Enolase1 alleviates cerebral ischemia-induced neuronal injury via its

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Enolase1 alleviates cerebral ischemia-induced neuronal injury via its enzymatic product Phosphoenolpyruvate Wei Jiang, Xibin Tian, Peng Yang, Jianglin Li, Le Xiao, Junqiang Liu, Chao Liu, Weihong Tan, and Haijun TU ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00103 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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ACS Chemical Neuroscience

Enolase1 alleviates cerebral ischemia-induced neuronal injury via its enzymatic product Phosphoenolpyruvate Wei Jiang, †,* Xibin Tian, †,* Peng Yang, †,* Jianglin Li,†,‡ Le Xiao, † Junqiang Liu, † Chao Liu,‡ Weihong Tan, †,‡,§ and Haijun Tu†,||# †

Institute of Neuroscience, State Key Laboratory of Chemo/Biosensing and

Chemometrics, College of Biology, Hunan University, Changsha, Hunan, 410082, China. ‡

Molecular Sciences and Biomedicine Laboratory (MBL), State Key

Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, China. §

Department of Chemistry, Department of Physiology and Functional

Genomics, Center for Research at the Bio/Nano Interface, UF Health Cancer Center, UF Genetics Institute and McKnight Brain Institute University of Florida, Gainesville, Florida 32611, United States ||

Shenzhen Research Institute, Hunan University, Shenzhen, Guangdong,

518000, China * These authors equally contributed to the work. # Corresponding

author contact information:

E-mail: [email protected]

1

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Abstract. Stroke is a leading cause of disability and the second leading cause of death among adults worldwide, while the mechanisms underlying neuronal death and dysfunction remain poorly understood. Here, we investigated the differential proteomic profiles of mouse brain homogenate with 3 h of middle cerebral artery occlusion (MCAO) ischemia, or sham, using Coomassie Brilliant Blue staining, followed by mass spectrometry. We identified Enolase1 (ENO1), a key glycolytic enzyme, as a potential mediator of neuronal injury in MCAO ischemic model. RT-PCR and Western blotting data showed that ENO1 was ubiquitously expressed in various tissues, distinct regions of brain, and different postnatal age. Immunohistochemical analysis revealed that ENO1 is localized in neuronal cytoplasm and dendrites. Interestingly, the expression level of ENO1 was significantly increased in the early stage, but dramatically decreased in the late stage, of cerebral ischemia in vivo. This dynamic change was consistent with our finding in cultured hippocampal neurons treated with oxygen/glucose

deprivation

(OGD)

in

vitro.

Importantly,

ENO1

overexpression in cultured neurons alleviated dendritic and spinal loss caused by OGD treatment. Furthermore, the enzymatic product of ENO1, phosphoenolpyruvate (PEP), was also synchronously changed along with the dynamic ENO1 level. The neuronal injury caused by OGD treatment in vitro

2


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or ischemia in vivo was mitigated by the application of PEP. Taken together, our data revealed that ENO1 plays a novel and protective role in cerebral ischemia-induced neuronal injury, highlighting a potential of ENO1 as a therapeutic target of neuronal protection from cerebral ischemia.

Keywords. Cerebral ischemia; middle cerebral artery occlusion (MCAO); Oxygen/glucose deprivation (OGD); Enolase1; Phosphoenolpyruvate (PEP); Neuronal protection

3



Introduction. Cerebral ischemia is caused by the obstruction of a cerebral artery by thrombus formation or endovascular embolus, resulting in synaptic failure, followed by neuronal death and dysfunction, as well as destruction of neural circuitry, in stroke brain regions 1. Based on the data from the World Health Organization, cerebral ischemic stroke is one of the leading cause of disability and the second leading cause of death among adults over 60 years of age worldwide 2. Unfortunately, treatment options for acute ischemic stroke (AIS) are quite limited. To date, recombinant tissue plasminogen activator (rtPA) is the only therapeutic drug approved by the U.S. Food and Drug Administration (FDA) to treat cerebral ischemia through dissolving the obstructive clot to restore blood flow 3, 4. However, rtPA for the treatment of acute ischemic stroke patients is only effective within 4.5 h after the onset of stroke symptoms 3, 5. Furthermore, owing to the strict clinical criteria for its use, rtPA is only administered to 5% - 10% of patients with acute stroke 2. In order to uncover potential targets of cerebral ischemia for therapy or neural protection, the critical key is to understand how brain cells react at the molecular level after cerebral ischemia onset. To date, the molecular and cellular mechanisms underlying synaptic loss and brain cell death and dysfunction caused by ischemia have been studied 6-13. Since the brain has a higher consumption of oxygen and glucose

4


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than other tissues, a series of neurochemical events occur over time when an area of the brain is affected by the onset of ischemia. First of all, cerebral ischemia results in dysfunction of energy-dependent ion transporter and depolarization of neurons and glia, in turn activating voltage-gated calcium channels localized at soma to dendritic and presynaptic membranes, followed by the release of excitatory amino acids, especially glutamate, into extracellular space

14, 15.

The accumulation of extracellular glutamate leads

to overstimulation of glutamate receptors including α-amino-3-hydroxy-5methyl-4-isoxazol propionic acid receptors (AMPARs), N-methyl-Daspartic acid receptors (NMDARs), and kainic acid receptors (KARs), on other neurons, in which the influx of sodium, potassium, and calcium ions go through the channels gated by glutamate 16, 17. As a consequence, theses neurons become depolarized, which leads to more calcium influx and more glutamate release, causing generation of free radical, activation of Ca2+dependent enzymes, and, ultimately, excitotoxicity 18. Then oxidative stress occurs when the focal ischemic brain produces the reactive mitochondrial oxygen radical caused by a high level of calcium and sodium 19. Oxidative stress causes brain-blood barrier injury through activation of matrix metalloproteinase (MMP), especially MMP9 20, 21. Apart from these events, the resultant ischemia-induced microvascular injury, post-ischemia inflammation, and necrotic and apoptotic cell death contribute to ischemic brain injury and dysfunction (Reviewed by Lo et al. 22). 5



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Several molecules are involved in cerebral ischemia-induced neuronal death

9, 13, 23-25.

For instance, death-associated protein kinase 1(DAPK1)

interaction with the N-methyl-D-aspartate receptor (also known as the NMDA receptor or NMDAR) NR2B subunits mediates brain damage in stroke, while DAPK1 deletion protects neurons against ischemia-induced death 9. Moreover, DAPK1 regulates spinal damage in stroke via its interaction with Tau, blocking DAPK1-Tau interaction through a membranepermeable peptide (MPP), and ultimately acting to protect the spine from damage and improving neurological functions affected by ischemia

10.

Cyclin- dependent Kinase 5 (CDK5) induces hippocampal CA1 cell death by directly phosphorylating NMDA receptor 2A subunit at Ser1232, following forebrain ischemia

13.

Interferon regulatory factor 4 (IRF4)

restores neuronal injury resulting from ischemia/reperfusion (I/R) injury. Moreover, neuron-specific IRF4 transgenic (IRF4-TG) mice exhibited reduced infarct lesions, and this effect was reversed in IRF4-knockout (KO) mice 24. However, the number of molecules involved in ischemic brain injury is still low, indicating that even more molecular mechanisms underlie the brain injury that results from ischemic stroke. Enolase, also known as phosphopyruvate hydratase, is a metalloenzyme responsible for the catalysis of the conversion of 2-phosphoglycerate (2-PG) to PEP in glycolysis 26. Enolase belongs to the large enolase superfamily and 6


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is evolutionally conserved and ubiquitously present in a wide range of organisms from bacteria to mammals. Mammals have three enolase isotypes, denoted α enolase, β enolase and γ enolase, which are encoded by ENO1, ENO3 and ENO2, respectively

27, 28.

ENO1 is found in a variety of tissues,

while ENO3 is exclusively expressed in muscle tissues, and ENO2 is specifically present in neurons and neuroendocrine tissues 29. ENO1 plays an essential role in growth, survival and the tumorigenic potential in glioblastoma cells30, promotes tumorigenesis and metastasis via regulating the AMPK/mTOR pathway in colorectal cancer31, and regulates the metabolic reprogramming and malignant phenotype of pulmonary artery smooth muscle cells through AMPK/Akt pathway32. Neuronal damage following spinal cord or ischemic brain injury is associated with an elevation of neuron-specific enolase (NSE), namely ENO2. Thus, ENO2 is a putative biomarker in ischemic brain damage and traumatic brain injury (TBI)29, 33, 34. However, less is known about the role of ENO1 in cerebral ischemia. In the current study, we performed differential proteomic analysis of mouse brain homogenate with 3 h of permanent MCAO ischemia or sham treatment using Coomassie Brilliant Blue staining, followed by mass spectrometry. As a consequence, we identified ENO1 as a potent mediator of cerebral ischemia-induced neuronal death in our mouse MCAO model. ENO1 was expressed in mouse brain and localized in the soma and dendrite. ENO1 expression level in brain cells was dynamically changed by ischemic 7



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insult, both in vivo and in vitro. Either ENO1 overexpression or application of its enzymatic product PEP in cultured hippocampus neurons or MCAO model by stereotactic injection mitigated neuronal injury. Our data demonstrated that ENO1 plays a critical role in neural protection in response to cerebral ischemia, indicating that it is a potential target for stroke therapy.

Results and Discussion. Characterization of ischemia-induced neuronal loss in the cerebral ischemic model Upon the lack or loss of blood flow during focal stroke, a complicated pathophysiological response occurs in ischemic brain regions. So far, multiple mechanisms have been attributed to this response, including cellular bioenergetics failure, excitotoxicity, mitochondrial dysfunction, protein misfolding, and inflammatory response, and all have been determined to lead to neuronal injury and death 35. Clinically, the window of opportunity for stroke treatment is mainly limited within 6 h after stroke onset. However, the timing of neuronal death after ischemia onset is unknown. Since neurons in the CA1 region of brain hippocampus are highly vulnerable to ischemia 13, we

attempted

to

investigate

CA1

region

of

hippocampus

by

immunohistochemistry staining followed by confocal imaging to evaluate

8


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the pathological process of neuronal loss resulting from cerebral ischemia using

a

cerebral

ischemic

mouse

model.

First,

we

performed

immunohistochemistry staining to label living neurons using an antibody against NeuN, a specific neuronal marker, the positive staining of which accounts for survival neurons. Simultaneously, we also utilized 4',6diamidino-2-phenylindole (DAPI), a nuclear DNA dye, to label nucleus, the positive staining of which represents the total number of cells in CA1. The ratio of NeuN to DAPI-positive stains indicates the percentage of survival neurons in CA1 regions, which reflecting the severity of neuronal loss 36. In the first two h of cerebral ischemia, we did not observe any significant difference in the ratio of NeuN to DAPI-positive stains in the hippocampal CA1 region of ischemia for 1 h (90.951.18%) or 2 h (91.080.83%) as compared to that of sham operation for 1 h (93.87 1.16 %) or 2 h (90.41  2.59%) (Figure 1a-b, i-j), respectively. These data suggest that the viability of neurons in ischemic regions of the brain is not obviously changed in the beginning phase of ischemia, even though many intricate pathophysiological events are undoubtedly occurring. After 3 h, we found that the ratio of NeuN/DAPI-positive stains in CA1 decreased to 58.046.86 % at 3 h (Figure 1e-f, k) and further decreased to 22.715.76 % at 4 h (Figure 1g-h, l) after cerebral ischemia onset, whereas this ratio in sham group remained at about 90% in an unchanged state 1-4 h after mice were subjected to sham operation

9



(Figure 1a-l), suggesting that ischemia induces the initiation of neuronal loss at around 3 h after ischemia onset in MCAO model.

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Figure 1. Characterization of temporal process of hippocampus CA1 neuronal loss in cerebral ischemia. (a-h) Representative confocal images of hippocampal CA1 neurons in brain slices after 1, 2, 3, 4 h of sham (a, c, e, g) or ischemia (b, d, f, h) with double staining of NeuN antibody and DAPI. Scale bar, 200 μm (Top images of a-h), 100 μm (Bottom images of a-h). (i-l) Quantification of neuron-positive cells in mouse slices with 1, 2, 3, or 4 h of sham (black column) or ischemia (red, magenta, blue, or cyan indicates 1, 2, 2, 3, and 4 h, respectively). n=4 mice (Sham 1h), 4 mice (Ischemia 1h); n=3 mice (Sham 2 h), 3 mice (Ischemia 2h); n= 4 mice (Sham 3h), 4 mice (Ischemia 3 h); n=4 mice (Sham 4h), 4 mice (Ischemia 4 h). Data are means ± standard error of the mean (SEM). n.s., not significant, **p < 0.01, ***p < 0.001. Two-tailed, unpaired Student’s t-test was performed.

As a medical emergency, cerebral ischemic stroke is a cerebrovascular accident that results in loss of blood flow in ischemic regions of brain, leading to high rates of mortality and morbidity. It has been well described that focal ischemia causes cellular bioenergetic failure in stroke areas, followed by excitotoxicity, ion imbalance, oxidative stress, blood-brain barrier dysfunction, microvascular injury, post-ischemic inflammation and, ultimately, brain cell death 6-13. Transient ischemia-reperfusion (I/R) induces selective upregulation of both CaMKIIδ and CaMKIIγ which play neuroprotective role in neuronal cells through activation of the NF-κB 12


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signaling pathway37. Moreover, transient receptor potential melastatinrelated 2 (TRPM2) channel accelerate postischemic ROS generation and delayed cytosolic Zn2+ accumulation and hippocampal CA1 pyramidal neuronal death by using in vitro and in vivo models of transient I/R38. However, the timing of neuronal death after permanent ischemia onset is less known. To address this question, the present work identified that neurons remained intact within 2 h of ischemia onset, but that brain cells began to die by 3 h, and more severe at 4 h after ischemia onset (Figures. 1a-l).

Identification of ENO1 as a potential molecule involved in ischemiainduced neuronal loss Once the loss of hippocampal neurons in CA1 began at 3 h after ischemia onset which reflecting neuronal loss was characterized, we attempted to identify molecules involved in the pathological machinery of neuronal loss and/or dysfunction in cerebral ischemia. To do this, proteins were extracted from ipsilateral brain hemisphere (Figure 2a) 3 h after mice were subjected to MCAO or sham operation and separated on SDS-PAGE gel, respectively, followed by Coomassie Brilliant Blue staining (Figure 2b). Interestingly, the protein bands sized at about 48 kDa in MCAO were weaker than those of sham (Figure 2b). These bands were cut and analyzed by Mass Spectrometry (MS), respectively (Figure 2c). We found that the peptides (Figure S1) enzymatically digested from both sham and MCAO bands were matched to 13



ENO1 (Figure 2d), and the abundance of ENO1 band of MCAO was 49.395.39% of control, suggesting that ENO1 protein level was decreased at 3 h after ischemia onset. We next compared the expression level of ENO1 between ischemic ipsilateral hemispheres and sham using Western blotting with an antibody against ENO1, and we found that the ENO1 protein level was significantly decreased to 52.575.17% of control at 3 h after ischemia (Figure 2e and f) as compared to sham, which is consistent with the MS result (Figure 2d). To further verify our observation, we also conducted immunohistochemistry staining with ENO1 antibody on frozen brain section. The ENO1-stained fluorescence intensity in ischemic section was obviously weaker than that in control (Figure 2g). Collectively, these results demonstrate that we had identified a putative novel protein molecule, the expression level of which had dramatically decreased at 3 h post-ischemia, indicating that ENO1 might play an important role in ischemia-induced neuronal loss. Although previous studies have demonstrated that DAPK, CDK5, Tau, and IRF4 are involved in NMDA receptor-mediated neuronal death after cerebral ischemia 9, 13, 23-25, the molecular mechanisms of cerebral ischemia causing brain cell injury and dysfunction are still not well understood. The key findings of this study present an intriguing insight into the previously unidentified, but essential, roles of ENO1, which appears to be involved in

14


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the pathological process of neuronal loss after ischemia onset.

Figure 2. ENO1 is downregulated after cerebral ischemia in

15



hippocampus of MCAO mice model. (a) A cartoon illustration for ipsilateral hemisphere of mouse focal ischemia for brain homogenate. Dotted circles indicate ipsilateral hemisphere of sham (S3) or ischemia (I3) for 3 h. (b) Representative Coomassie Brilliant Blue staining image of SDS-PGAE gel separation for ipsilateral hemisphere homogenate from mice with sham or ischemia for 3 h. Arrowhead indicates that the intensity of a selected band was weaker in ischemia (I3) than that in sham (S3). (c) Base peak chromatogram of mass spectrometry analysis from a selected protein band (arrowhead in (b)) after trypsin digestion. (d) Quantification of ENO1 protein from the selected protein band by mass spectrometry analysis. n=3 mice (S3), 3 mice (I3). (e) Representative immunoblotting images of ipsilateral hemisphere homogenate from mice with sham (S3) or ischemia (I3) for 3 h by probing with anti-ENO1 antibody. (f) Intensity quantitation of the ENO1 band in (d). n=4 mice (S3), 4 mice (I3). (g) Representative confocal images for hippocampal CA1 region neurons of brain slice with sham or ischemia for 3 h, followed by double staining with ENO1 antibody and DAPI. Scale bar, 20 μm. Data are means ± SEM. *p < 0.05, ***p < 0.001. Two-tailed, unpaired Student’s t-test was performed.

ENO1 is expressed in mouse brain and localized to neuronal cytoplasm and dendrites To characterize the expression of ENO1 in the different mouse tissues, we 16


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designed a pair of specific primers locate in the Exon 3 and 5, respectively, by which the product of polymerase chain reaction (PCR) is 184 base pair (bp) for the ENO1 gene to perform reverse transcriptional PCR (RT-PCR) after extracting total RNA from different tissues (Figure 3a). We found that ENO1 is expressed in brain, heart, liver, and kidney (Figure 3b), suggesting that ENO1 is ubiquitously expressed in various tissues. To see if ENO1 expression is restricted spatiotemporally in brain, the different regions and developmental stages of brain were dissected to extract total RNA followed by RT-PCR, respectively. We found ENO1 to be widely expressed in olfactory bulb, cortex, hippocampus, brainstem, and cerebellum (Figure 3c), as well as postnatal brains at seven days (P7), P14, P28, and P56 (Figure 3d). ENO1 protein level was also confirmed by Western blotting using antibody against ENO1 (Figure 3e – 3f). To determine the expression of ENO1 in neurons, ENO1 was detected in primary hippocampal cultured neurons by using immunoblotting with antibodies against ENO1 and MAP2, a neuronal marker protein, and human embryonic kidney 293 (HEK293) cells were employed as a positive control (Figure 3g). To assess the distribution of ENO1 in neurons, immunofluorescent staining was conducted in green fluorescent protein (GFP) transfected-positive neurons using anti-ENO1 antibody, and the co-localization of GFP (green) and ENO1 (red) was observed in the soma and the dendrite of cultured hippocampal neurons (Figure 3h), which is consistent with previous study 29. Taken together, these 17



results show that ENO1 is ubiquitously expressed in various regions of brain and different postnatal stages through adulthood. In particular, ENO1 may practically be distributed throughout the entire neuronal compartment, such as soma and dendrites.

Figure 3. ENO1 is expressed in mouse brain and localized to soma and dendrites of neurons. (a) Diagram of mouse ENO1 gene structure and fragment of RT-PCR. (b-f) RT-PCR analysis (b-d) and Western analysis (e-g) of ENO1 mRNA in C57BL/6J adult mouse tissues including brain, heart, 18


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liver, and kidney (b and e); different brain regions including olfactory bulb, cortex, hippocampus, brainstem and cerebellum (c and f) and brains at different ages from day 0 (P0) to day 56 (P56) (d and g), respectively. (g) Western analysis of ENO1 protein expression in cultured hippocampus neurons and HEK293 cells. (i) Representative confocal images of cultured hippocampal neurons with double immunostaining of GFP and ENO1. Scale bar, 20 μm, or 5 μm (4 x magnification), 3–5 independent experiments were conducted.

ENO1 expression level is dynamically changed in the brain of MCAO model in vivo and cultured neurons with OGD in vitro Since neuronal loss is initiated at 3 h post-ischemia (Figure 1e, f, and k), we asked if ENO1 expression level would dynamically change during insult of cerebral ischemia in MCAO model. To investigate this, we extracted the homogenate protein of ipsilateral hemisphere 1 or 2 h post-ischemia and subjected it to immunoblotting with an anti-ENO1 antibody. Interestingly, ENO1 protein level increased to 120.8 7.48% of control at 1 hour postischemia, but decreased to 57.4418.87% of control at 2 h post-ischemia as compared to the protein level of the corresponding shams in the MCAO model (Figure 4a and b). Cultured neurons treated with oxygen glucose deprivation (OGD) are widely used to investigate the pathogenesis and

19



potential treatment strategies for cerebral ischemic stroke in vitro (Figure S2). Therefore, we subsequently assessed whether ENO1 expression changed in the cultured hippocampal neurons after treatment with OGD. Using immunoblotting, we detected ENO1 expression level in cultured hippocampal neurons after OGD treatment for 1- 3 h. More specifically, we found that the ENO1 protein level in the cultured hippocampal neurons treated with OGD for 1 hour (131.210.06 % of control), or 2 h (202.62.57% of control) was significantly increased, but that it decreased in those neurons treated by OGD for 3 h (67.564.32% of control) as compared to corresponding controls, respectively (Figure 4c and d). Next, the subcellular distribution of ENO1 in cultured hippocampal neurons, with or without OGD treatment, was examined using immunofluorescent staining combined with quantitative analysis, using the anti-ENO1 antibody. The fluorescence intensity in the cytoplasm of the cultured neurons treated with OGD for 2 h was significantly increased to 178.713.72% of control, which is consistent with the immunoblotting result, while cultured neurons treated with OGD for 3 and 6 h resulted in a significant decrease to 68.233.15% and 43.645.68% of control in cytoplasmic fluorescence intensity, respectively (Figure 4e and f). In summary, ENO1 level dynamically changes during cerebral ischemia, the expression of which is increased in the early stage, but decreased in the late stage of ischemia. Previous studies revealed that 20


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resident brain cells mount a natural host defense, or ischemic tolerance, in response to ischemia or preconditioning stimuli, respectively 39, 40. As shown in the current study, the dynamic changes in the level of ENO1 likely represent intrinsic machinery to counter ischemic response in order to mitigate brain cell injury and death, the molecular mechanisms of which remain to be investigated.

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Figure 4. ENO1 expression is dynamically regulated after cerebral ischemia in vivo or OGD in vitro. (a) Representative immunoblotting images probed with the antibody against ENO1 for mouse ipsilateral hemisphere homogenate of sham or ischemia for 1 or 2 h. (b) Quantification 22


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of relative ENO1 protein level from immunoblotting images in (a). n=5 mice (Sham 1 h), 5 mice (Ischemia 1 h); n=4 mice (Sham 2 h), 4 mice (Ischemia 2 h). (c) Representative immunoblotting images of ENO1 for cultured hippocampal neurons treated with OGD or control for 1, 2, or 3 h. (d) Quantification of relative ENO1 protein level from immunoblotting images in (c). At least three independent experiments were conducted for all experiments. (e) Representative images of double staining with anti-ENO1 antibody and DAPI in cultured hippocampal neurons treated with control or OGD for 2, 3 or 6 h. (f) Quantification of the intensity of immunostaining (green) for ENO1 expression level in cultured hippocampal neurons treated with control or OGD for 2, 3 or 6 h. n= 23 neurons/3 cultures (Control), 22 neurons/3 cultures (OGD 2 h), 23 neurons/3 cultures (OGD 3 h), 30 neurons/3 cultures (OGD 6 h). Scale bar, 50 μm, or 20 μm (5 x magnification). Data are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Two-tailed, unpaired Student’s t-test was performed for (b) and (d). Oneway ANOVA test, followed by Tukey’s multiple comparison test was performed for (f).

Overexpression of ENO1 relieves ischemia-induced neuronal injury To test if ENO1 plays any roles during cerebral ischemia, the human ENO1 cDNA plasmid was first selected from the hORFeome V8.1 Lenti Collection 23



Library 41. To determine expression level of the ENO1-V5 tag (ENO1-V5) plasmid in cultured neurons, we transfected the ENO1-V5-expressing plasmid in HEK293 cells for 48 h and immunoblotted with antibodies against ENO1 and V5 tag, respectively. ENO1 was detected in HEK293 cells both with and without ENO1-V5 transfection. As expected, the V5-tagged ENO1 was expressed in HEK293 cells transfected with ENO1-V5 plasmid, but not in the mock HEK293 cells (Figure S3a and b). We then co-transfected ENO1-V5- and GFP-encoding plasmids in cultured hippocampal neurons at 10 days in vitro (DIV), and performed immunocytochemistry with V5 tag antibody at 14 DIV. We found that ENO1-V5 was stained in the GFPpositive neurons (Figure S3c). These data demonstrated that ENO1-V5 was successfully expressed in both HEK293 cells and cultured hippocampus neurons. Next, we examined the structural morphology of neuron including the total length of dendrites and spines numbers per 10 m in GFPexpressing cultured hippocampal neurons, which reflecting neuronal injury 11,

with or without ENO1 overexpression, followed by OGD treatment. We

found that the average total length of dendrite was dramatically decreased to 65.63.92m per neuron after OGD treatment for 2h, which was significantly relieved in ENO1-overexpressing cultured hippocampal neurons (99.363.19m) as compared to that of control (Figure 5a and b). While the average number of spines per 10 μm dendrites was dramatically

24


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decreased to 0.540.05 and 0.20.04 in the cultured hippocampal neurons with OGD treatment for 1h or 2h, respectively. Additionally, we observed that the average number of spines significantly restored to 1.060.06 and 0.790.08 in the ENO1 overexpression neurons after OGD treatment for 1h or 2h, respectively (Figure 5c and d). In conclusion, these data suggested that ENO1 plays a protective role in ischemia-induced neuronal injury. Previous studies revealed that ENO1 is an intracellular protein with a single-enzyme domain

42

and that it redundantly functions with ENO2

31.

However, the activity of ENO1 in the brain remains unknown. Our data showed that ENO1 overexpression in cultured hippocampal neurons alleviates neuronal spine loss and the length reduction of dendrite caused by OGD treatment in vitro (Figure 5A-D). To the best of our knowledge, this is the first study showing that overexpression of ENO1 protects neurons against ischemia-induced injury of dendrites and spines.

25



Figure 5. Overexpression of ENO1 in cultured hippocampal neurons relieves dendrite injury and spine loss during OGD. (a) Representative images of hippocampal neurons transfected with either an empty vector (control) or a vector encoding ENO1 together with pFUGW-GFP at 10 days in vitro (DIV) and analysis of dendrites at 14 DIV after OGD for 1 or 2 h. Scale bars, 20 μm. (b) Quantification of average total dendrite length of cultured neurons with (magenta) or without (black) ENO1 overexpression (ENO1O/E) in (a), n= 27 neurons/3 cultures (Normal/Control), 25 neurons/3 cultures (Normal/ENO1O/E); n=21 neurons/3 cultures (OGD/Control 1h); 26


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n=20 neurons/3 cultures (OGD/ENO1O/E 1h); n=19 neurons/3 cultures (OGD/Control 2 h); n=21 neurons/3 cultures (OGD/ENO1O/E 2h). (c) Representative images of spine density of hippocampal neurons transfected with either an empty vector (control) or a vector encoding ENO1 together with pFUGW-GFP after OGD for 1 or 2 h. Scale bars, 5 μm. (d) Quantification of spine density in cultured hippocampal neurons with (blue) or without (black) ENO1 overexpression (ENO1O/E) in (c). n= 27 neurons/3 cultures (Normal/Control), 25 neurons/3 cultures (Normal/ENO1O/E); n=21 neurons/3

cultures

(OGD/Control

1h);

n=20

neurons/3

cultures

(OGD/ENO1O/E 1h); n=19 neurons/3 cultures (OGD/Control 2 h); n=21 neurons/3 cultures (OGD/ ENO1O/E 2h). Scale bars, 10 μm. Data are means ± SEM. n.s., not significant, *p < 0.05, ***p < 0.001. Two-tailed, unpaired Student’s t-test was applied to compare the two groups between control and OGD. One-way ANOVA test, followed by Tukey’s multiple comparison test was applied to compare multiple control groups among normal, OGD 1 h, and OGD 2h.

The enzymatic product of ENO1, PEP, mitigates primary cultured neurons from OGD-induced neuronal injury Since ENO1 is a key glycolytic metalloenzyme in the cytoplasm of cells 26, 42(Figure

6a) and the overexpression of ENO1 in cultured neurons protects

neuronal cells from OGD-induced injury in present study, we hypothesized 27



Page 28 of 49

that the enzymatic product of ENO1, PEP, might also provide energetic resources required to perform a protective role against ischemia-induced neuronal injury. To test this hypothesis, PEP in ischemic ipsilateral hemisphere

hippocampus

was

first

examined

with

a

PEP

Colorimetric/Fluorometric Assay Kit. The concentration of PEP in the ischemic brain region at 1hour was significantly increased (11.310.20 ng/mg tissue), then gradually and significantly decreased back to 10.340.45 ng/mg tissue and 8.721.03 ng/mg tissue at 2 and 3 h, respectively, after MCAO treatment in vivo (Figure 6b and c). We subsequently tested whether PEP is sufficient to rescue cultured neurons injured by OGD insult. The cultured hippocampal neurons were treated by OGD without or with application

of

PEP

chemical,

Phosphoenolpyruvic

acid

(PEP)

cyclohexylammonium salt (P3637, Sigma, USA) (Figure 6d) for 1 or 2 h in vitro, followed by quantitative evaluation of total length of dendrites per neuron. We found that the average total length of dendrite significantly decreased after OGD treatment for 2h, which was significantly mitigated by the application of PEP chemical (Figures. 6e and f). Next, we also examined the neuronal survival by quantitative evaluation of NeuN-positive neurons, and found that the number of survival neurons decreased to 19.352.34 % with OGD treatment for 2 h, but this neuronal loss was significantly mitigated by the application of PEP chemical (27.212.62 %) (Fig. 6g and

28


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h). These results supported the notion that the ENO1 enzymatic product, PEP, protects neurons from OGD-induced neuronal injury.

29



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Figure 6. ENO1 enzymatic product PEP mitigates OGD-induced neuronal injury in cultured hippocampal neurons. (a) Chemical reaction equation of ENO1 catalyzing 2-phosphoglycerate (2-PG) to produce phosphoenolpyruvate (PEP) and water (H2O). (b) Experimental schematic for PEP quantification after sham or cerebral ischemia. (c) Summary graphs of concentration of PEP after sham or cerebral ischemia for 1 h, 2 h, and 3 h, respectively. At least three independent experiments were performed. (d) Chemical formula of phosphoenolpyruvic acid (PEP) cyclohexylammonium salt (P3637, Sigma, USA) applied in (e) - (h). (e) Representative images of hippocampal neurons transfected with pFUGW-GFP treated by OGD for 1 or 2 h with or without PEP chemical application. Scale bars, 20 μm.

(f)

Quantification of average total dendrite length per neurons treated with OGD for 1 or 2 h with or without PEP chemical application. n= 13 neurons /3 cultures (Control 1h), 15 neurons/ 3 cultures (OGD 1h), 14 neurons /3 cultures (OGD 1h + PEP); n=13 neurons/3 cultures (Control 2h), 13 neurons/3 cultures (OGD 2h), 13 neurons/3 cultures (OGD 2h + PEP). (g) Representative images for double staining with anti-NeuN antibody (green) and DAPI (blue) in cultured hippocampal neurons treated by OGD for 1 or 2 h with or without PEP chemical application. Scale bars, 50 μm. (h) Quantification of positive cells stained with anti-NeuN antibody in (e). Data are means ± SEM. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA test, followed by Tukey’s multiple comparison test was 30


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applied for (c), (f), and (h).

PEP reduces ischemia-induced neuronal loss and infarct size in MCAO model of Focal cerebral ischemia in vivo To corroborate the neuroprotective effect of compound PEP on experimental ischemic stroke, the PEP chemical was stereotactic injected into the CA1 region of the hippocampus after focal cerebral ischemia for 2 h (Figure 7a). Neuronal loss was observed in the hippocampal CA1 region at 3 h after the onset of focal cerebral ischemia. The effect of ischemia-induced neuronal loss was significantly suppressed by the application of PEP chemical (Figures 7b and c). Next, we evaluated the infarct size of ischemic brain sections at 24 h after MCAO ischemia without or with PEP administration by using 2,3,5-tetrazolium chloride (TTC) staining. We found that the infarct volume size of the ischemic lesion in PEP-treated animal was significantly smaller than in vehicle-treated animals (Figures 7d and e). These results combined with the data obtained in cultured hippocampus neuron by the application of PEP chemical (Figure 6e - h) demonstrated a neuroprotective effect of PEP in the experimental focal cerebral ischemia model.

31



Page 32 of 49

Figure 7. PEP relieves ischemia-induced neuronal loss and cerebral infarct in MCAO model in vivo. (a) Timeline of experiments for sham or MCAO

operation,

stereotactic

injection

of

PEP

chemical,

immunohistochemistry (IH), and brain slice TTC staining. (b and c) Representative images (b) and quantification of NeuN staining-positive cells (c) of hippocampal CA1 region neurons stained with NeuN (green) and DAPI (blue) from vehicle- and PEP-treated mice after cerebral ischemia for 3h. n=3 mice (Sham 3h), 3 mice (Ischemia 3h), 3 mice (Sham 3h + PEP). (d) Representative images of Six coronal sections (2mm each) from sham, vehicle and PEP-treated mice after MCAO stained with TTC. (e) Summary graphs of infarct size of vehicle and PEP-treated mice stained with TTC. N=3 mice (Ischemia 24h), 3 mice (Sham 24h + PEP). Data are means ± SEM. 32


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n.s., not significant, *p < 0.05. One-way ANOVA test, followed by Tukey’s multiple comparison test was applied for (c). Two-tailed, unpaired Student’s t-test was applied for (e).

Recent studies have uncovered that ENO1 plays an essential role in growth, survival and the tumorigenic potential in glioblastoma cells 31 and promotes tumorigenesis and metastasis via regulating the AMPK/mTOR pathway in colorectal cancer 43. Together with our data, these studies indicate that ENO1 plays multiple roles in various cells, raising the possibility that ENO1 mediates neuronal survival through a molecular mechanism distinct from that observed in cancer cells. It will be intriguing to investigate the detailed molecular mechanism of neuronal function in future studies. Moreover, previous study showed that neuron-specific enolase (NSE), namely ENO2, is elevated in acute spinal cord injury in rats, which accelerates neuronal damage

44.

In future studies, it will be interesting to test if ENO2 is also

involved in neuronal injury of cerebral ischemia.

Methods. Animals C57BL/6J male wild-type mice (P0 to P56) were used for this study. All animal experiments were conducted in strict accordance with the Guide for

33



the Care and Use of Laboratory Animals (Eighth Edition). All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Hunan University. Mice were separately housed in a temperature- and humidity-controlled room under a 12-h light-dark cycle with free access to food and water.

Middle Cerebral Artery Occlusion Model Focal cerebral ischemia was induced by intraluminal middle cerebral artery occlusion (MCAO)45. Briefly, mice (aged 56 days, 25 ± 2 g of body weight) were anesthetized with 5% isoflurane and maintained with 1% isoflurane (R511-22, RWD Life Science, Shen Zhen, China) in an oxygen/air mixture by using a gas anesthesia mask (R580S, RWD Life Science). The left common carotid artery (CCA) and the external carotid artery (ECA) were exposed by a ventral midline neck incision and clipped. The ECA was ligated with 5-0 silk suture, and a 2-cm long silicon-rubber-coated monofilament (MSMC24B104PK50, RWD Life Science) was advanced from the CCA through the internal carotid artery (ICA) up to the level of the anterior cerebral artery. The suture was inserted 9–11 mm from the bifurcation of CCA to occlude the middle cerebral artery (MCA) to induce permanent cerebral ischemia. As a control, the sham operated animals were treated identically, except that the middle cerebral artery was not occluded after the neck incision. 34


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Brain homogenate preparations and protein identification Brain homogenate was prepared as described before 46. One mouse ipsilateral hemisphere was homogenized in 3 ml of PBS, 0.1 mM EGTA (E0396, Sigma),

1

mM

PMSF

(10837091001,

Sigma),

1

μg/ml

pepstatin(10253286001, Sigma), 1 μg/ml leupeptin (L2884, Sigma) and 2 μg /ml aprotinin(A6103, Sigma) and adjusted to 1% Triton X-100（T9284， Sigma）final concentration. Proteins in the homogenate were extracted for 2 h at 4 °C, and insoluble debris was removed by centrifugation (2 h at 10,000 g). Mouse brain homogenate was excised manually from the Coomassie Brilliant Blue-stained gels and protein bands and then digested automatically using 6×5 LC-MS/MS peptide Reference Mix (Promega, Madison, WI, USA). Tryptic products were analyzed using the LTQ Orbitrap Velos Pro (Thermo Fisher Scientific). Each spectrum was internally calibrated with mass signals of trypsin autolysis ions to reach a typical mass measurement accuracy of ± 10 ppm. Raw data were searched in the SWISSPROT database. The Proteome Discoverer™ Software (version 2.0) was employed to do the match score between MS data and SwissProt database.

Western blotting Protein extracts were denatured at 95°C for 5 min and separated on 10% 35



sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels at 110 V for about 70 min. The proteins were transferred to nitrocellulose (NC, HATF00010, Millipore) filters at 80 V for 5 h. The NC membrane was initially blocked with 5% nonfat milk and 2% goat serum （ 16210064 , Thermo Fisher ） (v/v) in Tris-buffered saline with 0.1% Tween 20 (93773, Sigma) (TBS-T) at room temperature for 1 h. Monoclonal antibodies to β-actin (1:5000, HC201-02, TransGen Biotech), the V5 tag (1:500, HT401-02, TransGen Biotech) and polyclonal antibody to ENO1 (1:2000, CSB-PA007670GA01HU; CUSABIO Biotech) were employed for Western blot analyses as primary antibodies at 4°C overnight. After three washes of 5 min each with TBS-T, either goat anti-rabbit (HS101-01, TransGen Biotech) or anti-mouse （ HS201-01, TransGen Biotech ） immunoglobulin G (IgG) was added at a dilution of 1: 5,000 as the secondary antibody. The NC membrane was scanned with an imaging system (MicroChemi 4.2, DNR Bio Imaging Systems, Jerusalem, Israel).

RNA isolation and RT-PCR Brain samples were homogenized in a glass-Teflon® homogenizer according to the protocol supplied with the TransZol Reagent (ET101-01, TransGen Biotech, China). The concentration of RNA was measured with spectrophotometry, and the reaction volume consisted of 1 μg of total RNA, 36


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2×TS Reaction Mix, TransScript® RT/RI Enzyme Mix (TransGen Biotech), oligo (dT), gDNA Remover, and RNase-free H2O (to a final volume of 20 μl). The amplification program was as follows: 42 °C for 15 min, 85 °C for 5 s, and a final hold at 4 °C. RT-PCR was carried out in a C1000 Touch Thermal Cycler (BIO-RAD, CA, USA) RT-PCR system with qPCR Mix using the designed primers. ENO1 forward primer (5′-GTCTCACAGGCTG TTGAGCACATC-3′), and ENO1 reverse primer (5′- CACCAGCTTTGC AGACAGCCAG-3′). GAPDH forward primer (5′-ACTTCAACAGCA ACTCCCACTC -3′), and GAPDH reverse primer (5′- TAGGCCCCTCCT GTTATTATGG-3′). Polymerase chain reaction (PCR) was performed with the following protocol: 95°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 30 s (30 cycles); 72°C for 5 min, and a final hold at 4°C. PCR products were run on 3 % agarose gel.

HEK293 cell culture and transfection Human embryonic kidney 293 (HEK293) cells (CRL-11268, ATCC) were grown at 37°C supplied with 5% CO2 in an incubator (Thermo Fisher) with a humidified atmosphere as described before

23.

The cells were grown in

Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and penicillin-streptomycin (15140122, Gibco, Grand Island, NY, USA). The cells were washed once using PBS and digested with 0.05% TrypsinEDTA (25300054, Gibco, Grand Island, NY, USA) at 37°C for routine 37



passage of the cells. All HEK293 cell transfections were performed using the polyethylenimine method. The ratio of polyethylenimine (PEI, 1 mg/ml in ddH2O) (24765-2, Polyscience, USA) to DNA was 3:1. The PEI/DNA mixture was incubated for 30 min at room temperature before the mixture was added to the HEK293 cell cultures dropwise.

Primary hippocampal neuron culture and OGD treatment Cultured neurons were obtained from C57BL/6J mouse hippocampal cells, as described previously 43. Briefly, mouse hippocampal cells were dissected from postnatal day-0 wild-type mice, dissociated with 0.25% trypsin (25200056, Gibco, Grand Island, NY, USA), digested for 12 min at 37°C, plated on poly-D-lysine-coated glass coverslips (8 mm) at a density of 80,000 neurons per coverslip (Scope Cell Counter Basic, C.E.T. Corporation, Beijing, China), and maintained at 37°C in a humidified chamber of 95% air and 5% CO2. OGD experiments were performed using a specialized humidified chamber maintained at a constant temperature of 37 °C, which contained an anaerobic gas mixture (90% N2, 5% H2, and 5% CO2) 11. To initiate OGD, culture medium was replaced with deoxygenated, glucose-free Dulbecco’s modified Eagle’s medium (Life Technology, 11966-025), and culture was treated for various times (1, 2, 3 and 6 h). The normal culture was used as a control.

38


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Immunocytochemistry and confocal imaging Primary neuronal cultures were fixed for 12 min with 4% paraformaldehyde (16005, Sigma) and 4% sucrose (V900116, Sigma) in phosphate-buffered saline (PBS, a pH of 7.4), followed by permeabilization with 0.2% Triton X100 (T9284, Sigma) (v/v) in PBS. An initial blocking step was performed with PBS-MILK/NGS (PBS containing 5% milk and 3% normal goat serum) for 30 min at room temperature, followed by incubation overnight with antiENO1 antibody (1:4000, CSB-PA007670GA01HU; CUSABIO Biotech) diluted in PBS-MILK/NGS. After washing with PBS, cultures were incubated with Alexa Fluor® 546-conjugated goat anti-rabbit (A-11071, 1:500; Invitrogen, Eugene, OR, USA) antibody to detect ENO1. After washing with PBS, the samples were mounted with a mounting medium Fluoromount-G® mounting medium (0100-01, Southern Biotechnology Associates, Inc., USA). The transfected neurons were randomly chosen, and images were acquired using a confocal microscope (Olympus FV1000) with a 60× objective lens; the same settings were maintained for the images of all samples. Z-stacked confocal images were converted to maximum projections and analyzed with respect to the length and density of the dendrite and spine using Image J software. The integrated density (IntDen) of ENO1 was measured by NIH image program, the neighborhoods background of IntDen were measured with the same area. The protein expression of ENO1 were quantified by IntDen of neurons minus neighborhoods background of IntDen. 39



Immunohistochemistry Mice were perfused with saline solution prior to perfusing with 4% paraformaldehyde, post-fixed overnight in 4% paraformaldehyde at 4 °C, cryoprotected in 30% sucrose, and embedded in optimal cutting temperature compound

（ OCT, 25608-930, Tissue-Tek® O.C.T. Compound

Sakura）. Tissues sections were initial blocking in 0.5% Tween-20 (v/v) in PBS-MILK/NGS (PBS containing 5% milk and 3% normal goat serum) for 2 h at room temperature, followed by incubation with primary antibodies (NeuN, 1:1000, ab177487, Abcam; USA) overnight at 4 °C and then with Alexa Fluor488-conjugated goat anti-rabbit (1:500, A-11034, Life Science & Technology, USA) for 1 h at room temperature. After washing with PBST, the sections were mounted with Fluoromount-G® mounting medium (010001, Southern Biotechnology Associates, Inc., USA). Sections were analyzed by confocal fluorescence microscopy (Olympus FV1000) with a 60× objective lens.

Primary neuron culture and plasmid transfection Hippocampal neurons were transfected using the calcium phosphate transfection method after 10 days in vitro (DIV) and analyzed at 14 DIV as described before 47. Briefly, for each coverslip in a 48-well plate, 0.6 μg of 40


Page 40 of 49

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the total plasmid was mixed with 0.99 μl of 2 M CaCl2 solution and ddH2O was added to reach a final volume of 8 μl, and the DNA/CaCl2 solution was added slowly to 8 μl of 2×HBS (631312, CalPhos™ Mammalian Transfection Kit, TaKaRa). DNA/CaCl2/HBS solution was incubated at room temperature for 30 min and then added to the neuronal cell cultures and incubated for 30 min at 37 °C in CO2 incubator. The cells were washed once with medium containing MgCl2 and were maintained in CO2 incubator for 4 days before OGD or immunocytochemistry.

Phosphoenolpyruvate quantification and PEP chemical administration Phosphoenolpyruvate quantification were performed according to the manufacturer’s instructions (Sigma, USA). Briefly, hippocampus were frozen in liquid nitrogen then powdered thoroughly with mortar and pestle at –80 ℃. Transfer to a microcentrifuge tube. Add 100 μl of 3 M ice-cold perchloric acid and vortex until contents are thoroughly mixed. Neutralize by adding 10 μl of 3 M potassium bicarbonate until the pH reaches 6.5–7.5. Vortex between potassium bicarbonate additions. Centrifuge at 12,000 g for 3 minutes to remove insoluble debris. The sample preparations were reaction with a PEP Colorimetric/Fluorometric Assay Kit (MAK102, Sigma). For PEP chemical application, the culture medium was replaced with deoxygenated,

glucose-free

Dulbecco’s

modified

41


Eagle’s

medium


containing 1mM PEP chemical (P3637, Sigma), then subjected to OGD treatment for 1 h or 2 h. For stereotactic injection of PEP chemical, glass pipettes were pulled from borosilicate glass capillary tubes (World Precision Instruments) using a P-97 pipette puller (Sutter Instrument). Stereotactic injection (RWD 68526, RWD Life Science, Shen Zhen, China) of 1 μL of 1mM PEP were at coordinates 2.0mm posterior, 1.5 mm lateral and 1.9 mm ventral relative to bregma and stopped at the injection site for five minutes. Infarct Volume Evaluation The size of infarction was evaluated at 24 h after MCAO by using 2,3,5tetrazolium chloride (TTC) staining as described previously10. Briefly, mice were sacrificed under deep anesthesia at 24 h after MCAO. Brain was removed rapidly and cut into 2mm thick coronal sections by a Mouse Brain Slicers (68707, RWD Life Science), and the sections were immersed in 2% TTC solution for 15 min at 37℃. The size of infarct volume was determined by examining the areas of TTC-stained sections that did not stain with TTC by using the Image-J software.

Statistical analysis Data are presented as mean ± SEM. The data presented in this study were obtained from at least three independent experiments. To compare two groups, we performed two-tailed, unpaired Student’s -t tests. For comparison 42


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of more than two groups, we used one-way ANOVA tests, followed by Tukey’s multiple comparison tests. A value of p < 0.05 was taken as a significant difference between groups. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA).

Abbreviations. ENO1, Enolase1; MCAO, middle cerebral artery occlusion; RT-PCR, reverse transcription polymerase chain reaction ; OGD, oxygen/glucose deprivation; AIS, acute ischemic stroke; rtPA, recombinant tissue plasminogen activator ; FDA, Food and Drug Administration ; AMPAR, αamino-3-hydroxy-5-methyl-4-isoxazol propionic acid receptor; NMDAR, N-methyl-D-aspartic acid receptor; KAR, kainic acid receptor; DAPK1, death-associated protein kinase 1; CDK5, Cyclin- dependent Kinase 5; IRF4, Interferon regulatory factor 4; I/R, ischemia/reperfusion; KO, knockout ; O/E, overexpression; CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; DIV, Day in vitro.

Supporting Information. Three supporting figures containing peptides of ENO1 by MS, cultured hippocampal neurons 2 h after OGD treatment, and ENO1-V5 expressed in HEK293 cells and cultured hippocampal neurons. 43



Page 44 of 49

Author Information. Corresponding Author #

Mailing address: Institute of Neurosience, State Key Laboratory of

Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, Hunan, 410082, China. Telephone: 0086-731-84111402. E-mail: [email protected] ORCID Haijun Tu: 0000-0002-8960-856X Weihong Tan: 0000-0002-8066-1524 Author contributions * W.J., X.T., and P.Y. contributed equally to this work. W.J., X.T., P.Y., and H.T. designed the research; W.J., X.T., P.Y., J.L., L.X., J.L., and C.L. performed the research; W.J., X.T., P.Y., and H.T. analyzed data; W.J., W.T., and H.T. wrote the paper. Funding This work was supported by the National Natural Science Foundation of China (31540020, 31671048), the Free Exploration Foundation of Shenzhen Science

and

Technology

Innovation

Committee

(JCYJ20160530192506314), and the Provincial Natural Science Foundation 44


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of Hunan Province (2017JJ2041) to H. T. and Human Provincial Innovation Foundation For Postgraduate (CX2018B212). Notes The authors declare no competing financial interest.

Acknowledgements. The authors thank Dr. Jian Yi for the critical assistance for MCAO model at the initiation of project, thank Dr. Chen Zhang for the kind gift of plasmid encoding ENO1, and thank the facilities for Mass Spectra and confocal microscopy at SKLCBSC (Hunan University).

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For Table of Contents Use Only Enolase1 alleviates cerebral ischemia-induced neuronal injury via its enzymatic product Phosphoenolpyruvate Wei Jiang, †,* Xibin Tian, †,* Peng Yang, †,* Jianglin Li,†,‡ Le Xiao, † Junqiang Liu, † Chao Liu,‡ Weihong Tan, †,‡,§ and Haijun Tu†,||#

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Enolase1 alleviates cerebral ischemia-induced neuronal injury via its

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