REGENERATIVE MEDICINE

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1 REGENERATIVE MEDICINE TIMP3 Attenuates the Loss of Neural Stem Cells, Mature Neurons and Neurocognitive Dysfunction in Traumatic Brain Injury STUART L. GIBB, a,b YUHAI ZHAO, c DANIEL POTTER, a,b MICHAEL J. HYLIN, c ROBERTA BRUHN, a,b GYULNAR BAIMUKANOVA, a,b JING ZHAO, c HASEN XUE, d MOHAMED ABDEL-MOHSEN, a,b SATISH K. PILLAI, a,b ANTHONY N. MOORE, c EVAN M. JOHNSON, c CHARLES S. COX JR, d PRAMOD K. DASH, c SHIBANI PATI a,b Key Words. Tissue inhibitor of matrix metalloproteinase-3 Mesenchymal stem cell Neural stem cell Traumatic brain injury Hippocampus a Blood Systems Research Institute, San Francisco, California, USA; b Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA; c Department of Neurobiology and Anatomy and d Department of Pediatric Surgery and Institute for Molecular Medicine, The University of Texas Health Sciences Center at Houston, Houston, Texas, USA Correspondence: Shibani Pati, M.D., Ph.D., Blood Systems Research Institute, 270 Masonic Avenue, San Francisco, California, USA. Telephone: ; Fax: ; spati@bloodsystems.org. Received April 17, 2015; accepted for publication August 7, 2015; first published online in STEM CELLS EXPRESS August 24, VC AlphaMed Press /2014/$30.00/ /stem.2189 ABSTRACT Mesenchymal stem cells (MSCs) have been shown to have potent therapeutic effects in a number of disorders including traumatic brain injury (TBI). However, the molecular mechanism(s) underlying these protective effects are largely unknown. Herein we demonstrate that tissue inhibitor of matrix metalloproteinase-3 (TIMP3), a soluble protein released by MSCs, is neuroprotective and enhances neuronal survival and neurite outgrowth in vitro. In vivo in a murine model of TBI, intravenous recombinant TIMP3 enhances dendritic outgrowth and abrogates loss of hippocampal neural stem cells and mature neurons. Mechanistically we demonstrate in vitro and in vivo that TIMP3-mediated neuroprotection is critically dependent on activation of the Akt-mTORC1 pathway. In support of the neuroprotective effect of TIMP3, we find that intravenous delivery of recombinant TIMP3 attenuates deficits in hippocampal-dependent neurocognition. Taken together, our data strongly suggest that TIMP3 has direct neuroprotective effects that can mitigate the deleterious effects associated with TBI, an area with few if any therapeutic options. STEM CELLS 2015;33: SIGNIFICANCE STATEMENT TBI is the main cause of death worldwide in ages 1-44 with few if any effective therapeutic options to treat patients. Our data suggest that TIMP3, a protein released by Mesenchymal Stem Cells may be a worthy therapeutic candidate. We report that TIMP3 has direct and potent effects on neuronal survival and neurocognitive function post TBI. This study builds upon our previous work that showed TIMP3 is protective against blood brain barrier compromise in the disease. Our findings report a critical role of the Akt-mTORC1 pathway and that inhibition of this pathway abrogates the neuroprotective effect of TIMP3. INTRODUCTION Traumatic brain injury (TBI) is the number one cause of death and disability in the United States between the ages of 1 and 44 with approximately 53,000 deaths annually [1]. However, deaths are only a fraction of the actual number of cases. According to the CDC [2], there are approximately 1.7 million TBIs per year. In addition to US civilian cases, recent conflicts in Iraq and Afghanistan have resulted in an additional 230,0001 TBIs for military personnel [3]. Patients that survive TBI are faced with chronic post-injury symptoms such as motor and sensory deficits [4], impaired cognitive capability [5] and neuropsychological symptoms such as anxiety and depression [6]. Furthermore, there is a substantial increased risk of developing epilepsy [7, 8]. Aside from the initial gross structural damage, TBI pathology is greatly exacerbated by secondary effects that continue months to years later [9]. On a cellular level, the primary injury results in damage to the blood brain barrier (BBB) and to neurons. Traumatic axonal injury, the loss of connection between neurons causes dysfunction in neural circuitry and is thought to contribute to the long-lasting neurological deficits of TBI [1, 10]. The secondary wave of injury is dominated by the initiation of inflammatory responses by resident STEM CELLS 2015;33: VC AlphaMed Press 2015

2 Gibb, Zhao, Potter et al microglia and infiltrating immune cells [9, 11]. Continued disruption of the BBB allows for entry of blood-born immune cells into the brain and the subsequent release of inflammatory mediators. The hippocampus is a key structure in the brain known to be involved in learning and memory. Damage to the human hippocampus, known to occur following TBI [5, 12] results in significant cognitive deficits [4, 13]. The hilar neurons of the Figure 1. VC AlphaMed Press 2015

3 3532 TIMP3 Treatment is Neuroprotective Post-TBI dentate gyrus are especially vulnerable to TBI [6, 14 16]. Loss of these interneurons disrupts hippocampal circuitry and causes neurocognitive deficits [7, 8, 16]. Furthermore, loss of inhibitory neurons in the hilus of the dentate gyrus could potentially decrease the threshold for post-tbi epilepsy, a condition that develops in some patients [7, 8]. Moreover, experimental studies have shown that doublecortin (DCX) positive neural stem cells in the dentate gyrus die soon after TBI, which may also contribute to TBI-induced cognitive deficits. Unfortunately, effective treatments to reduce these deleterious consequences of TBI are currently not available. Rodent models of TBI can capture some of these deleterious consequences and display deficits in cognitive tasks that are hippocampal-dependent [17]. Treatment of TBI injured mice with mesenchymal stem cells (MSCs) has been shown to attenuate hippocampal-dependent cognitive deficits [17]. The specific mechanism of cognitive support provided by MSC treatment is unknown. Our lab has previously reported that MSC treatment has potent therapeutic effects on TBI injured mice by decreasing BBB permeability and enhancing vascular stability [18, 19]. We subsequently identified the MSC released protein, tissue inhibitor of matrix metalloproteinase-3 (TIMP3), as a critical factor mediating the protective effect on the BBB [19]. TIMP3 is one of four members of the TIMP family of proteins originally characterized as inhibitors of matrix metalloproteinases (MMPs), the principal extracellular matrix-degrading proteinases. Changes in extracellular matrix composition are necessary for tissue remodeling and repair, subsequently TIMPs are expressed throughout the body in all tissues [20, 21]. However, it is now appreciated that the activities of TIMPs are not restricted to MMP inhibition alone. To date, activities of TIMP3 known to be independent of metalloproteinase inhibition include induction of apoptosis in a number of cancer cell lines [22 24] and the inhibition of angiogenesis [21]. Most interestingly, we found that intravenous (IV) delivery of recombinant TIMP3 could recapitulate in part the beneficial effects of MSCs on the BBB [19]. Moreover, sirna inhibition of TIMP3 expression in MSCs abrogates their protective effects on post-tbi BBB permeability. Since the exact mechanisms of action of TIMP3 in TBI are unknown, we sought to determine if TIMP3 might have direct effects on neuronal function and survival, aside from its effects on the BBB post- TBI. The goal and design of this study was to determine if IV TIMP3 administration could ameliorate neuronal loss and attenuate neurocognitive deficits following TBI and if so, what would be the location and mechanism underlying such effects. To this end this study has demonstrated that IV TIMP3 can impart a neuroprotective effect and enhance neurite outgrowth both in vitro and in brain-injured animals in vivo by activating the Akt-mTORC1 signaling pathway. In an established mouse model of controlled cortical impact [19, 25] we demonstrate in vivo neuroprotection was associated with improved cognitive function. Taken together our data strongly suggests that TIMP3 has translational potential as a neuroprotective agent to mitigate the deleterious effects of TBI, a disease area with few effective therapeutic options. MATERIALS AND METHODS Study Design In order to study mesenchymal stem cell derived factor TIMP3 as a potential therapeutic in a mouse model of TBI we conducted multifaceted and temporally separated analysis. Analysis of phosphorylation events initiated by TIMP3 treatments were conducted on tissue from animal sacrificed 1 hour after the last injection (3 days post-tbi). Analysis of structural and cell type specific population changes were conducted 7 days post-tbi. This time point was chosen to (a) enable a clear assessment of the effects of IV TIMP3 treatment post-tbi and (b) avoid the inclusion of ongoing population changes in the acute phase of injury. The design of the behavioral experiments was intended to assess the effects of TIMP3 on several relevant measures of neurocognitive function post- TBI. Anxiety was conducted 3-days post-tbi. Learning and memory deficits were analyzed using the Morris water maze (MWM) on day 8 and 9 post-tbi and context-dependent fear discrimination conducted days post-tbi. In experimental studies of TBI, anxiety-like behaviors can be observed within days of injury [26, 27] as indicated by reduced exploratory behaviors in the elevated plus maze and the open field test. Furthermore, this task is not overly sensitive to TBI-induced motor disturbances and can be tested during the acute phase of injury. Performance in the MWM can be heavily influenced by motor dysfunction. As previous studies have shown that controlled cortical impact (CCI)-induced motor deficits typically subside by day 3 5 post-injury [28], water maze training and testing was carried out on days 8 and 9 post-injury. Both spatial learning and memory and contextual fear conditioning depend on the function of the hippocampus. As such, testing these behaviors simultaneously could lead to task interference. Thus, fear conditioning was carried out on day 14 postinjury, 4 days after the completion of the MWM task in order to minimize the potential for task interference. Figure 1. Intravenous TIMP3 penetrates into parenchyma post-tbi and preserves vulnerable neuronal populations of the hippocampus. (A) Coronal sections show target coverage following IV administration of infrared tagged TIMP3. Bar indicates the intensity of the distribution. Representative images shows penetrance into the ipsilateral hippocampus of TBI injured animals but not for sham controls n 5 2. (B) NeuN staining of post-mitotic mature neurons 7 days post-tbi. Representative images of ipsilateral hippocampi from the 3 groups show NeuN staining in the granule cell layer (GCL) and Hilus (HL) of the dentate gyrus. Highlighted areas show enhanced view of NeuN1ve hilar interneurons. Scale bar lm. Lower panel, quantification of NeuN1ve neurons in the HL. n 5 4, 4 section per animal, mean 6 s.e.m. TBI resulted in a significant reduction in NeuN1ve cells on the ipsilateral side which was attenuated by TIMP3 treatment (One-way ANOVA: F (2,9) , p , Bonferroni p and 0.012, respectively). (C) DCX staining of neural stem cells 7 days post-tbi. Representative images of ipsilateral hippocampi from the 3 groups showing DCX staining in the subgranule zone (SGZ) of the dentate gyrus. Highlighted areas show enhanced view of DCX positive immature neurons. Scale bar lm. Lower panel, quantification of DCX1ve neurons, n 5 4, 4 sections per animal, mean 6 s.e.m. TBI resulted in a significant reduction in ipsilateral DCX1ve cells compared to sham controls, which was attenuated by TIMP3 treatment (One-way ANOVA: F (2,9) , p , Bonferroni p and 0.032, respectively). (* 5 p < 0.05, ** 5 p < 0.01). VC AlphaMed Press 2015 STEM CELLS

4 Gibb, Zhao, Potter et al Statistical Analysis Data involving three or more groups were analyzed by oneway ANOVA with multiple comparisons using the Bonferroni post hoc test. Student s t tests (paired and unpaired) were used as appropriate. Statistical significance was set a priori at p <.05. Analyses were prepared using Microsoft Excel 14.5 and StataMP CCI Mouse Model The mouse CCI injury model was used as previously described [19]. In brief, mice were anesthetized with 5% isoflurane and 1:1 O 2 :N 2 mixture. Animals were mounted on a stereotaxic frame and were secured by ear bars and an incisor bar. Anesthesia was maintained with 2.0% isoflurane and 1:1 O 2 :N 2 mixture. A 5 mm diameter craniotomy was made midway between bregma and lambda on the right side, with the medial edge of the craniotomy 1 mm lateral to midline. Injury was produced using a magnetic impactor mounted at an angle of 1108 from the vertical plane. A single impact at a velocity of m/second was used inflict a moderate to severe injury ( mm impact depth). After injury, the incision was closed. Sham animals underwent identical surgeries except for impact injury. Core body temperature was monitored using a rectal thermometer and maintained at C with a heating pad throughout the procedure. For treatments, TIMP3 (60 lg/kg) or vehicle control (PBS) administration was performed at 1, 24, and 72 hours after injury via tail vein injections in a total volume of 125 ll. Animal research was performed with approval of the Institutional Animal Care and Use Committee at ISIS Services LLC (San Carlos, CA). Mice received humane care according to the criteria outlined by the National Research Council s Institute of Laboratory Animal Resources in the Guide for the Care and Use of Laboratory Animals. Neurocognitive testing conducted at UT Houston was in compliance with University of Texas Houston Institutional Animal Care and Use Committee. Further methodology can be found in the Supporting Information Materials section. RESULTS Intravenous TIMP3 Prevents TBI-Induced Loss of Postmitotic Neurons and Neural Stem Cells in the Dentate Gyrus of the Hippocampus TBI in known to cause the death of both postmitotic mature neurons and neural stem cells resulting in neurocognitive deficits [14 16, 29], importantly investigations into post-injury treatment with MSCs have shown an attenuation of these deficits [17, 30 32]. As we had previously reported a therapeutic effect for the MSC released protein TIMP3 in TBI on BBB permeability [19], we investigated if TIMP3 could also have neuroprotective effects. We first asked whether IV delivered TIMP3 could penetrate into the parenchyma. Using an infra-red tagged version of TIMP3, we demonstrate that post- TBI administration of IV TIMP3 can penetrate into the ipsilateral hippocampus of injured mice but not in sham controls (Fig. 1A). The pattern of penetrance, reflective of the injury allows for target coverage and contact of exogenously delivered TIMP3 with cells of the hippocampus and the potential for direct neuroprotective effects. The hilus of the dentate gyrus is home to a heterogeneous population of GABAergic inhibitory interneurons that filter information from the granule cell layer neurons before transmission onto the CA3 region. Loss of these interneurons disrupts hippocampal circuitry and causes neurocognitive deficits [16]. Staining of coronal tissue sections through the damaged dorsal hippocampal with notable cortical damage for the neuronal marker NeuN demonstrated a significant reduction in hilar neurons that was abrogated by IV TIMP3 treatment (Fig. 1B). There were no significant differences on the contralateral side for either group (representative images shown in Supporting Information Fig. 1A). To further examine the effects of TIMP3 on neuronal cells, we quantified the presence of neural stem cells by DCX staining. Neural stem cells exist in the subgranule zone (SGZ) of the hilus and give rise to immature neurons that then migrate into the granule cell layer and integrate into the brains neural circuitry [33]. Staining of ipsilateral sections from the dorsal hippocampus for DCX revealed a significant loss of positive stained cells post-tbi that was attenuated by IV TIMP3 treatment (Fig. 1C). There were no significant differences on the contralateral side (representative images shown in Supporting Information Fig. 1B). Although we do not rule out the possibility that some of these DCX positive cells arise secondary to enhanced neurogenesis post-tbi, the degree of dendritic arborization observed in the DCX1ve population, argue against this possibility (Supporting Information Fig. 1C). Zhao and colleagues have clearly demonstrated extensive arborization takes 3 4 weeks to occur [34], thereby suggesting that these cells assessed at day 7 are unlikely to be derived from newly born neurons. Another population of neurons that has previously been reported to show significant loss post-tbi are those of the granule cell layer. Witgen and colleagues using a fluid percussion injury mouse model of TBI reported a significant reduction in neurons 7 days post-tbi [16]. However, we did not find significant differences with our CCI model (Supporting Information Fig. 1D). TIMP3 Activates Akt-mTORC1 Signaling in Neurons In Vitro Having determined that TIMP3 can penetrate into the parenchyma and has a protective effect on hippocampal neurons, we sought to further understand this observation at the molecular level. Our initial goal was to delineate direct neuronal effects from possible indirect effects of TIMP3 on endothelial integrity and BBB permeability [19]. To determine this we directly treated neurons in vitro with TIMP3 and simultaneously assessed 39 unique signaling nodes using Pathscan antibody signaling arrays from Cell Signaling Technologies (Danvers, MA, com). Treatment of differentiated SH-SY5Y cells, a neuronallike human cell line, with TIMP3 for 15 minutes resulted in a significant activation of the pro-survival kinase Akt (Fig. 2A). To confirm our observation and exclude the possibility of a false-positive result, the experiment was repeated with analyses performed by Western blot (Fig. 2B) and by phosflow cytometry (Fig. 2C). Both techniques confirmed our initial findings. To relate these findings to our mouse model and VC AlphaMed Press 2015

5 3534 TIMP3 Treatment is Neuroprotective Post-TBI Figure 2. TIMP3 activates Akt-mTORC1 signaling in neurons. (A) Fluorescent antibody signaling array. 15 mins treatment with 1 lg/ml TIMP3 activates phospho-akt (Ser473) in SH-SY5Y cells. (Paired t-test P 5 < 0.001). n 5 4, mean 6 s.e.m. (B) Western blot. 15 mins treatment with 1 lg/ml TIMP3 activates p-akt (Ser473) in SH-SY5Y cells. (Paired t-test P 5 <0.05). n 5 4, mean 6 s.e.m. (C) Phosflow cytometry. 15 mins (Upper panel) and 45 minutes (Lower panel) of treatment with 1 lg/ml TIMP3 activates p-akt (Ser473) in SH-SY5Y cells. n 5 4, mean 6 s.e.m. (Paired t-test, P 5 < 0.05). (D) Fluorescent antibody signaling array. 15 mins treatment with 1 lg/ml TIMP3 activates p- Akt (Ser473) in primary hippocampal neurons. n 5 4, mean 6 s.e.m. (Paired t-test, P 5 < 0.01). (E) Western blot. 15 mins treatment with 1 lg/ml TIMP3 activates p-akt (Ser473) in primary hippocampal neurons. n 5 4, mean 6 s.e.m. (Paired t-test, P 5 < 0.01). (F) Signaling diagram indicating the steps of the Akt-mTORC1 pathway between Akt and S6 Ribosomal Protein (S6RP). (G) Fluorescent antibody signaling array shows 15 mins treatment with 1 lg/ml TIMP3 phosphorylates mtorc1 pathway target S6 Ribosomal Protein (S235/236) in SH-SY5Y cells (Left panel, Paired t-test, P 5 < 0.05) and primary hippocampal neurons (Right panel, Paired t-test, P 5 < 0.05). n 5 4, mean 6 s.e.m. (H) Western blot confirms 15 mins treatment with 1 lg/ml TIMP3 activates S6 Ribosomal Protein (S235/236) in SH-SY5Y cells (Left panel, Paired t-test, P 5 < 0.001) and primary hippocampal neurons (Right panel, Paired t-test, P 5 < 0.01). n 5 4, mean 6 s.e.m. (* 5 p < 0.05, ** 5 p < 0.01, *** p 5 < 0.001). VC AlphaMed Press 2015 STEM CELLS

6 Gibb, Zhao, Potter et al avoid cell lines versus primary cultures disparities, we repeated the experiment using primary hippocampal neurons. Pathscan analysis on homogenates of TIMP3 treated hippocampal neurons also determined that TIMP3 directly activates Akt in primary cells (Fig. 2D), which was additionally confirmed by Western blot analysis (Fig. 2E). Figure 3. VC AlphaMed Press 2015

7 3536 TIMP3 Treatment is Neuroprotective Post-TBI Activation of Akt is quintessential for the survival of neurons with its many breakpoints against the apoptotic machinery [35]. However, we noted additional hits from the Pathscan arrays downstream of Akt that did not fall into an anti-apoptotic category. One such observation was a terminal branch point of the mtorc1 pathway, S6 ribosomal protein (S6RP) (Fig. 2F). The activation of S6RP has been previously reported to be involved in regeneration of axons following injury [36] and axonal injury is a significant part in the pathology of TBI [10]. Increased array signals for phospho-s6rp were observed for both SH-SY5Y cells and isolated primary hippocampal neurons (Fig. 2G), which we also confirmed by Western blot (Fig. 2H). Intravenous TIMP3 Activates the Akt-mTORC1 Pathway In Vivo Post-TBI Using the TBI and IV TIMP3 delivery paradigm applied previously, we sought to determine whether TIMP3 could activate the Akt-mTORC1 pathway in vivo in the hippocampus post- TBI. Animals were sacrificed on day 3 of the procedure, 1 hour after the last TIMP3 injection based upon in vitro data on the point of maximal phosphorylation. As shown (Fig. 3A, 3B), Akt and S6RP phosphorylation levels in homogenates from the ipsilateral hippocampi post-tbi were found to be significantly higher following IV TIMP3 treatment than with TBI alone. No differences were observed between treatments for Akt and S6RP phosphorylation on the contralateral side (Supporting Information Fig. 2A, 2B). Previous studies have reported elevated S6RP phosphorylation in the hippocampus 24 hours post-tbi [37, 38]. However, in our experiments at 3 days post-injury we did not find significant differences in S6RP phosphorylation between sham and TBI samples (Supporting Information Fig. 2C). Previous work by Park and colleagues [37] showed increased S6RP phosphorylation in the hippocampus in microglia and astrocytes using a mouse model of TBI suggesting that this was part of the pathology of TBI. Chen and colleagues using a rat model had also observed increased S6RP phosphorylation post-tbi but in neuronal subfields [38]. Because of these mixed results we sought to determine the location or cell type where the mtorc1 pathway is activated following TIMP3 treatment. Animals were sacrificed 1 hour after the last dose of TIMP3 and tissue sections were stained for phosphorylated S6RP. As shown, (Fig. 3C) we observed strong staining for phosphorylated S6RP in the non-neuronal molecular layer of the ipsilateral hippocampus only in TBI mice with no treatment. This staining, predominantly colocalized to microglia was also present in astrocytes but to a lesser extent (Supporting Information Fig. 2D). Neither sham controls nor TIMP3 treated TBI mice showed this pattern of staining (Fig. 3C). However, in TIMP3 treated mice an increase in phospho-s6rp was found in neurons of the granule cell layer of the dentate gyrus, but not along the SGZ (Fig 3D). An increase in phospho-s6rp was also observed in neurons of the CA3 region (Fig. 3E). Interestingly, we observed that ipsilateral hippocampi from TBI mice showed an almost complete absence of phospho-s6rp staining in neurons of the dentate gyrus, levels that were below those observed in sham controls (Fig. 3D). Taken together, these results suggest that IV TIMP3 administered post-tbi promotes S6RP activity in hippocampal neurons in direct contrast to observations from TBI-alone mice and also prevents S6RP activity in microglia and astrocytes. TIMP3 Promotes Both Neuronal Survival and Neurite Outgrowth In Vitro Activation of the Akt-mTORC1 signaling pathway in neurons has the potential to promote both protection and neurite outgrowth. As post-tbi hypoxia is known to exacerbate neuronal death [39], we investigated the neuroprotective capability of TIMP3 using a hypoxic chamber. In our paradigm, exposure of SH-SY5Y cells to 5 hours of hypoxia followed by 18 hours of recovery at normoxia significantly reduced viability as assessed by MTT assay. However, pretreatment with TIMP3 for 1 hour significantly attenuated cell death (Fig. 4A). As inhibition of Akt causes death of SH-SY5Y cells [40], we could not directly assess the role of TIMP3-induced Akt activation through use of specific Akt inhibitors. However, inhibition of the mtor pathway in neurons has been shown to only result in reduced neurite outgrowth without a decrease in cell survival [41]. As expected, the mtorc1 inhibitor rapamycin did not affect the ability of TIMP3 to inhibit neuronal cell death (Fig. 4B). However, at a concentration that completely blocked S6RP phosphorylation, rapamycin pretreatment resulted in an elevated level of phospho-akt. This result, due to a feedback loop [42, 43] correlated with a mild but nonsignificant increase in neuroprotection (Fig. 4B, 4C). Taken together these data suggest that TIMP3 has a neuroprotective effect that involves Akt but not the mtorc1 pathway. Activation of the mtorc1 pathway in injured retinal ganglia neurons has been shown to induce neurite regeneration whereas pathway suppression results in a failure to regenerate neurites [36]. To determine if TIMP3 induced mtorc1 pathway activation could result in neurite outgrowth, we used a colorimetric neurite outgrowth assay. Culturing SH-SY5Y cells on semiporous membranes allows for neurites to grow through the pores and for direct assessment of neurite outgrowth. As shown, treatment of SH-SY5Y cells with TIMP3 resulted in a significant increase in neurite outgrowth that Figure 3. Intravenous TIMP3 activates the Akt-mTORC1 pathway in the hippocampus in vivo. TIMP3 administration (60 lg/kg) was performed via tail-vein injection at 1 hr, 24 hrs and 72 hrs post-tbi. Animals were sacrificed on day 3, 1 hr after last injection. (A) Western blot. TIMP3 treatment significantly elevates both phospho-akt (Ser473) and (B) phospho-s6 Ribosomal Protein (S235/236) in ipsilateral hippocampal homogenates. n 5 8, mean 6 s.e.m. (Unpaired t-test, P 5 <0.01). (C) Representative images showing the interface between the granule cell layer (GCL) and the molecular layer (Mol.layer) of the dentate gyrus proximal to the site of injury. Phosphorylated S6RP (S235/ 236) (green) in the non-neuronal molecular layer was only detected in TBI-injured animals. Scale bar lm. (D) Quantification of phospho-s6rp (S235/236) (green)/neun+ve (red) neurons in the GCL proximal to injury site. n 5 4, 4 sections per animal, mean 6 s.e.m. Analysis show a significant reduction with TBI (One-way ANOVA: F (2,9) , p , Bonferroni p ) and an elevation with TIMP3 treatment (p<0.0001). Scale bar lm. (E) Quantification of phospho-s6rp (S235/236) (green)/neun+ve (red) neurons in area CA3. n 5 4, 4 sections per animal, mean 6 s.e.m. Scale bar lm. Analysis show a reduction with TBI and a significant elevation with TIMP3 treatment. (One-way ANOVA: F (2,9) , p<0.0001, Bonferroni p and p<0.0001, respectively). (** 5 p<0.01, *** p 5 <0.001). VC AlphaMed Press 2015 STEM CELLS

8 Gibb, Zhao, Potter et al was completely abrogated by cotreatment with rapamycin (Fig. 4D). To visualize this increase on hippocampal neurons we used neurite chamber slides that allow for the plating of cell bodies in one chamber and neurites to grow through channels into the next chamber under a flow gradient. Seven days of culture with TIMP3 resulted in a clear increase in the density and complexity of neurites (Fig. 4E). In order to quantify differences between treatments on individual neurons, we analyzed sparsely populated neuronal cultures following 72 hours treatment with TIMP3. Sholl analysis conducted on Figure 4. VC AlphaMed Press 2015

9 3538 TIMP3 Treatment is Neuroprotective Post-TBI images of isolated neurons (See Materials and Methods section) demonstrated that treatment resulted in a significantly more complex neurite network (Fig. 4F). As with SH-SY5Y, the increase in neurite outgrowth during TIMP3 treatment was significantly attenuated by rapamycin treatment (Fig. 4G), representative images shown in Supporting Information Figure 3A. To further determine the involvement of mtor in the enhancement of neurite outgrowth by TIMP3 we investigated the effects of short hairpin RNA (shrna) against mtor. The sequence we choose had been previously reported to successfully knockdown mtor in hippocampal neurons [44]. As a control, we also created a scrambled control sequence (SCR) (Supporting Information Fig. 3B, 3C). Validation experiments using real time PCR demonstrated that the shrna sequence was capable of reducing mtor message levels by approximately 50% compared to SCR (Supporting Information Fig. 3D). Following transfection, neurons were cultured for 7 days. Successfully transfected neurons were identified by GFP expression before Sholl analysis was conducted. As shown in Figure 4H shrna against mtor resulted in a significant decrease in neurite outgrowth compared to SCR (representative images shown in Supporting Information Fig. 3E). Taken together, these results suggest that the mtor pathway is critical for the effects of TIMP3 and that both rapamycin and shrna are equivocal in their impact on TIMP3 promoted neurite outgrowth. Intravenous TIMP3 Treatment Preserves Neuronal Projections In Vivo in the Molecular Layer of the Dentate Gyrus Following TBI Given our observations on neuronal preservation (Fig. 1), neuronal signaling in vitro and in vivo and (Figs. 2, 3), neurite outgrowth in vitro (Fig. 4) and the studies reporting mtorc1/ S6RP activation leads to neurite outgrowth [36, 41, 45, 46] we sought to determine how IV TIMP3 treatment affects neurite outgrowth and neuronal connections in vivo post-tbi. To assess the effects of IV TIMP3, Western blot analysis was conducted to determine protein levels of the neuronal specific cytoskeletal marker b3-tubulin, a marker previously used to assess in vitro neurite outgrowth (Fig. 4D 4H). Homogenates from the ipsilateral hippocampi 7 days post-tbi showed a small but consistent and significant reduction in the total hippocampal level of b3-tubulin that was attenuated by IV TIMP3 treatment (Fig. 5A). To determine whether this was generalized to all regions of the hippocampus or was specific to any subregion, we conducted tissue staining of the injured dorsal hippocampus for b3-tubulin. Although we do not rule out subtle changes in other subfields we only observed specific changes in the molecular layer of the dentate gyrus (Fig. 5B). Quantification of the number of observable dendritic branches in the molecular layer proximal to injury revealed a significant reduction post-tbi that was attenuated by IV TIMP3 treatment. Thus, TIMP3 treatment preserves cytoskeletal morphology and circuitry of the hippocampus that could potentially improve hippocampal function and neurocognition following TBI. Pharmacological Inhibition of mtor and Akt Differentially Subvert the Protective Effects of Intravenous TIMP3 Since our observations in vitro demonstrate that TIMP3 mediated activation of the Akt-mTORC1 pathway promotes neurite outgrowth that can be subverted by rapamycin (Fig. 4), we sought to determine if the effects of pathway inhibition could be reproduced in vivo. Although we could not inhibit Akt with triciribine in vitro, in vivo studies have shown that inhibition of both mtorc1 (with rapamycin [47, 48]) and Akt (with triciribine [49, 50]) can be achieved with systemic administration. Our model of the effects of TIMP3 (summarized in Fig. 6A) suggests that mtorc1 inhibition with rapamycin would inhibit the preservation/reinnervation of neuronal projections (b3-tubulin staining) whereas Akt inhibition with triciribine would inhibit the preservation/reinnervation of neuronal projections but would also attenuate the survival of mature neurons (NeuN staining) and neuronal stem cells (DCX staining). To address these hypotheses, we repeated our TBI 1 TIMP3 experiments with three IV injections of 60 lg/kg TIMP3 as before (1, 24, and 72 hours post-tbi) but modified the design to include intraperitoneal injections of either 1 mg/kg triciribine or 1 mg/kg rapamycin 30 minutes prior to each IV TIMP3 delivery. As before, animals were sacrificed 7 days post-tbi in order to compare with our findings in Figures 1 and 5. The results summarized in Figure 6B are presented as percent change from TBI1TIMP3 to emphasize the effect of the inhibitors. In inhibitor treated mice significant alterations in NeuN, DCX and b3-tubulin staining were found. As predicted, TIMP3 promoted survival of NeuN positive mature neurons was attenuated by triciribine (white bars) but was unaffected by rapamycin treatment (checked bars). However, the effects of the inhibitors on survival and presence of DCX positive neural stem cells was not consistent with our Figure 4. TIMP3 protects neurons and promotes neurite outgrowth in vitro. (A) Hypoxia/MTT assay. SH-SY5Y cells, 5 hrs hypoxia 1 18 hrs normoxia reduces viability (One-way ANOVA (F (2,9) , p<0.0001, Bonferroni p < ) which was prevented by 1 hr pre-treatment with 1 ng/ml TIMP3 (p < ). n54, mean 6 s.e.m. Images representative of result. Scale bar: 100 lm. (B) MTT Assay. 1 nm of mtorc1 inhibitor did not affect viability. n54, mean 6 s.e.m. (C) Western blot. Akt (Ser473) phosphorylation was not inhibited with rapamycin. n54, mean 6 s.e.m. (D) Neurite outgrowth assay. Colorimetric changes due to increased neurite production. Cartoon depicts set up of treatment. 48 hrs treatment of SH-SY5Y cells with 1 lg/ml TIMP3 increased neurite outgrowth. n54, mean 6 s.e.m. (One-way ANOVA: F (2,9) , p , Bonferroni p < ) which was attenuated by rapamycin treatment (p < ). (E) Neurite chamber assay. Cartoon depicts location of images. Upper images (1) indicate 7 days treatment of hippocampal neurons with TIMP3 increased neurite outgrowth. Lower images (2) indicate comparable cell densities. Scale bar: 250 lm. (F) Sholl analysis. 72 hrs treatment of hippocampal neurons with 1 lg/ml TIMP3 increased neurite complexity. 20 neurons analyzed per treatment, n53, mean 6 s.e.m. (Paired t-test, P 5 <0.05). Images representative of result. Scale bar: 50 lm. (G) Sholl analysis. 72 hrs treatment of hippocampal neurons with 1 nm rapamycin inhibited the effects of TIMP3. n53, mean 6 s.e.m. (Paired t-test, P 5 <0.05). (H) Sholl analysis. shrna against mtor inhibited the effects of TIMP3 versus scrambled control sequence (SCR). Hippocampal neurons were cultured for 7 days post-transfection. n53, mean 6 s.e.m. (Paired t-test, P 5 <0.05). (* 5 p < 0.05, *** p 5 < 0.001). VC AlphaMed Press 2015 STEM CELLS

10 Gibb, Zhao, Potter et al Figure 5. Intravenous TIMP3 preserves neuronal projections in vivo in the molecular layer of the dentate gyrus following TBI. TIMP3 administration (60 lg/kg) was performed via tail-vein injection at 1 hr, 24 hrs and 72 hrs post-tbi. Animals were sacrificed on day 7. (A) Western blot. Samples from ipsilateral hippocampus show a reduction in neuronal cytoskeletal protein b3-tubulin 7 days post-tbi that was attenuated by TIMP3 treatment. Ubiquitous cytosolic protein GAPDH was used as a loading control. n54, mean 6 s.e.m. (One-way ANOVA: F (2,9) , p<0.0001, Bonferroni p < ). (B) Representative images showing the interface between the granule cell layer (GCL) and the molecular layer (Mol.layer) of the dentate gyrus proximal to the site of injury. Sections were stained for b3-tubulin (green) and NeuN (red) for location. Scale bar lm. Quantification of the number of observable b3-tubulin1ve dendritic branches in the molecular layer greater than 50 lm in length. n54, 4 section per animal, mean 6 s.e.m. Data show a reduction with TBI (Oneway ANOVA (F (2,9) , p<0.0001, Bonferroni p < ) that was attenuated by TIMP3 treatment (p < ). (*** p 5 < 0.001). hypothesis. The lack of change in DCX positive staining with triciribine is possibly due to a differential sensitivity of neural stem cells to Akt inhibition compared to the more vulnerable postmitotic neurons. The observation of significantly increased staining for DCX positive neural stem cells with rapamycin (an indication of enhanced proliferation) has been previously reported [51] and is therefore consistent with successful delivery of rapamycin. Also consistent with our hypothesis was the observation that both triciribine and rapamycin subverted the preservation/reinnervation of neurites in the molecular layer of the dentate gyrus (b3-tubulin staining). (Representative images for the effects of the inhibitors on NeuN, DCX, and b3-tubulin staining are shown in Supporting Information Fig. 4.) For both triciribine and rapamycin no significant changes in any of the markers were observed on the contralateral side (data not shown). Taken together, our observations suggest that the Akt-mTORC1 pathway is critically involved in the effects of TIMP3 on neurons and their projections in vivo post-tbi. IV TIMP3 Abrogates Hippocampal-Dependent Neurocognitive Decline Following TBI Having determined that TIMP3 can directly initiate a neurotrophic signaling cascade in neurons not only in vitro but most importantly in vivo post-tbi, we sought to determine on VC AlphaMed Press 2015

11 3540 TIMP3 Treatment is Neuroprotective Post-TBI Figure 6. Pharmacological inhibitors differentially subvert the protective effects of IV TIMP3. (A) Diagram depicts an overview of the experimental design and predicted results. (B) Quantification of tissue staining from the ipsilateral hippocampus 7 days post-tbi. Bar chart shows percent change from TIMP3 treatment post-tbi. Triciribine as predicted attenuated both neuronal survival (NeuN) and neurite outgrowth (b3-tubulin). Rapamycin as predicted only attenuated neurite outgrowth. DCX neuronal stem cells demonstrate a mixed reaction to the effects of triciribine and rapamycin treatment. For each stain n54 per group, 4 sections per animal, mean 6 s.e.m. NeuN: (One-way ANOVA: F (3,12) , p ), (Bonferroni. TBI, p , TBI1TIMP31Triciribine, p , and TBI1TIMP31Rapamycin, p 5 1.0), DCX: (One-way ANOVA: F (3,12) , p ), (Bonferroni p , p 5 1.0, and p , respectively). b3-tubulin (One-way ANOVA: F (3,12) , p ), (Bonferroni p < , p , and p , respectively). (* 5 p < 0.05, ** 5 p < 0.01, *** p 5 < 0.001). a systems level if IV TIMP3 could attenuate neurocognitive decline that is associated with TBI. Using our mouse model, we examined the effects of post-injury TIMP3 administration on tasks that strongly involve the hippocampus. As before, TIMP3 was delivered in three temporally separated doses followed by testing on the elevated plus maze (EPM) [52], MWM [53], and context-dependent fear discrimination [53] tasks. Experiment design is illustrated schematically in Figure 7A. Classically the hippocampus is known to be associated with learning and memory but it is also one of the principal structures involved in regulating stress and anxiety [54]. To assess anxiety, mice were tested in the EPM. This task juxtaposes the innate curiosity of mice against their natural fear of open spaces, with exploration time in the open and closed arms used as indices of anxiety. Previous studies using the EPM demonstrate that lesions of the dorsal hippocampus are anxiogenic [52] whereas lesions of the ventral hippocampus are anxiolytic [55]. However, our analysis on time spent in each section (Fig. 7B) conducted 3 days post-tbi showed that VC AlphaMed Press 2015 none of the groups were significantly different to each other in any section. Although our CCI model of TBI model centers the injury above the dorsal hippocampus we do not rule out damage to the ventral hippocampus that may mask manifestation of an anxiety-like result. We next examined spatial learning and memory using an abbreviated version of the MWM task [56]. Although sham animals effectively learned the location of the hidden platform (indicated by decreased latencies over the course of training), TBI injured mice displayed a dysfunction in spatial learning (Fig. 7C) although there was no difference in learning between the TBI alone and TBI 1 TIMP3 groups. When tested for long-term spatial memory, TBI alone mice were found display a significant deficit in their ability to locate the hidden platform, as indicated by fewer platform crossings (Fig. 7D). This deficit was abrogated by post-injury IV TIMP3 administration. Consistent with this observation, proximity analysis of the swimming paths of the injured animals (concentric circles of increasing diameter centered on the platform) showed that TIMP3-treated animals spent significantly more time searching in the immediate vicinity of the platform than TBI alone animals (Fig. 7E) indicating preservation of spatial memory with TIMP3 treatment. Context-dependent fear discrimination is a more complex form of learning that requires the ability to form distinct memories about two similar, but distinct, environments (also known as pattern separation). Performance in this task has been shown to correlate with the number of DCX-positive cells and the rate of neurogenesis in the hippocampus [57, 58]. Training consists of placing the animal in two similar cages, one in which a footshock is delivered (shock cage) and one in which no footshock occurs (safe cage) (Fig. 7F left panel). Testing is carried out 24 hours later (Day 1) by assessing freezing behavior, after which a reinforcement shock is delivered in the shock cage followed by a second test carried out 24 hours later (Day 2). Figure 7F (Right panels) shows that when tested for their fear memory, sham control mice quickly learn to discriminate between the two contexts, showing significant differences in freezing after only one day of training whereas TBI alone mice never learn to discriminate between the two contexts. Although requiring an additional day of training than sham-operated controls, TBI injured mice receiving IV TIMP3 treatment learned to discriminate between the two contexts, demonstrating a significant difference in contextual freezing by day 2 of testing. Taken together, these results demonstrate that post-injury IV TIMP3 administration is able to ameliorate post-tbi hippocampal dysfunction. DISCUSSION In this study, using an established CCI mouse model of TBI, we demonstrate that IV TIMP3 attenuates neuronal cell loss in the hippocampus post-tbi. In support of a functional effect of neuroprotection we find that IV TIMP3 ameliorates neurocognitive dysfunction post-tbi. Likely underlying the preservation of neurocognitive ability is our finding that TIMP3 treatment preserves several key features of the hippocampal cytoarchitecture that are otherwise lost in TBI. IV TIMP3 treatment is associated with the preservation of vulnerable neural populations and neurites in the molecular layer of the dentate STEM CELLS

12 Gibb, Zhao, Potter et al Figure 7. Intravenous TIMP3 treatment abrogates hippocampal-dependent neurocognitive decline post-tbi. (A) Diagram depicts experimental design. TIMP3 (60 lg/kg) was administered via tail-vein injection at 1 hr, 24 hrs and 72 hrs post-tbi, TBI alone and sham mice received PBS injections. n510 per group. Elevated Plus Maze (EPM) was performed on day 3 following the last injection, Morris Water Maze (MWM) over days 8 and 9 and Context-dependent fear discrimination task over days (B) Elevated Plus Maze. Data represented as % of 300 second time trial spent in each segment of the maze. No significant differences were observed between any of the groups in any of the segments (One-way ANOVA: (Open F (2,21) 50.95, p50.4), (Mid F (2,21) 53.02, p50.07), (Closed F (2,21) 50.12, p50.89) (C E) Morris Water Maze test. (C) During training TBI-injured mice did not learn the location of the hidden platform as effectively as sham mice (One-way ANOVA, F (2,21) , p<0.001). (D) TBI-alone mice but not IV TIMP3 treated mice display deficits in Long-term spatial memory (One-way ANOVA, F (2,21) 54.33, p50.027). (E) TIMP3 treated mice spend more time in the platform vicinity than TBI alone mice (One-way ANOVA, F (2,34) 53.35, p50.047). (F) Context-dependent fear discrimination. Habituation and training was conducted on day 14, and testing conducted on day 15 (task day 1) and day 16 (task day 2). Sham mice learn to distinguish between the two contexts immediately after training (One-way ANOVA, F (1,7) , p50.003). Although delayed, IV-TIMP3 treated mice learn to discriminate between the two contexts by day 2 of testing (One-way ANOVA, F (1,7) , p50.013) whereas TBI-alone mice do not learn to discriminate over the entire test period (One-way ANOVA, F (1,7) 50.27, p50.619). (* 5 p < 0.05). gyrus. Through a multifaceted approach we have demonstrated that TIMP3 treatment activates signaling cascades in neurons, specifically the Akt-mTORC1 pathway, that provides protection against TBI-induced insults such as hypoxia. Our in vitro and in vivo data suggest that the Akt-mTORC1 signaling cascade is a critical mediator of the effect of TIMP3 on neuronal survival and preservation of neurite integrity post-tbi. Moreover, we found that pharmacological and genetic inhibition of these signaling cascades can subvert the effects of TIMP3. This is the first study to report that IV TIMP3, VC AlphaMed Press 2015

13 3542 TIMP3 Treatment is Neuroprotective Post-TBI administered post-tbi, results in neuroprotection and neurocognitive improvement. Our previous study demonstrating the protective effects of IV TIMP3 on BBB permeability induced by TBI [19] and this study elucidating the direct neuroprotective effects of TIMP3 suggest that TIMP3 may have pleiotropic beneficial effects and therapeutic potential in the treatment of patients suffering from traumatic brain injury. Previous studies have reported activation of the AktmTORC1 pathway within the first 24 hours of TBI-alone [37, 38, 59]. However, design differences in these studies have led to a lack of a consensus on whether this is harmful or beneficial to recovery. Chen and colleagues [38] reported significantly increased phospho-s6rp in hippocampal neurons suggestive of a repair mechanism whereas Park and colleagues [37] reported the increase was located to microglia and astrocytes and not neurons thus suggestive of a role in inflammation. We too found an increase in microglial staining for phospho-s6rp post-tbi but only in TBI-alone mice and not those that had received IV TIMP3 treatment, hence suggesting that IV TIMP3 has an anti-inflammatory effect in TBI. To our knowledge this is the first report to detail this effect of TIMP3 in the brain, although our current data cannot delineate between direct suppression of pathway activation in microglia versus a lack of microglia activation as an indirect result of neuroprotection by TIMP3. Our results in this study have allowed us to hypothesize that the observed activation of the Akt-mTORC1 pathway, that accompanies TIMP3 treatment is the mechanism that imparts neuroprotection and neurite outgrowth. In agreement with our in vitro results, our in vivo studies demonstrate that IV TIMP3 treatment preserves or increases neurite outgrowth. This finding potentially underlies the attenuation of neurocognitive decline and is also consistent with previous studies that have linked the mtorc1 pathway to the generation and/or repair of neuronal projections and connections [36, 41, 45, 46]. Furthermore, our in vitro results show pharmacological and genetic inhibition of the mtorc1 pathway abrogates the effects of TIMP3 on neurite outgrowth in primary hippocampal neurons. Interestingly, pharmacological inhibition reproduced the same effects in vivo in our TBI mouse model. In these in vivo studies, our readout of TIMP3 function was dentate gyrus neurite outgrowth post-tbi and survival of NeuN positive hilar interneurons. We had hypothesized that since Akt is upstream of mtor, treatment with the Akt inhibitor triciribine would result in a decrease in both the survival of neuronal populations and the preservation/enhancement of neurites, whereas we anticipated that rapamycin would only affect neurites. Our results indicated that this was indeed the case. However, our results on the DCX positive neural stem cell population were not as robust and we did not see an inhibition on the effects of TIMP3 with triciribine. Given the limited understanding of the role of Akt activation on DCX positive cells [60], we put forth the hypothesis that the DCX positive neural stem cells are not as sensitive or vulnerable to Akt inhibition as NeuN positive postmitotic neurons. Correct information processing in the brain depends on a balance between neuronal excitation and inhibition. On a molecular level, TBI is known to render the dentate gyrus hyperexcitable due to a loss of inhibitory interneurons [16]. We show here that TIMP3 preserves both neuronal projections and inhibitory hilar neurons in the dentate post-tbi VC AlphaMed Press 2015 which could help preserve a normal balance in signaling and connectivity, thereby potentially preventing the deleterious consequences of TBI induced hyperexcitability such as epilepsy [7, 8, 61]. In our study, we have demonstrated on a systems level that IV TIMP3 delivered in three temporally separated doses post-tbi can attenuate neurocognitive deficits in hippocampaldependent tasks. Our MWM result suggests that TIMP3 was able to prevent some long-term post-tbi memory deficits as TIMP3 mice had correctly recalled the location of the platform. Interestingly, the most potent effects of the treatment were found to be improvement in context-dependent fear discrimination. Recent studies have shown that integration or preservation of newborn neurons into the hippocampal circuitry is critically involved in the formation of certain kinds of learning and memory, specifically context discrimination [57, 58]. Moreover, selective elimination of hippocampal progenitor cells impairs the ability of rodents to discriminate in these paradigms. Since TIMP3 attenuates the loss of neural stem cells in the dentate gyrus it is plausible that this protective effect in part contributes to the observed improvement in context-dependent fear discrimination. However, a caveat of our present study is that neither pharmacological nor genetic inhibition of the mtor pathway could be used to our studies on behavioral testing. The confounding effects of long-term global pathway inhibition with a pharmacological approach or the additional invasive cerebral surgery required for viral mediated shrna delivery would confound interpretation of both approaches for this study. Although we provide evidence in this study for the neuroprotective and therapeutic potential of TIMP3, it should be noted that several studies have reported TIMP3 to be pro-apoptotic. This notion stems from the observation that overexpression of TIMP3 promotes apoptosis in a variety of cancer cells [22 24]. Furthermore, several studies report this is also the case for neurons. Zhou et al. reported an increase in micrornas mir-21 and mir-222 in cultured dorsal root ganglion neurons post-injury correlates with decreased expression of TIMP3 and decreased apoptosis [62]. Walker and Rosenberg using an in vivo model of ischemia also suggest that TIMP3 is pro-apoptotic to neurons by comparing delayed neuronal death between wild type and TIMP3 knockout mice. Mechanistically Walker and Rosenberg suggest that TIMP3 stabilizes a death receptor pathway by inhibiting TACE. However, both studies do not provide direct evidence that TIMP3 initiates an apoptotic cascade but rather used correlative evidence to link TIMP3 to cell death. In contrast, our study did not observe any indication of apoptosis with exogenous TIMP3 treatment either in vitro or in vivo. In further support against an a pro-apoptotic effect of TIMP3, we found through use of a novel CT scan-based methodology (Supporting Information Fig. 5) that the contusion volume did not change with TIMP3 treatment post-tbi. If apoptotic cell death had been an issue with IV TIMP3 a change in contusion volume would have been observed in TIMP3 treated mice. Moreover, it should also be noted that TIMP3 has recently been reported to also be a potential therapy for myocardial infarction without any evidence of cardiomyocyte toxicity [63]. Taken together, the effects of TIMP3 are likely dose-dependent, cell type specific and disease specific. STEM CELLS