Impairment of neuronal tyrosine phosphatase STEP worsens post-ischemic inflammation and brain injury under hypertensive condition

Abstract

Hypertension is associated with poor outcome and higher mortality in patients with ischemic stroke. The impairment of adaptive vascular mechanisms under hypertensive condition compromises collateral blood flow after arterial occlusion in patients with acute ischemic stroke resulting in hypoperfusion. The increased oxidative stress caused by hypoperfusion is thought to be a trigger for the rapid evolution of ischemic infarct volume under hypertensive condition. However, the cellular factors and pathways that contribute to the exacerbation of ischemic brain injury under hypertensive condition is not yet understood. The current study reveals that predisposition to hypertension leads to basal loss of function of the neuron-specific tyrosine phosphatase STEP, which plays a crucial role in neuroprotection against excitotoxic insult. The findings further show that a mild ischemic insult in hypertensive rats triggers an early onset and sustained activation of the neuronal extracellular signal regulated kinase (ERK MAPK), a member of the mitogen activated protein kinase family and a substrate of STEP. This leads to rapid increase in the activation of neuronal NF-κB, expression of neuronal cyclooxygenase-2 and subsequent biosynthesis of the pro-inflammatory mediator prostaglandin E2, resulting in rapid morphological transformation of microglia to the pro-inflammatory state and subsequent exacerbation of ischemic brain injury. Restoration of STEP signaling with intravenous administration of a STEP-derived peptide mimetic reduces the pro-inflammatory response in neurons, activation of microglia, and ischemic brain injury. The findings suggest that the basal loss of STEP function under hypertensive condition contributes to the exacerbation of ischemic brain injury by enhancing post-ischemic inflammatory response. The study not only presents a novel role of STEP in regulating neuroimmune communication but also highlights the therapeutic potential of a STEP-mimetic in mitigating ischemic brain damage under hypertensive condition.

Keywords: Cyclooxygenase-2; ERK MAPK; Hypertension; Microglia; NFκB; Neuroinflammation; STEP; Tyrosine phosphatase.

Fig. 1

Predisposition to hypertension accelerates the progression of ischemic brain injury. (A - G) SD and SHR rats were subjected to MCAO (60 min) followed by reperfusion for the specified time periods (6, 12–24 h). (A) Neurological severity score was assessed 24 h after MCAO on a 5-point scale and represented as individual data points with median value. (B) Representative photomicrographs of TTC-stained brain slices (2 mm) show the extent of brain damage 24 h after the onset of ischemia. (C) Bar graphs represent quantitative analysis of total infarct volume (mean ± SD, n = 5–6/group). (D) Line graph represents total infarct area within each slice (mean ± SD, n = 5–6/group). (E-G) Representative photomicrographs of coronal brain sections at 6, 12 and 24 h after ischemia stained with Fluoro-Jade C. (H) Bar graphs represent quantitative analysis of infarct volume at 6, 12 and 24 h respectively (mean ± SD, n = 3). Significant difference between means was assessed by student t-test and presented as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 2

Loss of basal STEP function in SHR rats is associated with sustained increase in neuronal ERK MAPK phosphorylation during ischemia and reperfusion. (A) Cortical tissue lysates from SD and SHR rats with equal amount of protein were processed for immunoblot analysis under non-reducing condition (without β-mercaptoethanol) using anti-STEP antibody to evaluate STEP dimer formation (upper panel). The same lysates were also processed for immunoblot analysis under reducing condition (with β-mercaptoethanol) using anti-STEP antibody to assess total STEP expression level (lower panel). Bar graphs represent quantitative analysis of STEP dimer formation (mean ± SD, n = 6). (B) STEP was immunoprecipitated from equal amount of cortical tissue lysates of SD and SHR rats using anti-STEP antibody and the immune complexes were processed either for tyrosine phosphatase activity assay using pNPP as a substrate or immunoblotting with anti-STEP antibody to ensure equal pull down of STEP. Bar graphs represent quantitative analysis of STEP phosphatase activity (mean ± SD, n = 6) (C) SD and SHR rats were subjected to sham surgery or MCAO (I) for the specified time periods (10 and 60 min). In some experiments 60 min MCAO was followed by reperfusion (RPF) for the specified time periods (3, 6 and 18 h). Tissue lysates with equal amount of protein from the ipsilateral cortex were analyzed using anti-phospho-ERK1/2 (pERK1/2) MAPK antibody (upper panels). Equal protein loading was confirmed by re-probing the blots with anti-ERK2 antibody (lower panels). Bar graphs represent quantitative analysis of pERK levels (mean ± SD, n = 4). (A-C) Significant difference between means was assessed by student t-test and presented as *p < 0.01, **p < 0.001 and ***p < 0.0001. (D) SD and SHR rats were subjected to sham surgery or 60 min MCAO followed by reperfusion for 6 h and then processed for immunohistochemical staining with anti-phospho-ERK1/2 (pERK - red) antibody, a neuron specific antibody, anti-NeuN (green) and nuclear stain DAPI (blue). Arrows in the representative photomicrographs demonstrate localization of phosphorylated ERK in NeuN positive cells in the ipsilateral cortex

Fig. 3

Activation of neuronal NF-κB in SHR rats following ischemia and reperfusion. (A-J) SD and SHR rats were subjected to sham surgery, or MCAO (60 min) followed by reperfusion (RPF) for 6 h. (A-H) Tissue extracts from the ipsilateral cortex were processed to measure mRNA levels of NFκB1 and RelA by quantitative real-time-PCR. Transcript levels were normalized against reference genes β-actin or HPRT. Bar graphs represent relative expression of NFκB1 and RelA genes (mean ± SE, n = 5–6). (I) Tissue lysates with equal amount of protein from the ipsilateral cortex were analyzed using anti-IκB antibody (upper panels). Equal protein loading was confirmed by re-probing the blots with anti-β-tubulin antibody (lower panel). Bar graps represent quantitative analysis of IκB protein levels (mean ± SD, n = 4). (A-I) Significant difference between means was assessed by student t-test and presented as *p < 0.05, **p < 0.01 and ***p < 0.001. (J) Coronal brain sections through the ipsilateral cortex of SD and SHR rats (MCAO 60’/RPF 6 h) were processed for immunohistochemical staining with anti-phospho-NFκB (pNFκB - red) antibody, anti-NeuN (green) antibody and nuclear stain DAPI (blue). Arrows in the representative photomicrographs demonstrate nuclear localization of phosphorylated NFκB in NeuN positive cells in the ipsilateral cortex

Fig. 4

Increase in neuronal COX-2 expression and prostaglandin E2 level in SHR rats following ischemia and reperfusion. (A-F) SD and SHR rats were subjected to sham surgery or MCAO (60 min) followed by reperfusion (RPF) for 6 h. (A-D) Tissue extracts from the ipsilateral cortex (MCAO-60’/RPF 6 h) were processed to measure mRNA levels of COX-2 by quantitative real-time-PCR. Transcript levels were normalized against housekeeping genes β-actin or HPRT. Bar graphs represent relative expression of COX-2 gene (mean ± SE, n = 5–6). (E) Coronal brain sections through the ipsilateral cortex of SD and SHR rats (MCAO-60’/RPF 6 h) were processed for immunohistochemical staining with anti-COX-2 antibody (red), anti-NeuN antibody (green) and nuclear stain DAPI (blue). Arrows in the representative photomicrographs demonstrate localization of COX-2 in NeuN positive cells in the ipsilateral cortex. (F) PGE₂ level was measured by enzyme immunoassay in the supernatants from tissue lysates extracted from ipsilateral cortex of SD and SHR rats (MCAO-60’/RPF 6 h). Bar graphs represent quantitative analysis of PGE₂ level (mean ± SD, n = 3). (A-D, F) Significant difference between means was assessed by student t-test and presented as *p < 0.01

Fig. 5

Treatment with STEP mimetic alleviates post-ischemic inflammatory response in neurons of SHR rats. (A) Diagram of STEP₆₁ indicating the positions of the phosphatase domain, putative proteolytic sites (PEST), transmembrane domain (TM), polyproline rich regions (PP), kinase interacting motif (KIM), kinase specificity sequence (KIS) and known phosphorylation sites. The STEP mimetic (TAT-STEP-myc peptide) was generated from STEP₆₁. The peptide was rendered cell-permeable by fusion to the 11 amino acid protein transduction domain (TAT) of the human immunodeficiency virus-type I at the N-terminus and has a myc-tag at the C-terminus. The serine residue in the KIM domain was mutated to alanine to allow the peptide to bind constitutively to its substrates. The threonine and serine residues in the KIS domain were mutated to glutamic acid to render the peptide resistant to degradation. (B-L) SHR rats were subjected to MCAO (60 min) followed by reperfusion (RPF) for 6 h. TAT-STEP-myc peptide (TAT-STEP) was administered in a subset of SHR rats at the onset of reperfusion. (B) ERK MAPK phosphorylation and (G) IκB protein level were evaluated by immunoblot analysis of tissue extracts from ipsilateral cortex. Blots were re-probed with (B) anti-ERK and (G) anti-β-tubulin antibodies (lower panels). Bar graphs represent mean ± SD (n = 3). (C-F, I, J) Tissue extracts from the ipsilateral cortex were processed to measure mRNA levels of (C, D) NFκB1, (E, F) RelA and (I, J) COX-2 by quantitative real-time PCR. Bar graphs represent relative expression of NFκB1, RelA and COX-2 normalized against reference genes β-actin or HPRT (mean ± SE, n = 6). (H, K) Coronal brain sections through the ipsilateral cortex were processed for immunohistochemical staining with (H) anti-pNFκB (red) antibody, anti-NeuN antibody (green) and nuclear stain DAPI (blue); and (K) anti-COX-2 antibody (red), anti-NeuN antibody (green) and nuclear stain DAPI (blue). Arrows in the representative photomicrographs demonstrate (H) nuclear localization of pNFκB in NeuN positive cells and (K) localization of COX-2 in NeuN positive cells. (I, J). (L) PGE₂ level was measured by enzyme immunoassay using supernatants obtained from ipsilateral cortex. Bar graphs represent quantitative analysis of PGE₂ level (mean ± SD, n = 3). (B-G, I, J, L) Significant difference between means was assessed using student t-test and presented as *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 6

Enhanced morphological transformation of microglia to amoeboid form in SHR rats following ischemia and reperfusion. SD and SHR rats were subjected to sham surgery, or MCAO (60 min) followed by reperfusion (RPF) for 6 h. (A-D) Coronal brain sections through the ipsilateral cortex were processed for immunohistochemical staining with anti-Iba1 antibody. Representative photomicrographs in the left panels show microglial morphology in (A) SD sham, (B) SHR sham, (C) SD MCAO-60’/RPF-6 h and (D) SHR MCAO-60’/RPF-6 h. Amplified images of selected Iba-1 positive microglia (in yellow box) and skeletonized pattern of the microglia are shown in the right panels. (E-M) Microglial cells (n = 45–50 cells) imaged from the ipsilateral cortex of SD and SHR rats following MCAO / reperfusion were processed for assessment of morphological parameters by (E-I) fractal and (J-M) skeletal analysis. Scatter plots with mean ± SD presents the eight parameters: fractal dimension, lacunarity, cell area, cell perimeter, cell circularity, number of branches, number of junctions, maximum branch length and longest shortest path. (N-U) Tissue extracts from the ipsilateral cortex were processed to measure mRNA levels of (N-Q) CD68 and (R-U) TNFα by quantitative real-time PCR. Bar graphs represent relative expression of CD68 and TNFα normalized against reference genes β-actin or HPRT (mean ± SE, n = 6). Significant difference between means was assessed by student t-test and presented as *p < 0.01, **p < 0.001 and ***p < 0.0001

Fig. 7

STEP-mimetic alleviates post-ischemic microglial activation in SHR rats. SHR rats were subjected to MCAO (60 min) and reperfusion (RPF) for 6 h. STEP-mimetic (TAT-STEP-myc) was administered in a subset of SHR rats at the onset of reperfusion. (A-B) Immunohistochemical staining of microglia with anti-Iba1 antibody. Representative photomicrographs in the left panels show changes in microglial morphology in SHR rats treated with or without the STEP-mimetic. The right panels show amplified images of a select Iba-1 positive microglia (in yellow box) and skeletonized pattern of microglial morphology. (C-K) Microglial cells (n = 45–50 cells) from the ipsilateral cortex were processed for assessment of morphological parameters by (C-G) fractal and (H-K) skeletal analysis. Scatter plots with mean ± SD presents the eight parameters: fractal dimension, lacunarity, cell area, cell perimeter, cell circularity, number of branches, number of junctions, maximum branch length and longest shortest path. (L-O) Tissue extracts from the ipsilateral cortex were processed to measure mRNA levels of (L, M) CD68 and (N, U) TNFα by quantitative real-time PCR. Bar graphs represent relative expression of CD68 and TNFα normalized against reference genes β-actin or HPRT (mean ± SE, n = 6). (C-O) Significant difference between means was assessed by student t-test and presented as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 8

STEP-mimetic attenuates exacerbation of ischemic brain injury in SHR rats. (A) SHR rats were subjected to MCAO (60 min) and reperfusion (RPF) for 24 h. STEP-mimetic (TAT-STEP-myc) was administered in a subset of SHR rats at the onset of reperfusion. (A) Neurological severity score was assessed 24 h after MCAO on a 5-point scale and represented as individual data points with median value. (B) Representative photomicrographs of TTC-stained brain slices (2 mm) show the extent of brain damage 24 h after ischemia and reperfusion. (C) Bar graphs represent quantitative analysis of the total infarct volume (mean ± SD, n = 6). (D) Line graphs represent area of infarction within each slice. Significant difference between means was assessed by student t-test and presented as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 9

Schematic diagram representing impairment of STEP function under hypertensive condition causing upregulation of post-ischemic inflammatory response and exacerbation of brain injury. (A) Hypertension diminishes the basal activity of the neuroprotectant STEP in the brain. (B) The impairment of STEP function in SHR rats facilitates prolonged activation of neuronal ERK MAPK (substrate of STEP) following a mild ischemia and reperfusion. This in turn triggers an early onset of neuronal NF-kB activation, cyclooxygenase 2 (COX-2) expression, and prostaglandin E2 (PGE₂) release resulting in upregulation of microglial activation and subsequent exacerbation of ischemic brain injury