recombinant WNT3A showed positive effect in promoting neuronal regeneration via in vitro, ex vivo, and in vivo TBI models. Intranasal administration of WNT3A protein to TBI mice increased the number of NeuN+ neurons without affecting GFAP+ glial cells, compared to control mice, as well as retained motor function based on functional behavior analysis. Our findings demonstrated that WNT3A, 8A, 9B, and 10A promote regeneration of injured cortical neurons. Among these WNTs, WNT3A showed the most promising regenerative potential in vivo, ex vivo, and in vitro. Keywords: neuronal regeneration; traumatic brain injury; WNT3A; cortical neurons 1. Introduction Traumatic brain injury (TBI) is a major cause of morbidity and lifelong disability, making it a critical healthcare concern worldwide. Globally, an estimated 69 million individuals per year suffer from TBI, and according to the Centers for Disease Control and Prevention, it accounts for over 30% of injury-related deaths in the United States [1–3]. There is currently no effective treatment that can stimulate regeneration, partly due to the heterogeneity of pathophysiology, severity, and outcomes, so much of the damage/dysfunction in TBI patients becomes permanent [4]. Among over 200 registered ongoing or completed clinical trials for TBI, administration of chemical compounds (e.g., sertraline, amantadine, minocycline), transplantation of autologous bone marrow-derived cells, and other interventions may partially rehabilitate impaired consciousness and/or improve neurocognitive dysfunction. Nevertheless, a large number of these trials have been terminated (ClinicalTrials.gov). So, the medical burden of TBI highlights the pressing need to develop better treatments for brain injury. WNT signaling is an evolutionarily conserved pathway that regulates axis specification of the neural plate, neural tube morphogenesis, dendrite and axon development, and synaptic plasticity [5–7]. Aberrant WNT signaling has previously been implicated in Alzheimer’s disease and Parkinson’s Int. J. Mol. Sci. 2020, 21, 1463; doi:10.3390/ijms21041463 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1463 2 of 12 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 12 disease [8–10]. In this study, we determined the expression of a number of WNT genes during regeneration7]. Aberrant of injured WNT signaling cortical has neurons. previouslyIn been vitro implicatedand in vivoin Alzheimer’sstudies disease examined and Parkinson’s the regenerative disease [8–10]. In this study, we determined the expression of a number of WNT genes during potentialregeneration of WNT3A of uponinjured traumatic cortical neurons. brain injury. In vitro and in vivo studies examined the regenerative potential of WNT3A upon traumatic brain injury. 2. Results 2. Results 2.1. Identification of WNT Genes as RAGs for Axonal Regeneration of Cortical Neurons 2.1. Identification of WNT Genes as RAGs for Axonal Regeneration of Cortical Neurons To evaluate neuronal regeneration, rat cortical neurons were cultured in vitro. On DIV (day in vitro) 8, primaryTo evaluate cortical neuronal neurons regeneration, were injured rat cortical by neurons scraping were lines cultured through in vitro. the cultureOn DIV with(day in a p20 tip vitro) 8, primary cortical neurons were injured by scraping lines through the culture with a p20 tip (Figure1A). It was estimated that 10% neurons were injured via this approach. An anti-mitotic reagent, (Figure 1A). It was estimated that 10% neurons were injured via this approach. An anti-mitotic AraC, wasreag addedent, AraC, to thewas cultureadded to medium the culture to inhibitmedium proliferation to inhibit proliferation of glial cells of glial [11 ,12cells]. Regenerating[11,12]. neuritesRegenerating were tracked neurites for 72 were h after tracked injury. for Regeneration72 h after injury. of Regeneration injured cortical of injured neurons cortical was neurons most was prominent within 48most h (injuredprominent DIV10, within 48 iDIV10) h (injured after DIV10, the iDIV10 injury) after (Figure the 1injuryB). Immunofluorescence (Figure 1B). Immunofluorescence staining using anti-microtubule-associatedstaining using anti-microtubule protein-associated 2 (MAP2, protein marker 2 (MAP2, for dendrites) marker for and dendrites anti-Tau) and (axonal anti-Tau marker) + antibodies(axonal demonstrated marker) antibodies that the demonstrated re-growing that neurites the re were-growing mostly neurites Tau +wereaxons mostly (Figure Tau1C). axons (Figure 1C). Figure 1. Neurite re-growth of primary cortical neurons in an in vitro traumatic brain injury (TBI) Figure 1. Neurite re-growth of primary cortical neurons in an in vitro traumatic brain injury (TBI) model. (A) Schematic flow chart of experimental design. (B) Trajectory of single neurite re-growth model. (A) Schematic flow chart of experimental design. (B) Trajectory of single neurite re-growth was tracked from 0 h (blue) to 72 h (red) after injury using tracking function of AxioVision software was tracked(left). Quantitative from 0 h (blue) results to of 72 average h (red) length after ofinjury re-growing using neurite tracking (blue functionbars) and average of AxioVision cumulative software (left). Quantitativelength (line graph results) are of shown average from length iDIV8 to of iDIV11 re-growing (right). neurite (n = 3, track (blue 12 bars)neurites/exper and averageiment. cumulative) (C) length (lineCortical graph) neurons are were shown fixed fromon iDIV8 iDIV8 to iDIV11 to iDIV11 and were (right). subjected (n =to 3,immunofluorescence track 12 neurites staining/experiment.) (C) Corticalwith neuronsanti-Tau ( weregreen, fixedaxonal on marker iDIV8), anti to iDIV11-MAP2 ( andred, dendritic were subjected marker) toantibodies immunofluorescence, and DAPI (blue, staining with anti-Tau (green, axonal marker), anti-MAP2 (red, dendritic marker) antibodies, and DAPI (blue, nuclei). Merged images are shown. White dashed lines indicate the borders of the injured gap. Scale bar, 50 µm. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 12 Int. J. Mol. Sci. 2020, 21, 1463 3 of 12 nuclei). Merged images are shown. White dashed lines indicate the borders of the injured gap. Scale bar, 50 µm. To identify candidate regeneration-associated genes (RAGs) during regeneration of injured cortical To identify candidate regeneration-associated genes (RAGs) during regeneration of injured neurons, our previous RNA-seq data suggest a subset of WNT genes as candidate RAGs (data not cortical neurons, our previous RNA-seq data suggest a subset of WNT genes as candidate RAGs (data shown),not inshown addition), in addition to pathway-focused to pathway-focused superarray superarray analysis analysis of 88 ofWNTs 88 WNTsand and WNT WNT target target genes, genes given, that ourgiven previous that our data previous has suggested data has thatsuggested WNT that signaling WNT issignaling implicated is implicated in neuronal in neuronal regeneration (dataregeneration not shown). (data Superarray not shown analysis). Superarray showed analysis that showed 48 genes that 48 (54.5%) genes ( were54.5%) up-regulated. were up-regulated. Among them,AmongWNT3 them,, WNT3A, WNT3 WNT4,, WNT3A, WNT5A, WNT4, WNT5B,WNT5A, WNT5B, WNT6, WNT7A,WNT6, WNT7A, WNT8A, WNT8A, WNT8B, WNT8B, WNT9A, WNT9A, WNT9B, and WNT10BWNT9B, andexpression WNT10B expression were up-regulated were up-regulated (fold change (fold change> 1.5) > 1.5 during) during regeneration regeneration (Figure(Figure 2A). Increased2A). expressionIncreased expression of WNT3A, of WNT8A,WNT3A, andWNT8A,WNT9B and onWNT9B iDIV10 on compared iDIV10 compared to DIV10 to was DIV10 validated was by qPCR,validated with the by expression qPCR, with of theWNT3A expressionand ofWNT9B WNT3Ashowing and WNT9B significant showing increase significant (Figure increase2B). (Figure Although the fold2B). increase Although of theWNT fold genesincrease is notof WNT high, genes local is concentrationnot high, local concentration of secreted WNTs of secreted in the WNTs brain
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