CRMP2 improves memory deficits by enhancing the maturation of neuronal dendritic spines after traumatic brain injury

Journal Pre-proof CRMP2 improves memory deficits by enhancing the maturation of neuronal dendritic spines after traumatic brain injury

Yi-Yu Sun, Liang Zhu, Zhao-Liang Sun, Dong-Fu Feng PII:

S0014-4886(20)30084-4

DOI:

https://doi.org/10.1016/j.expneurol.2020.113253

Reference:

YEXNR 113253

To appear in:

Experimental Neurology

Received date:

18 September 2019

Revised date:

12 February 2020

Accepted date:

17 February 2020

Please cite this article as: Y.-Y. Sun, L. Zhu, Z.-L. Sun, et al., CRMP2 improves memory deficits by enhancing the maturation of neuronal dendritic spines after traumatic brain injury, Experimental Neurology (2020), https://doi.org/10.1016/j.expneurol.2020.113253

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© 2020 Published by Elsevier.

Journal Pre-proof CRMP2 improves memory deficits by enhancing the maturation of neuronal dendritic spines after traumatic brain injury

Yi-Yu Sun1,†, Liang Zhu1,†, Zhao-Liang Sun1, Dong-Fu Feng1,2,* 1

Department of Neurosurgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of

Medicine, Shanghai 201999, China 2

Institute of Traumatic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 201999, China †

Yi-Yu Sun and Liang Zhu contributed equally to this article.

*

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Corresponding author at: Department of Neurosurgery, No.9 People's Hospital, Shanghai Jiaotong University

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School of Medicine, Shanghai, 201999, China. Institute of Traumatic Medicine, Shanghai Jiaotong University

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School of Medicine, Shanghai, 201999, China.

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ABSTRACT

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Our recent study investigated the role of collapsin response mediator protein-2 (CRMP2) on dendritic spine morphology and memory function after traumatic brain injury (TBI). First, we examined the density and morphology

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of dendritic spines in Thy1-GFP mice on the 1st day (P1D) and 7th day (P7D) after controlled cortical impact injury (CCI). The dendritic spine density in the hippocampus was decreased on P1D, in which mainly mushroom-type and

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thin-type spines were lost. The density of dendritic spines was increased on P7D, most of which were of the thin type. Next, we explored the expression of CRMP2 on P1D and P7D. CRMP2 expression was decreased on P1D,

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but the levels of the CRMP2 breakdown product were increased. On P7D, the expression pattern was the opposite. Then, we constructed CRMP2 overexpression and knockdown plasmids and transfected them into cultured neurons in vitro. CRMP2 increased the dendritic spine density of cultured neurons and the proportion of mushroom-type spines, while CRMP2-shRNA reduced the dendritic spine density and the proportion of mushroom-type spines. To determine the role of CRMP2 in dendritic spines after TBI, we stereotactically injected the CRMP2 overexpression and knockdown viruses into the hippocampus and found that CRMP2 increased the dendritic spine density and the proportion of mushroom-type spines after TBI. Meanwhile, as suggested by the morphological changes, fear conditioning behavioral experiments confirmed that CRMP2 improved memory deficits after TBI.

KEYWORDS: CRMP2, traumatic brain injury, dendritic spine, learn and memory function

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Abbreviations:

CRMP2 collapsin response mediator protein-2 TBI traumatic brain injury CCI cortical impact injury

Introduction Traumatic brain injury (TBI) is a worldwide public health and socioeconomic problem as well as a major cause of death in adults. Approximately 5.3 million and 7.7 million individuals in the United States and Europe suffer

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from TBI-related sequelae.(Langlois and Sattin, 2005; Tagliaferri et al., 2006) Learning and memory dysfunction are common complications after TBI.(Azouvi et al., 2009; Hoskison et al., 2009)

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Two-photon microscopy has revealed that the stability and plasticity of dendritic spines are closely related to

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learning and memory.(Grutzendler et al., 2002) After TBI, spared neurons present significant morphological and functional changes, including a bead-like appearance of dendritic spines and a decrease in dendritic spine number,

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especially the number of mushroom-type dendritic spines.(Winston et al., 2013; Winston et al., 2016) Dendritic spines are key to synapse formation.(Frotscher et al., 2014; Kasai et al., 2010) Therefore, the decrease in spine

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number after TBI results in a decrease in synapse number and plasticity, thereby affecting learning and memory functions after TBI.(Gao et al., 2011)

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Collapsin response mediator protein-2 (CRMP2) is a microtubule-associated protein(Arimura et al., 2005; Gu et al., 2010; Hensley et al., 2010; Kawano et al., 2005) also known as Ulip2, CRMP-62, TOAD-64, DRP2 or

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DPYSL2 expressed in dendritic spines (Makihara et al., 2016). In central nerve system, it has been found to be

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related with axonal growth, neurotransmitter release and synaptic physiology (Chi et al., 2009; Hou et al., 2009). Deletion of CRMP2 leads to decrease of spine density in cortical neurons and aberrant behaviors which are symptoms of neuropsychiatric disorders (Makihara et al., 2016; Nakamura et al., 2016). Brain-specific deletion of CRMP2 leads to impaired learning and memory (Zhang et al., 2016). Besides, it is proved to have neuroprotective effect (Hensley et al., 2011; Nagai et al., 2016). At present, changes in the morphology of dendritic spines are believed to be caused by cytoskeleton remodeling.(Bellot et al., 2014; Brody et al., 2015; Lei et al., 2016) Although the morphology of dendritic spines is closely related to learning and memory function,(Gipson and Olive, 2017; Segal, 2017) no research thus far has focused on the potential relationship between CRMP2 and the morphological changes of dendritic spines after TBI. Moreover, whether CRMP2 can improve learning and memory after TBI is still unknown. Therefore, we studied the effect of CRMP2 on dendritic spine morphology and learning and memory function after TBI.

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Materials and methods Animals

All procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine and carried out in accordance with the guidelines of the National Institutes of Health regarding animal care. Thy1-GFP mice were donated by professor Xu Nan-Jie, researcher at the Shanghai Jiao Tong University School of Medicine. ICR male mice and E18 mice were purchased from Shanghai Slac Experimental Animal Center.

Plasmid and AAV The Institutional Biosafety Committee of the Shanghai Jiao Tong University School of Medicine approved the

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procedure concerning adeno-associated virus. The shRNA was constructed using the pAKD-CAG-eGFP-U6-

CRMP-2

shRNA

sequences

were

as

follows:

shRNA-1:

CTAATGATGCAATTATGTA,

shRNA-2:

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CCCAACCACTCCAGACTTT.

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shRNA vector. Packages of AAV9-shRNA viruses were provided by Obio Technology, Shanghai, China. The

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Cell culture and transfection

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Neuro2a cells were maintained in DMEM containing 10% FBS. Transfection of indicated plasmids is performed using lipo3000 (Invitrogen) according to the instructions. After 72 hours transfection, cells were lysed

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and collected for WB.

Primary hippocampal neurons were dissected from E18 mice. Briefly, cells were dissociated mechanically

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(trituration through a Pasteur pipette) and enzymatically (with papain and DNase I and resuspended in Neurobasal medium) and plated on poly-D-lysine-coated coverslips. Then, cells were cultured in Neurobasal medium containing

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2% NuSerum, 5% B27, penicillin/streptomycin (100 U/mL; 50 g/mL) and 0.1 mM L-glutamine (Invitrogen). After 48 h of culture, the neurons were infected with pAKD-CAG-eGFP-U6-shRNA using a Ca2+ transfection system (Invitrogen) and seeded at a density of 1 × 105 cells on PDL-coated coverslips in 24-well plates.

Stereotactic injection and controlled cortical impact (CCI) model of TBI ICR male mice were injected with virus 4 weeks after birth. For intracranial viral infection, 1 µl of concentrated AAV was injected into CA1 area (0.2 µl/min) at the following coordinates relative to the bregma: posterior, -2.0 mm; lateral, 1.5 mm; ventral, 1.8 mm. 4 weeks after injection ICR mice and Thy1-GFP male mice (8 weeks old) suffered CCI to model moderate TBI. Mice were anesthetized with 3.5% chloral hydrate (0.1 ml/10 g) by intraperitoneal injection. The animals were mounted on a stereotaxic instrument (RWD Life Science, China) in a prone position and fixed by auxiliary ear and incisor bars. Using sterile procedures, the animals received a midline

Pre-proof cranial skin incision and unilateral (on the leftJournal side) craniotomy (3.5 mm in diameter) 1.5 mm lateral to midline and 1.5 mm posterior to the bregma, directly over the forelimb sensorimotor cortex. The dura mater was kept intact over the cortex. The cortical impact was delivered at a velocity of 3.0 m/s with a depth of 1.0 mm below the cortical surface and an impact duration of 180 ms. After CCI, medical grade cyanoacrylate gel was applied to the exposed dura mater and skull surface. The skin incision was sutured with an absorbable suture, and antibiotic ointment was applied in the suture area. The TBI mice were then wrapped in a heating pad and transferred to a clean cage. All TBI mice were housed individually. Sham injury animals underwent the same craniotomy and postoperative care procedures.

Western blot analysis

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Hippocampal neurons and cultured neurons were harvested, dissected, and solubilized at 4°C in RIPA lysis

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buffer (Beyotime, P0013) supplemented with 1 mM PMSF and protease inhibitor cocktail (Thermo, 78425). The total protein lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-CRMP-2 (1:1000,

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ab129082) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000, Sigma-Aldrich, G8795) antibodies and HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (A0208 and A0216) purchased

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from Beyotime. Analysis of the data was performed using Quantity One software. The mean density of each band

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was normalized to the GAPDH signal in the same sample.

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Immunofluorescence

For immunofluorescence, frozen sections (30 µm-thick) and cell slides were blocked with blocking buffer (1

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× PBS plus 0.3% Triton X-100 and 10% normal goat serum, Jackson ImmunoResearch, 122346) at room temperature for one hour and then incubated with primary antibodies (anti-CRMP-2, 1:500, ab129082) in blocking

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buffer containing 2% goat serum overnight at 4°C. After 3 washes (10 min for each wash) in 1 × TBST, the sections or cells were incubated in the fluorescence-conjugated secondary antibodies goat anti-rabbit Alexa-488 and goat anti-rabbit Cy3 (Thermo, A32731, A10520, 1:500, dilution in 1 × PBS) plus DAPI for 1 h at room temperature. After 3 washes, the chamber slides were then mounted with mounting medium and imaged. Photographed using a confocal microscope (Leica TCS SP8). Fluorescence microscopic images were imported into ImageJ (NIH) for analysis, and all the parameters used were kept consistent during capture.

Quantification and classification of spines To quantify the shape of spine accurately, the antibody signal threshold was defined as three times brightness to the background, and brightness/contrast adjustment within linear ranges was made using ImageJ when necessary, a procedure was adapted from Xu’s study(Xu et al., 2011). Approximately 15–30 neurons from 3 to 4 mice were

Journal Pre-proof analysed. Spines were classified by NeuronStudio software package and an algorithm with the following cutoff values: AR_thin(crit) = 2.5, head-to-neck ratio (HNR(crit)) = 1.3, and head diameter (HD(crit)) = 0.3 μm. Protrusions with length 0.2–3.0 mm and maximum width 3 mm were counted. Acquisition of the images as well as morphometric quantification was performed under ‘blinded’ conditions

Fear conditioning experiments A fear conditioning behavioral paradigm, which can reflect learning and memory function, was conducted 7 days after CCI. Each group containing 10 mice. Mice were habituated to the conditioning chamber (25 × 25 × 25 cm) for 1 h after transport to the behavioural room before any tests were conducted. On the first day, mice were placed in the conditioning boxes to explore freely for 20 min and then returned to its home cage. On the second day,

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the mice were placed in the chamber for 3 min to explore freely, after which an auditory conditioned stimulus (30 s,

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70 db, 3000 Hz) and mild footshock (2 s, 0.5 mA, constant current) were presented, the mouse was taken out 30 s after termination of the foot shock and returned to its home cage. After the experiments, the chamber was cleaned

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with 70% ethanol after each mouse. On the third day, mice were placed in novel conditioning boxes that completely different from the previous conditioning boxes to explore freely for 3 min and then the sound cue was played for 1

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min. During this process, freezing time and freezing number were recorded by digital video cameras mounted above

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the conditioning chamber. Freezing was scored only when the mouse stand still for at least 1s. FreezeFrame and

Transmission electron microscopy

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FreezeView software (Ugo Basile, Italy) were used for recording and analyzing the freezing behavior respectively.

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The brain was dissected, fixed with Karnovsky's fixative (a mixture of 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 24 h at 4°C and embedded in Epon-Araldite

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(Merck,45359). Semithin sections (1-2 µm) of the brain were prepared and stained with toluidine blue; ultrathin sections (60-90 nm) were stained with uranyl acetate and lead citrate. The tissues were examined with a Tecnai™ G² Spirit BioTWIN electron microscope.

Statistical analysis The results are presented as Mean ± S.D. Statistical differences were determined by Student’s t-test for twogroup comparisons or ANOVA followed by Tukey’s test for multiple comparisons among more than two groups

Results Morphological changes of dendritic spines after TBI We performed CCI on Thy1-GFP mice (Fig. 1A). Through immunofluorescence experiments, we found that

Journal Pre-proof dendrites of neurons swelled, fractured and formed a bead-like structure (Fig. 1B) in the cortex and hippocampus 1 day after injury. Dendritic spine density was significantly reduced in the injured side, from 13.91±0.21 to 7.9±0.05 per 10um. Meanwhile, the loss of thin-type dendritic spines was dominant, accounting for 17.98%±0.25 at 1 day post injury. At 7 days after TBI, the neurons in the injured side began to repair (Fig. 1B), showing a reduction in the bead-like structure of neuron processes and an increase in dendritic spine density; however, the increase in dendritic spines was mainly represented by an increase in the thin type (Fig. 1C). Transmission electron microscopy was used to observe changes in the membrane (indicated by red arrows), synapses (indicated by yellow arrows), and mitochondria (indicated by stars) in the injured area (Fig. 1D). One day after TBI, the neurons in the injured area were permeable and the neurite and mitochondrial swelling ratios were increased, accompanied by a decrease in synapse density. Seven days after TBI, neuronal repair had begun, the membranes were more intact, and the neurite

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and mitochondrial swelling ratios had decreased, while synapse density had increased (Fig. 1E).

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Changes in CRMP2 expression after TBI

The amount of CRMP2 protein in the injured hippocampus was detected by Western blot on the 1st and 7th

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day after injury (Fig. 2A). Compared with the sham group, the TBI group exhibited a decrease in CRMP2 expression and an increase in levels of the CRMP2 degradation product (that is calpain-cleaved CRMP2) one day after injury

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but an increase in CRMP2 expression and a decrease in the degradation product at 7 days after injury (Fig. 2B). We

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further detected the difference in CRMP2 expression levels between the injured side and the contralateral side by immunofluorescence and found high immunofluorescence intensity on the injured side 7 days after injury (Fig. 2C).

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At 7 days after TBI, CRMP2 expression was significantly elevated on the injured side and was mainly located in the cortex and hippocampus near the injury site (Fig. 2D). Analysis of the fluorescence intensity revealed a negative

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correlation between the intensity of CRMP2 fluorescence and the distance from the injury point (Fig. 2F). The brightness of CRMP2 in the injured site was significantly higher than that in the control side, and CRMP2 was

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mainly distributed in the neuronal cytoplasm and dendrites. (Fig. 2E). CRMP2 promotes dendritic spine growth and maturation in vitro We constructed a mouse CRMP2 overexpression (CRMP2-OE) plasmid and CRMP2-shRNA-2 plasmid. Neuro2a cells were transfected with the plasmids to verify their effect on CRMP2 expression (Fig. 3A). As a result, CRMP2-OE significantly increased the expression of CRMP2 in cells, and CRMP2-shRNA-2 significantly reduced it (Fig. 3B). Three plasmids, the empty vector plasmid, CRMP2-OE and CRMP2-shRNA-2, were transfected into cultured neurons, which were observed after 14 days (Fig. 3C). CRMP2 increased dendritic spine density in neurons (Fig. 3D), mainly by increasing the number of thin and mushroom-type spines (Fig. 3E). CRMP2-shRNA, on the other hand, reduced dendritic spine density (Fig. 3D). To further verify the validity of the plasmids, we injected AAV containing relevant plasmids to ICR mice, as shown in Fig 3F and 3G it can affect the expression of CRMP2 effectively in vivo.

Journal Pre-proofafter TBI CRMP2 promotes dendritic spine regeneration and maturation CCI was performed 4 weeks after stereotactic injection of AAV9 into the hippocampus of ICR mice. Seven days after injury, we performed immunostaining to calculate the dendritic spine density and classification (Fig. 4A). The dendritic spine density of the TBI + vector group (n=30) was comparable to that of the sham + vector group. However, most of the spines were of the thin type, and mushroom-type dendritic spine density was still lower than that in the sham + vector group. Compared with the TBI + vector group, the group injected with the CRMP2-shRNA (n=30) virus exhibited a lower dendritic spine density (Fig. 4B), mainly showing a reduction in the density of the thin type and mushroom type (Fig. 4C). Furthermore, some spines still presented a swollen state. The density of dendritic spines was higher in the TBI + CRMP2-OE group than in the TBI + vector group (n=30; Fig. 4B), and mushroom-type dendritic spines were dominant. (Fig. 4C)

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CRMP2 promotes recovery of learning and memory function in mice after TBI

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Changes in dendritic spines in the hippocampal region after injury may affect learning and memory function. Fear conditioning is a form of associative learning in which subjects exposed to an aversive stimulus associate a

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neutral stimulus with defense responses, after which this neutral stimulus thus becomes a conditioned stimulus that elicits a conditioned response. Seven days after TBI, we performed fear conditioning behavior experiments on the

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above four groups of experimental mice. The freezing number (Fig. 4D) and freezing time (Fig. 4E) were lower in

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the TBI + vector group than in the sham + vector group. Compared with the TBI + vector group, the TBI + shRNA group exhibited significantly fewer freezing bouts (Fig. 4D) and shorter freezing duration (Fig. 4E), while the pattern

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Discussion

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was opposite in the TBI+CRMP2-OE group (Fig. 4D, 4E).

In vivo studies have found that animals present impaired brain functions to some degree after TBI, (Azouvi et

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al., 2009; Hart and Sander, 2017) accompanied by a decrease in CRMP2 expression.(Campbell et al., 2012; Zhang et al., 2007) Thus, whether rescuing CRMP2 expression could restore brain function appeared to be a particularly meaningful question. Interestingly, Dr. Khanna's team fused a CRMP2 peptide (CBD3) to the HIV-Tat protein to generate an agent that can prevent neural death after TBI in mice.(Brittain et al., 2011) Moreover, Hiroki and his colleague developed a CRMP2-binding compound, edonerpic maleate, which recovered motor function after TBI in macaque monkeys.(Abe et al., 2018) However, the relationship between CRMP2 and learning and memory function after TBI is still unknown. This study revealed that increasing CRMP2 expression led to a significant recovery in the memory function of mice suffering from TBI, thereby broadening the role of CRMP2 in neural repair. Changes in spine morphology may underlie memory formation. Dendritic spines are tiny protrusions from dendritic shafts, which consist of a dense network of cytoskeletal and scaffolding molecules and numerous surface receptors. Based on their morphology, dendritic spines are traditionally divided into three subtypes: stubby spines,

Journal Pre-proof thin spines and mushroom spines.(Gipson and Olive, 2017; Hering and Sheng, 2001; Segal, 2017; Yuste and Bonhoeffer, 2004) Studies have shown a close relationship between spine morphology and function. Thin-type spines are often referred to as ‘immature’ spines due to their dynamic retraction and extension capabilities and limited contribution to synaptic transmission.(Bourne and Harris, 2008; Holtmaat et al., 2005) The long-standing stable dendritic spines are usually mushroom type with a larger spine head, the size of which is positively correlated with synapse activity.(Holtmaat et al., 2005; Paulin et al., 2016; Trachtenberg et al., 2002) Spines suffer degeneration after TBI, usually revealed by a decrease in spine density and a reduction in spine complexity.(Campbell et al., 2012; Gao et al., 2011; Winston et al., 2013; Winston et al., 2016) In addition to a decrease in spine density, we found that there was a relatively greater loss of thin-type dendritic spines than of mushroom-type spines. This phenomenon indicates that thin-type spines are more vulnerable than mushroom-type spines, considering their dynamic

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characteristics. Unlike the proportion of thin- and mushroom-type spins, the proportion of stubby-type spines was

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higher in the TBI group than in the sham group at 1 day after TBI. The same phenomenon was observed in other papers.(Gao et al., 2011; Ratliff et al., 2019; Zhao et al., 2016) Stubby-shaped spines are considered to represent

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either a transitional growth stage between an early and mature spine or the stage during retraction of a mature spine for elimination.(Fischer et al., 1998; Petrak et al., 2005; Portera-Cailliau et al., 2003) Under the adverse environment

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of TBI, most neurons suffer varying degrees of damage, and their spines make corresponding adjustments, such as

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retraction, potentially explaining the increase in stubby-type spines on day 1 after injury. The morphological changes of dendritic spines are mostly caused by cytoskeleton remodeling. CRMP2 can

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affect spine structure and synaptic physiology by regulating microtubule polymerization, actin bundling and vesicle trafficking.(Arimura and Kaibuchi, 2007; Gibbs et al., 2015; Ip et al., 2014) CRMP2 has been widely accepted as a

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neuroprotective molecule after TBI. In our study, CRMP2 expression was decreased at 24 h after TBI and increased 1 week after injury. However, there is no uniform understanding of its changes after TBI. Wilson et al. found that

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the expression of CRMP2 in the injured hippocampus was increased 24 h after TBI,(Wilson et al., 2014) while Zhang et al. found that the amount of CRMP2 protein was decreased and the amount of degradation products was increased at 24 h after injury.(Zhang et al., 2007) Variations in the parameters of CCI cause varying degrees of damage,(Siebold et al., 2018; Zhang et al., 2014) which lead to varying functional deficits.(Xiong et al., 2013) Differences in damage strength between these studies (the former used a 4 mm bone window/1.5 mm impact depth /3.0 m/s impact speed; the latter used a 7 mm bone window/1.6 mm impact depth/3.5 m/s impact speed) may have caused the contradictory results. Additionally, the immunofluorescence results in our study reflected a link between injury intensity and CRMP2 expression. This link may be due to the hyperactivation of N-methyl-D-aspartate receptors (NMDARs) caused by stress in TBI, resulting in a large amount of Ca2+ influx(Ikonomidou et al., 2000) that activates Ca2+-dependent calcium proteases, which in turn shear CRMP2 to produce an approximately 55 KD degradation product.(Zhang et al., 2007) The more calpain produce, the more CRMP2 is degraded.

Previously, Haruko and Zhang found thatJournal CRMP2 -/-Pre-proof mice present impaired learning and memory function and aberrant dendritic spine development.(Nakamura et al., 2016; Zhang et al., 2016) However, to the best of our knowledge, no research has focused on the role of CRMP2 in spine morphology after TBI. In vitro, we demonstrated that CRMP2 not only increased the spine density of injured hippocampal neurons but also improved the proportion of mushroom-type spines. Furthermore, memory function recovery was accompanied by an increase in mushroomtype spines in vivo after TBI. These results may be explained in two ways. On the one hand, as the inflammation and stress faded, the decrease in injury-induced Ca2+ influx may have resulted in less calpain activation, thus allowing the level of CRMP2 to recover gradually. The inactivation of glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) following TBI may account for the decreased phosphorylation of CRMP2, which indirectly promotes the maturation of spines.(Wilson et al., 2014) On the other hand, according to reports, CRMP2

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can bind to actin, which is crucial for synaptic amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR)

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delivery,(Gu et al., 2010; Hensley et al., 2010; Hensley et al., 2011) and AMPAR recruitment is closely related to mushroom-type spine formation in hippocampal neurons,(Matsuo et al., 2008; Matsuzaki et al., 2001) which are

TBI. However, further mechanisms need to be explored.

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often labeled ‘memory spines’. This effect on actin may explain why CRMP2 accelerates memory recovery after

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In summary, we examined the influence of CRMP2 on spine morphology after TBI and found that CRMP2 not

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only increased spine density by also promoted spine maturation after TBI. Furthermore, CRMP2 restored memory

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function after TBI, indicating that CRMP2 may have a broad therapeutic value.

Acknowledgments

Author contribution

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This work was supported by National Natural Science Foundation of China (81372047, 81772059)

Dong-Fu Feng designed research; Yi-Yu sun and Liang Zhu performed research and wrote the paper; ZhaoLiang sun performed data analysis

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Figure 1. Verification of brain injury model and changes in dendritic spines after TBI. (A) Cortical brain slice diagram of Thy1-GFP mice 1 day after CCI. (B) Immunofluorescent images of dendritic spines in hippocampus CA1 at 1 day and 7 days after injury. (C) The statistical results of the above three groups of dendritic spines and their classification percentages. (n=30 neurons from five mice in each group). M=mushroom, S=stubby, T=thin. (D) Electron micrographs of dendritic spines in the hippocampal CA1 region at 1 day postinjury, 7 days after injury, and in the sham injury group. Right panels are magnified pictures from dotted box in left panel

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Red arrows indicate the outline of the cells, yellow arrows indicate PSD, and blue stars indicate mitochondria. (E) Statistical results for PSD density and mitochondrial integrity in (D). (n=30 neurons from five mice in each group).

Figure 2. Expression changes of CRMP2 after TBI

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*P < 0.05, **P < 0.01, and ***P < 0.001

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(A) Western blot results of changes in expression levels of CRMP2 and its degradation products in the sham-

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injured group, 1 day and 7 days after injury. CRMP2 and CRMP2 BDP are normalized to the corresponding GAPDH. (B) is a chart of (A). (C) Immunofluorescence of CRMP2 expression 7 days after injury. (n=5 to 6 slices per group)

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(D) is the diagram of CRMP2 in (C). Other contain midbrain and hypothalamus regions (E) Amplification of the distribution of CRMP2 on the lesion side and contralateral (C) in CA1 hippocampus neurons at the cellular level.

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(F) The intensity of CRMP2 in the (C) and the distance from the injury point presents a negative correlation. *P <

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0.05, **P < 0.01, and ***P < 0.001

Figure 3. CRMP2 promotes neuronal dendritic spines density and maturation (A) Protein expression levels after CRMP2 overexpression and knockdown in vitro. (B) is a statistical analysis of (A), showing that CRMP2 overexpression plasmid can significantly increase the expression of CRMP2 protein, while shRNA-2 can significantly reduce the expression of CRMP2 protein. (C) The empty vector, CRMP2-OE, shRNA-2 plasmids were transferred into cultured neurons to observe the effects on dendritic spines. (n=30 neurons from five mice in each group). (D, E) are dendritic spine densities and classification percentage statistics in (C). (F) Protein expression levels after CRMP2 overexpression and knockdown in vivo. (G) is a statistical analysis of (F). M=mushroom, S=stubby, T=thin. *P < 0.05, **P < 0.01, and ***P < 0.001

Journal Pre-proof Figure 4. CRMP2 promotes regeneration and maturation of dendritic spines after TBI (A) The result of immunofluorescence staining showed the changes of dendritic spines 7 days after injury in the groups of Sham+vector, TBI+vector, TBI+shRNA, and TBI+CRMP2 within CA1 hippocampus neurons. (B, C) are dendritic spine density and percentage statistics of dendritic spine classification of (A). (n=30 neurons from five mice in each group). (D, E) is a statistical graph of freezing in the four groups of conditional fear behavior experiments (n=10 animals per group). M=mushroom, S=stubby, T=thin. *P < 0.05, **P < 0.01, and ***P < 0.001

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Highlights Reveal the changing relationship between CRMP2 expression and the morphology of spines.

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CRMP2 increases spine density and promotes recovery of mushroom type spines after TBI.

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CRMP2 improved memory deficit after TBI.

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