Increased expression of phosphotyrosine after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus

To investigate the role of tyrosine kinase underlying glial cell proliferation after axotomy, the localization of phosphotyrosine was studied immunohistochemically in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after nerve transection in adult rats. An anti-phosphotyrosine antibody weakly stained the cytoplasm of the neurons and some glial cells on the control side of both nuclei, while preferentially staining the plasma membrane of perineuronal microglial cells and neurons weakly on the severed side 2 days after axotomy and intensely between 3 and 7 days. Some of the microglial cells reacted positively with both anti-bromodeoxyuridine and antiphosphotyrosine antibodies, suggesting that tyrosine kinase is involved in microglial cell proliferation. Proliferation of numerous microglial cells was observed in the severed nuclei between 2 and 4 days after axotomy, while only a few were detected on days 5 and 7. These findings suggest that tyrosine kinase is involved in not only the proliferation of perineuronal microglial cells but also in some retrograde neuronal reactions such as differentiation and regeneration.     

蛛网膜下腔出血后大鼠基底动脉p38MAPK信号传导途径研究

目的探讨蛛网膜下腔出血(SAH)后大鼠基底动脉中p38丝裂原活化蛋白激酶(p38MAPK)信号传导通路的活化情况以及与脑血管痉挛(CVS)的关系。方法通过枕大池二次注血方法制作大鼠SAH模型,以免疫组化方法和逆转录酶-多聚酶链反应分析,分别从蛋白、基因水平分析SAH后基底动脉中p38MAPK信号传导通路的活化情况。结果 SAH后大鼠基底动脉逐渐出现痉挛。基底动脉磷酸化p38MAPK表达逐渐增加,3 d时达高峰并持续至第5 d,14 d时恢复正常。p38MAPK基因表达在注血后1 d明显增加,逐渐增加,于5 d时达高峰,14 d仍维持较高水平。结论SAH后大鼠基底动脉中p38MAPK信号传导通路激活,可能诱导CVS的发生。     

Inhibitors of p38 mitogen-activated protein kinase enhance proliferation of mouse neural stem cells

The p38 mitogen-activated protein kinase (MAPK) is induced in response to environmental stress. Although p38 MAPK has been implicated in diverse cellular processes, including cell proliferation, differentiation, and survival of differentiated cells in the central nervous system (CNS), the expression profile and roles of p38 MAPK in the developing brain remain largely unknown. In the present study, we demonstrate that p38 MAPK is expressed predominantly in nestin-positive cells in the cerebral cortex in embryonic day 10 (E10) brain and that expression of the protein decreases gradually during development. To investigate the roles of p38 MAPK in the embryonic brain, two selective p38 MAPK inhibitors, SB202190 and SB203580, were added to the primary neuronal cultures from E10-E14 brains. After 7 days of exposure to these inhibitors, but not SB202474, a negative analog of SB203580, numerous large neurospheres were present. MAPK inhibitors also selectively increased the growth rate of neural stem cells (NSCs) purified from secondary neurospheres and the number of bromodeoxyuridine-positive NSCs. Thus, p38 MAPK inhibitors are potent stimulators of NSC proliferation, and p38 MAPK may be an intrinsic negative regulator of NSC proliferation during early brain development.     

Exogenous nitric oxide negatively regulates the S‐nitrosylation p38 mitogen‐activated protein kinase activation during cerebral ischaemia and reperfusion

S. H. Qi, L. Y. Hao, J. Yue, Y. Y. Zong and G. Y. Zhang (2013) Neuropathology and Applied Neurobiology 39, 284–297 Exogenous nitric oxide negatively regulates the S‐nitrosylation p38 mitogen‐activated protein kinase activation during cerebral ischaemia and reperfusion Aims: A number of studies have suggested that nitric oxide (NO) plays an important role in the reactive phosphorylation of p38MAPKα (p38). However, whether S‐nitrosylation of p38 is activated by NO and the details remain unclear. The aim of the present work was to assess the activation of p38, the S‐nitrosylation site and the p38 signalling pathway in rat hippocampus and in HEK293 cell induced by exogenous NO. Methods: Primary hippocampal cultures, HEK293 cells and rat model of cerebral ischaemia/reperfusion (brain ischaemia was induced by four‐vessel occlusion procedure) were used in this study. Biotin‐switch method and immunoblotting were performed to study the S‐nitrosylation and phosphorylation of p38, and neuronal loss was observed by histology. Results: Endogenous NO increased p38 phosphorylation and S‐nitrosylation, and the activation of p38 was dependent on the S‐nitrosylation of Cys‐211, which was critical for the NO‐mediated activation of p38. The exogenous NO donor sodium nitroprusside, S‐nitrosoglutathione, 7‐nitroindazole, the inhibitor of the neuronal nitric oxide synthase, inhibited the activation of p38 signal pathway induced by cerebral ischaemia/reperfusion and attenuated the damage in rat hippocampal neurones. Moreover, the N‐methyl‐D‐aspartate receptor (NMDAR) is probably involved in the p38 activation process of S‐nitrosylation and phosphorylation. Conclusion: Endogenous NO induces the S‐nitrosylation and phosphorylation of p38 and mediates p38 signalling pathway by NMDAR, and as exogenous NO inhibits this process and is neuroprotective in rat cerebral ischaemia/reperfusion, it may make a contribution to stroke therapy.     

Mitogen- and stress-activated protein kinase 1: convergence of the ERK and p38 pathways in Alzheimer's disease

Two of the earliest manifestations of the selective neurodegeneration that occurs in Alzheimer's disease (AD) involve the oxidative modification of various biomacromolecules and the reexpression of a multitude of cell cycle-related proteins. Taken together with the proximal and ectopic increases in activated components of the ERK and p38 pathways, involved in mitotic and cellular stress signaling, respectively, there is a clear and important role for mitotic and oxidative insults in the pathogenesis of AD. Despite the mounting evidence, however, for the causal role of mitogenic abnormalities and oxidative stress in AD pathogenesis, the effect of the converging relevant pathways due to chronic stimulation in AD remains largely unknown. To delineate further the mechanism by which mitogenic and stress signaling cascades converge, we focused on one of the downstream effectors of activated ERK and p38, mitogen- and stress-activated kinase 1 (MSK1). Activated MSK1, phosphorylated at residues Ser376 and Thr581, was upregulated in vulnerable neurons in AD when compared to that in age-matched controls, whereas MSK1 phosphorylated at residue Ser360 was not increased in AD. Furthermore, activated MSK1 phosphorylated at Thr581 colocalized strongly with activated p38 but only weakly with activated ERK, whereas MSK1 phosphorylated at Ser376 colocalized strongly with activated ERK but only weakly with activated p38, suggesting potential preferential phosphorylation sites for the two upstream effectors.     

Early activation of p38 mitogen activated protein kinase is associated with interferon-alpha-induced depression and fatigue

Cytokine-induced stimulation of p38 mitogen activated protein kinase (MAPK) has been shown to influence behaviorally-relevant pathophysiologic pathways including monoamine neurotransmission and neuroendocrine function and thus may contribute to behavioral changes that occur during chronic administration of the innate immune cytokine, interferon (IFN)-alpha. Accordingly, in the current study, phosphorylation (activation) of intracellular p38 MAPK in peripheral blood lymphocytes was analyzed by flow cytometry every 2 h for 12 h following the initial injection of IFN-alpha in eleven patients with chronic hepatitis C. Hourly assessments of plasma concentrations of adrenocorticotropic hormone, cortisol and interleukin-6 were also obtained. Symptoms of depression and fatigue were measured at baseline and after 4 and 12 weeks of IFN-alpha treatment. Acute administration of IFN-alpha significantly increased the percentage of lymphocytes staining positive for intracellular phosphorylated p38 (p-p38). IFN-alpha-induced increases in p-p38 were significantly greater in patients that developed clinically significant depressive symptoms [Montgomery-Asberg Depression Rating Scale (MADRS) score ? 15] during the first 12 weeks of IFN-alpha treatment. Increases in the percentage of p-p38-positive lymphocytes following the first IFN-alpha injection also highly correlated with depression severity at weeks 4 (r = 0.85, p = 0.001) and 12 (r = 0.70, p = 0.018). Similar relationships were observed for fatigue. Examination of relationships between p-p38 induction and factors previously reported to predict IFN-alpha-induced depressive symptoms revealed strong associations of p-p38 with baseline MADRS (r = 0.82, p = 0.002) and cortisol responses to the initial injection of IFN-alpha (r = 0.91, p = 0.000). Taken together, these findings indicate that sensitivity of p38 MAPK signaling pathways to immune stimulation is associated with depressive symptoms during chronic IFN-alpha treatment.     

Implications of mitogen‐activated protein kinase signaling in glioma

Gliomas are the most common primary central nervous system tumors. Gliomas originate from astrocytes, oligodendrocytes, and neural stem cells or their precursors. According to WHO classification, gliomas are classified into four different malignant grades ranging from grade I to grade IV based on histopathological features and related molecular aberrations. The induction and maintenance of these tumors can be attributed largely to aberrant signaling networks. In this regard, the mitogen‐activated protein kinase (MAPK) network has been widely studied and is reported to be severely altered in glial tumors. Mutations in MAPK pathways most frequently affect RAS and B‐RAF in the ERK, c‐Jun N‐terminal kinase (JNK), and p38 pathways leading to malignant transformation. Also, it is linked to both inherited and sequential accumulations of mutations that control receptor tyrosine kinase (RTK)‐activated signal transduction pathways, cell cycle growth arrest pathways, and nonresponsive cell death pathways. Genetic alterations that modulate RTK signaling can also alter several downstream pathways, including RAS‐mediated MAP kinases along with JNK pathways, which ultimately regulate cell proliferation and cell death. The present review focuses on recent literature regarding important deregulations in the RTK‐activated MAPK pathway during gliomagenesis and progression. © 2015 Wiley Periodicals, Inc.     

Effects of the PPARγ activator pioglitazone on p38 MAP kinase and IκBα in the spinal cord of a transgenic mouse model of amyotrophic lateral sclerosis

Emerging evidence suggests the involvement of programmed cell death and inflammation in amyotrophic lateral sclerosis (ALS). To assess molecular pathological effects of the anti‐inflammatory peroxisome proliferator‐activated receptor‐gamma (PPARγ) agonist pioglitazone in ALS, we verified changes in the population of neurons, astrocytes, and microglia in the ventral horns of spinal cord lumbar segments from the pioglitazone‐treated and non‐treated groups of mice carrying a transgene for G93A mutant human superoxide dismutase‐1 (SOD1) (ALS mice) and non‐transgenic littermates (control mice), performed immunohistochemical and immunoblot analyses of PPARγ, active form of phosphorylated p38 mitogen‐activated protein kinase (p‐p38) and inhibitor of nuclear factor‐kappaB (NF‐κB)‐alpha (IκBα) in the spinal cords, and compared the results between the different groups. Image analysis revealed that optical density of NeuN‐immunoreactive neurons was significantly lower in the non‐treated groups of presymptomatic and advanced ALS mice than in the non‐treated groups of age‐matched control mice and was recovered with pioglitazone treatment, and that optical densities of GFAP‐immunoreactive astrocytes and Iba1‐immunoreactive microglia were significantly higher in the non‐treated group of advanced ALS mice than in the non‐treated group of control mice and were recovered with pioglitazone treatment. Immunohistochemical analysis demonstrated that immunoreactivities for PPARγ and p‐p38 were mainly localized in neurons, and that IκBα immunoreactivity was mainly localized in astrocytes and microglia. Immunoblot analysis showed that pioglitazone treatment resulted in no significant change in nuclear PPARγ‐immunoreactive density, a significant decrease in cytosolic p‐p38‐immunoreactive density, and a significant increase in cytosolic IκBα‐immunoreactive density. Our results suggest that pioglitazone protects motor neurons against p38‐mediated neuronal death and NF‐κB‐mediated glial inflammation via a PPARγ‐independent mechanism.     

Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury

Neuropathic pain is an expression of pathological operation of the nervous system, which commonly results from nerve injury and is characterized by pain hypersensitivity to innocuous stimuli, a phenomenon known as tactile allodynia. The mechanisms by which nerve injury creates tactile allodynia have remained largely unknown. We report that the development of tactile allodynia following nerve injury requires activation of p38 mitogen-activated protein kinase (p38MAPK), a member of the MAPK family, in spinal microglia. We found that immunofluorescence and protein levels of the dually phosphorylated active form of p38MAPK (phospho-p38MAPK) were increased in the dorsal horn ipsilateral to spinal nerve injury. Interestingly, the phospho-p38MAPK immunofluorescence in the dorsal horn was found exclusively in microglia, but not in neurons or astrocytes. The level of phospho-p38MAPK immunofluorescence in individual microglial cells was much higher in the hyperactive phenotype in the ipsilateral dorsal horn than the resting one in the contralateral side. Intrathecal administration of the p38MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), suppresses development of the nerve injury-induced tactile allodynia. Taken together, our results demonstrate that nerve injury-induced pain hypersensitivity depends on activation of the p38MAPK signaling pathway in hyperactive microglia in the dorsal horn following peripheral nerve injury.     

p38丝裂原活化蛋白激酶途径对大鼠脑缺血再灌注后脑组织基质金属蛋白酶-9表达及脑水肿形成的影响

目的探讨p38丝裂原活化蛋白激酶(MAPK)途径对大鼠脑缺血再灌注后脑组织基质金属蛋白酶-9(MMP-9)表达及脑水肿形成的影响。方法 54只SPF级雄性SD大鼠,随机分为假手术组(Sham组)、缺血再灌注组(I/R组)和p38抑制剂组(SB组)。采用改良线栓法制备大鼠大脑中动脉缺血再灌注模型。再灌注24 h后对大鼠进行神经功能缺损评分,Evans Blue法测定血-脑屏障通透性;干湿比重法测定脑组织含水量,采用Western blot检测缺血周边区脑组织磷酸化p38(p-p38)和MMP-9的表达。结果与Sham组相比,I/R组大鼠神经功能缺损加重(P0.05);与I/R组相比,SB组大鼠神经功能缺损明显减轻(P0.05)。与Sham组比较,I/R组血-脑屏障通透性及脑含水量明显增加(均P0.05);与I/R组相比,SB组血-脑屏障通透性及脑含水量降低(均P0.05)。与Sham组相比,I/R组大鼠缺血周边区脑组织p-p38、MMP-9的表达明显上调(均P0.05);与I/R组相比,SB组大鼠缺血周边区脑组织p-p38、MMP-9的表达明显下调(均P0.05)。结论 p38 MAPK参与了大鼠脑缺血再灌注后脑水肿的形成,机制可能为大鼠脑缺血再灌注后激活p38MAPK使缺血周边区脑组织MMP-9的表达上调,破坏血-脑屏障通透性,导致脑水肿发生。     

Expression of mRNA for Akt, serine-threonine protein kinase, in the brain during development and its transient enhancement following axotomy of hypoglossal nerve

Byin situ hybridization histochemistry, expression of mRNAs for the two species of serine/threonine protein kinase Akt, Akt1 and Akt2, were examined in the mouse brain during normal development and in the hypoglossal nucleus following axotomy. On the embryonic days, the gene expression for Akt1 and Akt2 was detected at high levels throughout the entire neuroaxis, then decreased gradually to adult levels during postnatal development. In the adult brain, the gene expression for Akt1 and Akt2 was weak in almost all neurons with no difference of expression levels. The expression level of Akt1 mRNA in the affected hypoglossal nucleus increased dramatically after 48 h to 7 d following axotomy of the hypoglossal nerve, whereas no change was seen in the level of Akt2 mRNA. The present findings suggest that Akt may contribute some important roles not only in neurogenesis, but also in regeneration of injured neuron.     

p38丝裂原活化蛋白激酶在实验性蛛网膜下腔出血后早期脑损伤中的作用

目的探讨p38丝裂原活化蛋白激酶(p38MAPK)在蛛网膜下腔出血(SAH)后早期脑损伤(EBI)中的作用。方法成年雄性SD大鼠随机分配至对照组、SAH组及p38MAPK干预组,每组18只。采用血管内穿刺法制作SAH模型,干预组于术前30 min经侧脑室注射p38MAPK特异性抑制剂SB203580,造模后24 h处死。观察各组大鼠脑含水量和神经功能评分,RT-PCR及免疫组化检测脑组织p38MAPK表达。结果与对照组相比,SAH组大鼠脑含水量(t=-196.35,P0.01)及p38 MAPK的mRNA水平(t=-24.75,P0.01)均明显升高,神经功能评分明显减低(t=201.08,P0.01)。与SAH组相比,干预组脑含水量(t=75.67,P0.01)及p38 MAPK的mRNA水平(t=9.43,P0.01)均明显下降,神经功能评分明显升高(t=-81.68,P0.01)。免疫组化示SAH组及干预组均有p38MAPK表达,但干预组较SAH组表达水平明显下降(t=-3.37,P0.01)。结论 p38 MAPK在EBI形成机制中起重要作用,有望成为防治EBI的药物作用新靶点。     

Ultrastructure of the dorsal motor nucleus of the vagus nerve in monkey with a comparison of synaptology in monkey and cat

Neurons of the dorsal motor nucleus of the vagus nerve were studied following injections of horseradish peroxidase into the vagus nerve in a monkey (Macaca fascicularis). In frozen sections, the dorsal motor nucleus appeared to be completely filled by labeled medium-sized (20-30 micron in long axis) neurons. Labeled dendrites from these neurons often extended outside the borders of the nucleus into the nucleus of the tractus solitarius. In 1 micron thick plastic sections and ultrathin sections of the dorsal motor nucleus, two distinct types of neurons were observed with the light and electron microscope. Medium-sized neurons with abundant cytoplasm and an oval nucleus were retrogradely labeled with HRP, while small (10-15 micron in long axis) neurons with a paucity of organelles and an invaginated nucleus remained unlabeled. Medium-sized neurons outnumbered the small neurons by approximately five to one. The synaptic organization of the dorsal motor nucleus in monkey was studied and compared with that in cat. The porportions of different types of axosomatic synapses were similar in both species. Terminals containing round vesicles and making symmetrical or asymmetrical contact with the postsynaptic structure were more common than synaptic terminals containing pleomorphic vesicles. In both species, there was a slightly greater synaptic density on the medium-sized neurons than on the small neurons. The synaptic density in the monkey dorsal nucleus was greatest on the smallest dendrites in the neuropil and least on the somata.     

Mitogen‐activated protein kinase phosphatase‐2 deletion modifies ventral tegmental area function and connectivity and alters reward processing

Mitogen‐activated protein kinases (MAPKs) regulate normal brain functioning, and their dysfunction is implicated in a number of brain disorders. Thus, there is great interest in understanding the signalling systems that control MAPK functioning. One family of proteins that contribute to this process, the mitogen‐activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in foetal development, the immune system, cancer and synaptic plasticity and memory. In the present study, we performed an unbiased investigation using MKP‐2^?/? mice to assess whether MKP‐2 plays a global role in modulating brain function. Local cerebral glucose utilization is significantly increased in the ventral tegmental area (VTA) of MKP‐2^?/? mice, with connectivity analysis revealing alterations in VTA functional connectivity, including a significant reduction in connectivity to the nucleus accumbens and hippocampus. In addition, spontaneous excitatory postsynaptic current frequency, but not amplitude, onto putative dopamine neurons in the VTA is increased in MKP‐2^?/? mice, which indicates that increased excitatory drive may account for the increased VTA glucose utilization. Consistent with modified VTA function and connectivity, in behavioural tests MKP‐2^?/? mice exhibited increased sucrose preference and impaired amphetamine‐induced hyperlocomotion. Overall, these data reveal that MKP‐2 plays a role in modulating VTA function and that its dysfunction may contribute to brain disorders in which altered reward processing is present.     

Synaptic remodeling in the dorsal motor nucleus of the vagus nerve following vagal-hypoglossal nerve anastomosis in the cat

Horseradish peroxidase (HRP) retrograde tracing techniques and morphometric analyses were performed to investigate synaptic remodeling associated with neuronal and glial changes in the dorsal motor nucleus of the vagus (DMV) of cats after vagal-hypoglossal nerve anastomosis (VHA). At 25 days postoperation (dpo), in the early target-reinnervation stage, there were 50% fewer presynaptic boutons containing round vesicles (R) or round and large dense-cored synaptic vesicles (R+D) contacting HRP-labeled DMV motoneurons. The loss of R boutons was maintained throughout the remaining postoperative intervals up to 500 dpo, whereas R+D boutons were further reduced at 123 dpo but were restored at 315 dpo, so that, by 500 dpo, 71.4% of them had gained access to the DMV motoneurons. Boutons containing pleomorphic synaptic vesicles (P) were completely disconnected from the DMV motoneurons at 25 dpo and did not reappear even in the long-term reinnervation stage. Loss and recovery of presynaptic boutons occurred in parallel with changes in astroglial ensheathment of the DMV motoneurons. It is suggested that synaptic remodeling associated with astroglial ensheathment in the DMV may be influenced by some retrogradely transported factors/signals derived from the newly acquired target organ, viz. tongue skeletal musculature. Our results further suggest that the observed changes in boutonal configurations may be attributable to modified functions of the DMV motoneurons induced by VHA.     

p38 mitogen-activated protein kinase modulates expression of tumor necrosis factor-related apoptosis-inducing ligand induced by interferon-gamma in fetal brain astrocytes

This study describes the involvement of the p38 mitogen-activated protein kinase (MAPK) during interferon-gamma (IFN-gamma) signaling in fetal brain astrocytes. In some pathological conditions of brain, p38 MAPK transduces stress-related signals, increases expression of proinflammatory cytokines, and induces cellular damage or apoptosis. In astrocytes, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression level was increased by IFN-gamma. AG490, a JAK inhibitor, blocked TRAIL expression induced by IFN-gamma. SB203580, a specific p38alpha and p38beta2 MAPK inhibitor, decreased the TRAIL expression induced by IFN-gamma. The phosphorylation of the Ser727 site of STAT1, but not the Tyr701 site, was inhibited by SB203580. These results suggest that p38 MAPK modulates STAT1 phosphorylation in IFN-gamma signaling in fetal brain astrocytes.