RF #2015 P-01 PI: Azizul Haque, PhD Grant Title: Targeting Enolase in Spinal Cord Injury 12-month Technical Progress Report Progress Report (First Six Months): Enolase is one of the most abundantly expressed cytosolic proteins implicated in ischemia and hypoxia. It is a key glycolytic multifunctional enzyme also known as a neurotrophic factor, hypoxic stress protein, c- Myc binding protein and transcription factor, and a strong plasminogen binding protein often overexpressed in neuronal cells following brain and spinal cord injury (). is a devastating neurotrauma whose progressive pathological changes include complex and evolving molecular cascades. To date, only few studies have focused on the role of neuron-specific enolase (NSE) on spinal cord protein content, and the changes induced after, and insights into the role of NSE in -mediated dysfunctions have not been fully elucidated. Neuronal damage is also associated with elevations of NSE and a calcium binding protein B (S100B). Our hypothesis was that inhibition of enolase may provide greater attenuation of secondary damage following due to the reduction of activated glial cells and protection of neurons. Since enolase is a multifunctional protein, blocking this enzyme with a novel small molecule inhibitor ENOblock may also reduce S100B after, and induce neuroprotection. To test these hypotheses, we proposed the following specific aim: Aim 1: Investigate the role of enolase in activation of glial cells, and induction of inflammation, axonal damage and neurodegeneration in acute (48h) and chronic (14d, 6w) in rats; and determine whether enolase inhibitor ENOblock attenuates NSE/S100B protein levels as well as other parameters contributing to neuroprotection in vivo in rat model. Our initial study examined NSE levels in spinal cord segments from three sham operated and three rats, which detected higher levels of NSE expression in animals as compared to sham controls (Figure 1 in the original proposal, not shown here). We also examined matrix metalloproteinase-9 (MMP-9) in LPS (1µg/ml) treated microglial cell line BV2. This study showed that LPS treatment increased NSE and MMP-9 B-cell Astrocyte TNF-α IL-1β TGF-β IFN-γ NSE T-cell MCP-1 MIP-1α IFN-γ Microglia Neuron (Neuronal apoptosis) Macrophage The potential adverse effects of cell surface enolase in the alterations of neuronal, glial, and immune cell functions, and induction of inflammatory responses following injury. NSE or γ-enolase expression is elevated in microglia and astrocytes following. Similarly, α-enolase expression is increased in macrophages and B-cells following activation or injury. Rapid translocation of enolase from cytosol to the cell surface drives a number of events such as production of reactive oxygen species (ROS), nitric oxide (NO), inflammatory cytokines (e.g., TNF-α, IL-1β, IFN-γ, TGF-β) and chemokines (e.g., MCP-1, MIP-1α), which contribute to neuronal cell death. Macrophages, microglia, and B-cells can enhance antigen presentation, T cell activation, and production of inflammatory cytokines, which may aggravate inflammatory process and neuronal death after injury. proteins in BV2 cells, and that ENOblock treatment significantly reduced the expression levels of these proteins as compared to vehicle control (Figure 2 in the original proposal, not shown here), suggesting that
ENOblock may attenuate activation of glial cells after injury. The following diagram implicates some of the adverse effects of cell surface NSE after (Haque et al, submitted). Since this pilot award began, we mainly focused on ex vivo and in vivo studies using an established rat model. Specifically, we examined the role of ENOblock in a male Sprague-Dawley (SD) rat acute (48h) Figure 1. Spinal cord injured animals were treated with vehicle alone or ENOblock (100µg/kg, iv), and blood samples were obtained 48h post-injury. NSE levels in naïve, sham operated and sera were measured by ELISA. Statistical analyses were performed by Student s t- test. results were first compared with sham surgery, sham/enoblock or naïve animals (p<0.001). *p < 0.05 compared to /ENOblock. N=2-3. model. We determined the effect of ENOblock on NSE levels in rat serum as well as spinal cord tissues following injury. Briefly, pooled serum samples from naïve, sham operated/vehicle treated, and ENOblock treated sham and animals, were tested for NSE by ELISA. These data suggested that ENOblock treatment markedly inhibited NSE levels in serum samples as compared to controls (Figure 1). We also measured serum cytokines and chemokines by using Discovery cytokine/chemokine arrays. Interestingly, expression levels of several inflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines (MIP-1α, IP-10) were markedly decreased in ENOblock treated animals as compared to vehicle control (Table 1). We also measured eotaxin, EGF, fractalkine, IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-10, IL-12(p70), IL- 13, IL-17A, IL-18, GRO/KC, G-CSF, GM-CSF, MCP-1, Leptin, LIX, MIP-2, RANTES, and VEGF animals were treated with vehicle alone or ENOblock (100µg/kg, iv), and blood samples were obtained 48h post-injury. Serum cytokines and chemokines were analyzed by using Discovery rat cytokine/chemokine arrays. N=2-3. Large group sizes are required to perform statistical analyses, which will be accomplished in the coming months. in naïve, sham/vehicle, sham/enoblock, /vehicle and /ENOblock groups using rat cytokine array/chemokine array 27-plex. Currently, we are analyzing these data to determine ENOblock s effect on these cytokines/chemokines in rats, which may reveal novel insights into the role of NSE in. During the past six months, we have also made significant progress in testing the effects of ENOblock on NSE and MMPs (2 and 9) in spinal cord tissues following. Data showed that ENOblock treatment decreased NSE, and MMPs 2 and 9 protein expression in spinal cord samples as determined by Western blot analysis (Figure 2). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression. Thus, our hypothesis remains unchanged and
additional experiments are underway to determine the effect of ENOblock on these factors in both acute and chronic. A 47kDa Naïve Sham vehicle Sham ENO vehicle ENO NSE B 4.5 NSE 4 MMP-2 3.5 MMP-9 72kDa 42kDa 92kDa 42kDa MMP-2 Actin MMP-9 Actin Relative density 3 2.5 2 1.5 1 0.5 0 Figure 2. ENOblock treatment decreases the expression of NSE and MMPs in acute in rats. Spinal cord injured animals were treated with vehicle alone or ENOblock (100µg/kg, iv) and compared to sham surgery alone. (A) Spinal cord samples (S-4) from different groups (naïve, sham and ) were analyzed by Western blotting for NSE, MMP-2 and MMP-9 proteins. Actin was used a loading control. Representative blots showing protein levels in treated and untreated groups (n=3). (B) Densitometric analysis confirmed significant inhibition of NSE and MMPs protein expression in ENOblock treated rats as compared with vehicle treated animals. *p < 0.01 compared to sham surgery + vehicle or ENOblock. Studies are also underway to determine whether ENOblock (100µg/kg) attenuates S100B protein levels as well as other parameters contributing to neuroprotection in vivo in rat model. Primary injury to spinal cord would lead to secondary injury causing gliosis, inflammation, and neurodegeneration but treatment of animals with ENOblock may inhibit these inflammatory events in, inducing neuroprotection. Ongoing studies are testing the status of macrophage infiltration (ED2), microgliosis (OX42), astrogliosis (GFAP) in spinal cord sections obtained from vehicle or ENOblock treated animals. Immunohistochemical analyses of spinal cord samples for other markers such as NSE, S100B, NeuN are also in progress. Dose-dependent studies with ENOblock are also in progress where the primary endpoint will be defined by improved locomotor function (according to the BBB scale) and supported by evaluation of changes in lesion volume, axonal damage, and angiogenic markers as proposed in the original application. The data obtained so far suggest that reduction of NSE by ENOblock may have potential therapeutic implications in. Since this pilot award began in July 2015, we published one review article in the journal Reviews in the Neurosciences, and submitted another review article for publication in the journal Metabolic Brain Disease. We also submitted an abstract to the 47th Annual American Society for Neurochemistry (ASN) Meeting in 2016 for presenting our work on. Finally, we thank RF for supporting this pilot project. Publications: Manuscript(s) published: 1. Chakrabarti M, Das A, Samantaray S, Smith JA, Banik NL, Haque A, Ray SK (2015) Molecular mechanisms of estrogen for neuroprotection in spinal cord injury and traumatic brain injury. Rev Neurosci. 2015 Oct 13. pii: /j/revneuro.ahead-of-print/revneuro-2015-0032/revneuro-2015-0032.xml. doi: 10.1515/revneuro-2015-0032. [Epub ahead of print] Manuscript(s) submitted:
2. Haque A, Ray SK, Cox A, and Banik NL (2015) Neuron specific enolase: a promising therapeutic target in acute spinal cord injury. Metab Brain Dis. Abstract(s) accepted: 1. Haque A, Bryant MJ, Bouchard M, Cox A, Matzelle D, and Banik NL. Targeting neuron specific enolase in reducing secondary damage in rat spinal cord injury model. 47th Annual American Society for Neurochemistry (ASN) Meeting (March 19-23, 2016) Denver, Colorado. Progress Report (Last Six Months): To date, the role of NSE in -mediated alterations of key protein profiles and dysfunctions remains unclear. Initially (first six months), we measured serum NSE levels and reported the results that ENOblock treatment inhibited NSE levels as compared to controls (Fig. 1). Recently (last six months), we tested NSE expression levels in spinal cord tissues obtained from two shamoperated/vehicle, three /vehicle and two /ENOblock rats, which detected higher levels of NSE expression in spinal cord tissues of /vehicle animals as compared to sham controls (Fig. 3). Treatment of rats with ENOblock (100 µg/kg) markedly reduced NSE protein expression in NSE Figure 3. Spinal cord injured animals were treated with vehicle alone or ENOblock (100µg/kg, iv), and spinal cord tissues were obtained 48h post-injury. Immunohistochemical analyses were performed for NSE proteins. Sham operated animals were used as a negative control. N=2-3. spinal cord tissues as compared to /vehicle control. Elevation of NSE triggers MMP activation and degradation of extracellular matrix proteins. MMP-9 plays an important role in blood-spinal cord barrier dysfunction and induction of inflammation. MMP-9 has been shown to be increased rapidly after a moderate contusion, reaching a maximum at 24h. Our data suggest that MMP- 9 expression is markedly elevated in tissues after 48h, and that it is downregulated by ENOblock treatment (Fig. 4A), suggesting that inhibition of MMP-9 by ENOblock could be a therapeutic strategy for attenuation of inflammatory processes in. Primary injury to spinal cord would lead to secondary injury causing gliosis, inflammation, and neurodegeneration, but treatment of animals with ENOblock may alter these inflammatory Figure 4. Spinal cord injured SD rats were treated with vehicle (DMSO, 0.01% in saline) alone or ENOblock (100µg/kg, twice, iv), and spinal cord sections were obtained 48h post-injury. Immunohistochemical analysis was performed for (A) MMP-9 and (B) Iba1 (microglial marker). N=2-3. Representative figures show that MMP-9 and Iba1 protein expression in rat spinal cords were downregulated by ENOblock treatment. events after, inducing neuroprotection. Recovery after is in part attributed to secondary
events that govern both detrimental and reparative processes. The elevation of MMP-9 in rats may promote microglial activation and release of inflammatory cytokines/chemokines as observed in Table 1, leading to neuronal cell death and disability. Immunohistochemistry of rat tissues showed that microglia/macrophage-specific calcium-binding protein Iba1, which is an activation marker of microglia, was markedly upregulated in acute rats (Fig. 4B). Interestingly, Iba1 protein expression was downregulated by treatment with ENOblock, suggesting that reduction of NSE by ENOblock may have potential therapeutic implications in acute. Experiments are in progress to determine the status of S- 100B protein levels, macrophage infiltration (ED2), microgliosis (OX42/Iba1), astrogliosis (GFAP) in spinal cord sections obtained from vehicle or ENOblock treated rats. A metabolic hormone profile following ENOblock treatment is also in progress. Preliminary data here suggest that c- peptide, leptin, and PYY molecules are altered in rats, where ENOblock may have therapeutic functions. Overall, we made significant progress in this pilot proposal in generating preliminary data for subsequent grant submission. Our study also showed that administration of ENOblock at 15 min following in rats significantly reduced inflammatory responses when tested 48 hours after therapy. Since early administration of ENOblock had beneficial effects, we slightly changed our research strategy and wanted to investigate if administration of ENOblock at 1h, 2h and 4h post- has better beneficial effects in rats (Chart I). These studies are more clinically relevant as opposed to going for 14 days and 6 weeks studies that were originally proposed. While longer time (14 days and 6 weeks) studies are underway, time-dependent effects of ENOblock (Chart I) are also in progress. In addition, during this award period, the PI presented data in the ASN meeting (March 19-23, 2016) in Denver, Colorado. The PI also published three related review articles, one abstract, and two other unrelated review articles. The PI also submitted an application for RF investigator award, which was funded. The PI also prepared an application for submission to the NIH. Again, we thank RF for supporting this pilot project.
Publications: Manuscripts published or accepted (related): 1. Chakrabarti M, Das A, Samantaray S, Smith JA, Banik NL, Haque A, Ray SK (2016) Molecular mechanisms of estrogen for neuroprotection in spinal cord injury and traumatic brain injury. Rev Neurosci. 2016 Apr 1; 27(3):271-81. 2. Haque A, Ray SK, Cox A, and Banik NL (2016) Neuron specific enolase: a promising therapeutic target in acute spinal cord injury. Metab Brain Dis. Metab Brain Dis. 2016 Jun; 31(3):487-95. 3. Haque A, Ray SK, Cox A, Banik NL (2016) Challenging Pathophysiology in Spinal Cord Injury and Promising Stem Cell Therapy. Proceedings of Neurosciences (in press). Manuscripts published (unrelated): 4. Capone M, Bryant JM, Sutkowski N, Haque A (2016) Fc Receptor-Like Proteins in Pathophysiology of B-cell Disorder. J Clin Cell Immunol. 2016 Jun;7(3). pii: 427. Epub 2016 Jun 17. 5. God JM and Haque A. 2016. Multiple defects impair the HLA class II antigen presentation capacity of Burkitt lymphoma. J Clin Cell Immunol. 7:4. DOI: 10.4172/2155-9899.1000e119. The Value to the State of South Carolina There are many individuals in our state of South Carolina. Current therapeutic strategies often fail to address cellular damages and molecular changes occur in. Thus, development of new therapeutic approaches should help better manage and is highly relevant to the lives of our South Carolina citizens. During the past 12 months, we investigated the potential role of a novel NSE inhibitor ENOblock in a relevant animal model. Results obtained indicate that ENOblock treatment attenuates inflammatory events after, suggesting that ENOblock therapy is beneficial to animals. While dose-dependent studies of ENOblock after are in progress, our current data in preclinical model suggest that ENOblock therapy may significantly impact individuals in our state of South Carolina.