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, 33 (45), 17814-26

Neuroprotective Role of a Brain-Enriched Tyrosine Phosphatase, STEP, in Focal Cerebral Ischemia

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Neuroprotective Role of a Brain-Enriched Tyrosine Phosphatase, STEP, in Focal Cerebral Ischemia

Ishani Deb et al. J Neurosci.

Abstract

The striatal-enriched phosphatase (STEP) is a component of the NMDA-receptor-mediated excitotoxic signaling pathway, which plays a key role in ischemic brain injury. Using neuronal cultures and a rat model of ischemic stroke, we show that STEP plays an initial role in neuroprotection, during the insult, by disrupting the p38 MAPK pathway. Degradation of active STEP during reperfusion precedes ischemic brain damage and is associated with secondary activation of p38 MAPK. Application of a cell-permeable STEP-derived peptide that is resistant to degradation and binds to p38 MAPK protects cultured neurons from hypoxia-reoxygenation injury and reduces ischemic brain damage when injected up to 6 h after the insult. Conversely, genetic deletion of STEP in mice leads to sustained p38 MAPK activation and exacerbates brain injury and neurological deficits after ischemia. Administration of the STEP-derived peptide at the onset of reperfusion not only prevents the sustained p38 MAPK activation but also reduces ischemic brain damage in STEP KO mice. The findings indicate a neuroprotective role of STEP and suggest a potential role of the STEP-derived peptide in stroke therapy.

Figures

Figure 1.
Figure 1.
Activation of p38 MAPK and STEP during MCAO and reperfusion. SD (a–e) and Wistar (g–j) rats were subjected to MCAO for (a, b, g, h) 15, 30, 60, or 90 min or (d, e, i, j) 90 min followed by reperfusion for 3, 6, or 12 h. Tissue punches from the ipsilateral striatum were analyzed by immunoblotting with (a, d, g, i) anti-phospho-p38 MAPK (top) and reprobed with anti-p38 MAPK (bottom); (b, e, h, j) anti-STEP antibody (top, STEP61; middle, STEP33) and then reprobed with anti-tubulin antibody (bottom). c, STEP was immunoprecipitated from striatal lysates (sham and 90 min MCAO) using anti-STEP antibody and processed for immunoblot analysis with p-ser 221 antibody (top). The blot was then reprobed with anti-STEP antibody (bottom). f, SD rats were subjected to 90 min MCAO followed by reperfusion for 6 h and then processed for immunohistochemistry with anti-phospho-p38 MAPK and NeuN antibodies and DAPI staining. Bar diagrams represent mean ± SEM obtained from 4 animals per group. a, *p < 0.01, from 15 min MCAO. **p < 0.001, from 15 min MCAO. b, *p < 0.001, from phosphorylated STEP in sham. #p < 0.001, from dephosphorylated form of STEP in sham. d, *p < 0.001, from I/R-6 h. e, *p < 0.05, from I/R-6 h.
Figure 2.
Figure 2.
Downregulation of STEP precedes ischemic brain damage. SD rats were subjected to right MCAO for 90 min followed by reperfusion for specified time periods (3, 6, and 12 h). a, Immunohistochemistry of coronal sections through the striatum with anti-STEP antibody. b, Fluro-Jade C staining (a marker for cellular degeneration) of adjacent sections.
Figure 3.
Figure 3.
TAT-STEP-myc peptide blocks phosphorylation of p38 MAPK and attenuates OGD-induced neuronal cell death. Neuron cultures were exposed to (a, b) OGD for 10 and 30 min or (c, d) OGD for 2 h (OGD/R-0 h) followed by reoxygenation for 4 h (OGD/R-4 h). Immunoblot analysis of cell lysates with (a, c) anti-phospho-p38 MAPK (top) and reprobed with anti-p38 MAPK (bottom), (b, d) anti-STEP antibody (top- STEP61; middle, STEP33), and then reprobed with anti-tubulin antibody (bottom). e, Schematic diagram of the TAT-STEP-myc peptide (TAT-STEP) indicating the positions of the three phosphorylation sites in the KIM and the KIS domains that were mutated either to alanine (S221A) or to glutamic acid (T231E and S244E). f, Neuronal cultures treated with TAT-STEP-myc peptide for the specified time periods were processed for immunocytochemical staining with anti-myc antibody and DAPI. g, Immunoblot analysis of neuron cultures preincubated (incub) with TAT-STEP-myc before exposure to OGD for 10 min. h, Immunoblot analysis of neuron cultures exposed to OGD for 10 min where TAT-STEP-myc peptide was applied at the onset of OGD (co-appl). i, Immunoblot analysis of neuron cultures exposed to OGD for 2 h followed by reoxygenation for 4 h, where TAT-STEP-myc peptide was applied at the onset of OGD (co-appl). g–i, Blots were analyzed using anti-phospho-p38 MAPK antibody (top) and then reprobed with anti-p38 (middle) or anti-myc (bottom) antibodies. a–d, Bar diagrams represent mean ± SEM (n = 4). a, *p < 0.001 from 10 min OGD. b, *p < 0.01 from phosphorylated STEP at 0 min OGD. #p < 0.01 from dephosphorylated form of STEP at 0 min OGD. c, *p < 0.0001 from OGD/R-4 h. d, *p < 0.05 from OGD/R-4 h. j, Representative photomicrographs of neurons exposed to OGD for 2 h in the presence of TAT-STEP-myc peptide (4 μm) showing pyknotic DNA stained with Hoechst 33342, 24 h later. Arrows indicate pyknotic nuclei. Percentage of neurons with pyknotic nuclei is represented as mean ± SEM from 12 cultures in four separate experiments (control 15.4 ± 1.1%, OGD 2 h 62.9 ± 2.5%, and TAT-STEP-myc + OGD 2 h 19.1 ± 1%). *p < 0.001 from untreated control. #p < 0.001 from OGD alone.
Figure 4.
Figure 4.
Administration of TAT-STEP peptide at the onset of insult significantly reduces ischemic brain damage. a, Immunohistochemical analysis with anti-myc antibody for detection of the STEP peptide in the brain after intravenous injection of vehicle or TAT-STEP-myc peptide (3 nmol/g). Representative photomicrographs, demonstrating myc-positive cells (indicated by arrows) in coronal sections through the striatum (top) and the cortex (bottom). Scale bar, 20 μm. b, Intravenous administration of TAT-STEP peptide followed by immunoprecipitation of the peptide from striatal and cortical lysates with anti-myc antibody. The immune complex was processed for immunoblot analysis with anti-p38 MAPK antibody (top) and then reprobed with anti-myc antibody (bottom). c–e, Rats were preinjected with TAT-STEP-myc (n = 8), TAT-myc (n = 4), or vehicle (n = 10), before 90 min of MCAO. c, Representative photomicrographs of TTC-stained brain slices (2 mm) showing brain infarct 24 h after the onset of ischemia. d, Quantitative analysis of the total infarct volume (vehicle 30.6 ± 3.5%, TAT-myc 29.1 ± 4.3%, vs TAT-STEP-myc 15.4 ± 3.3%). e, Total infarct area within each slice is represented as mean ± SEM. *p < 0.05 from vehicle-treated control.
Figure 5.
Figure 5.
Administration of TAT-STEP-myc peptide at the onset of reperfusion significantly reduces ischemic brain damage. a–c, Rats were subjected to MCAO for 90 min followed by injection of vehicle or TAT-STEP-myc (3 nmol/g). a, Representative photomicrographs of TTC-stained brain slices (2 mm) showing brain infarct 24 h after the onset of ischemia. Quantitative analysis of the (b) total infarct volume (vehicle 29.3 ± 2.5% vs TAT-STEP-myc 14.4 ± 2.7%) and (c) area of infarction within each slice is represented as mean ± SEM (n = 7). *p < 0.05 from vehicle-treated control. d, Rats subjected to MCAO for 90 min were injected with vehicle (I-90′/R-6 h) or TAT-STEP-myc peptide (I-90′/R-6 h + TAT-STEP-myc) at the onset of reperfusion. Immunoblot analysis of striatal lysates obtained from the ipsilateral side, 6 h after the insult. Blots were probed with anti-phospho-p38 MAPK (top) and reprobed with anti-p38 MAPK (bottom). Bar diagram represents mean ± SEM obtained from 4 animals. *p < 0.01 from phosphorylated p38 MAPK in vehicle (I-90′/R-6 h) treated animals.
Figure 6.
Figure 6.
Delayed administration of TAT-STEP-myc peptide significantly reduced ischemic brain damage. Rats were subjected to 90 min MCAO, and the peptide was administered 6 h after the onset of the insult. a, Representative photomicrographs of TTC-stained brain slices (2 mm) showing brain infarct at 24 h after the onset of ischemia. Quantitative analysis of the (b) total infarct volume (vehicle 33 ± 1.9% vs TAT-STEP-myc 23.6 ± 2.6%) and (c) area of infarction within each slice is represented as mean ± SEM (n = 10). *p < 0.05 from vehicle-treated control.
Figure 7.
Figure 7.
Neurobehavioral evaluation of WT and STEP KO mice. a, Spontaneous locomotion in the WT and STEP KO mice was assessed for 180 min. The number of photobeam breaks are represented as mean ± SEM (n = 11–18). p > 0.259. b, Forced swim task test (3 min) to assess fatigue-ability and motivation to escape. The initial length of active escape swimming (p > 0.915) and the total duration of escape (p > 0.275) directed behaviors across the duration of the test are represented as mean ± SEM (assessed in 11 WT and 9 KO mice). c, Balance and coordinated alternation of fore and hindpaw were evaluated using the rotarod. The mean ± SEM of three rotarod performances are presented for n = 6–8 for each strain. p > 0.713. d, Gait and balance were evaluated using a balance beam task. Performance scores were quantified as described in Materials and Methods. A score of 1 indicated strong coordination and gait, whereas a score of 6 indicated very poor performance. Scores for three traverses across the beam are represented as mean ± SEM (n = 9–12). p > 0.394 (Mann–Whitney). e, Spontaneous alteration in a Y-maze was used to evaluate working motor memory. Ability to spontaneously alternate in three consecutive sessions within the Y-maze is represented as mean ± SEM (n = 11 or 12). p > 0.974 (Mann–Whitney). f, Exploratory and general anxiety was measured using a nose poke exploration task. Latency to first explore a hole (p > 0.280) and the number of holes explored (p > 0.608) in the 5 min test are represented as mean ± SEM (n = 7–11). g, Social avoidance was assessed using a social interaction test. Time spent in the 10 min test in the side containing the novel mouse relative to time spent in the side containing a novel object is represented as mean ± SEM (n = 6–9). p > 0.891.
Figure 8.
Figure 8.
Genetic deletion of STEP exacerbates ischemic brain damage in mice. a, Representative photomicrographs of WT and STEP KO mice brain subjected to MCAO for 30 min and then perfused transcardially with India ink for visualization of cerebral vasculature. b, Quantitative analysis of changes in cerebral blood flow in WT and STEP KO mice before, during, and after 30 min of MCAO (n = 4 per genotype). c–g, WT and KO mice were subjected to 30 min of MCAO followed by 24 h of reperfusion. c–e, Postischemic neurological dysfunctions tested at 24 h after MCAO are represented as mean ± SEM (n = 7). c, Neurological severity score (NSS) tested on a 5 point scale was observed to be 0.5 ± 0.1 in WT mice compared with 2.6 ± 0.6 in STEP KO mice. *p < 0.05 (Mann–Whitney). d, Rotarod testing was done to measure the latency time for a mouse to fall from a rotating cylinder and was observed to be 31.25 ± 7.5 s for WT mice compared with 9.8 ± 5.14 s for STEP KO mice. *p < 0.05. e, The ability to stay and walk on a beam was tested using the beam balance test with a score range of 1–6 and was observed to be 0.66 ± 0.3 for WT mice compared with 5 ± 0.77 for STEP KO mice. *p < 0.05 (Mann–Whitney). f, Representative photomicrographs of coronal sections stained with Fluoro-Jade C, a marker for cellular degeneration. g, Total infarct volume was 15.03 ± 2.64% for WT mice compared with 43.9 ± 7.07% in STEP KO mice. *p < 0.003. n = 7.
Figure 9.
Figure 9.
Administration of TAT-STEP peptide at the onset of reperfusion reduces p38 MAPK phosphorylation and ischemic brain damage in STEP KO mice. a, Immunoblot analysis of striatal lysates from the ipsilateral side of WT mice, after 10 or 30 min MCAO (n = 3/group). b, Immunoblot analysis of striatal lysates from the ipsilateral side of STEP KO mice, after 10 or 30 min MCAO (n = 3/group). c, Immunoblot analysis of striatal lysates from the ipsilateral side of WT mice and STEP KO mice, after 30 min MCAO and 3 h reperfusion (n = 3/group). d, Immunoblot analysis of striatal lysates from the ipsilateral side of STEP KO mice subjected to 30 min MCAO followed by administration of TAT-STEP-myc peptide (3 nmol/g) and reperfusion for 3 h (n = 3/group). a–d, Blots were probed with anti-phospho-p38 MAPK (top) and reprobed with anti-p38 MAPK (bottom) or anti-STEP antibody (bottom). e, STEP KO mice were subjected to 30 min MCAO, and the peptide was administered at the onset of reperfusion. Representative photomicrographs of coronal brain sections, 24 h after the onset of ischemia, stained with Fluoro-Jade C. f, Total infarct volume was 43.9 ± 7.07% for STEP KO mice compared with 19.3 ± 2.87% in STEP KO mice. *p < 0.05. n = 5.

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