The O-GlcNAc Modification of CDK5 Involved in Neuronal Apoptosis Following In Vitro Intracerebral Hemorrhage.

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in regulating mammalian CNS development. Studies of CDK5 have focused on its phosphorylation, although the diversity of CDK5 functions in the brain suggests additional forms of regulation. Here we expanded on the functional roles of CDK5 glycosylation in neurons. We showed that CDK5 was dynamically modified with O-GlcNAc in response to neuronal activity and that glycosylation represses CDK5-dependent apoptosis by impairing its association with p53 pathway. Blocking glycosylation of CDK5 alters cellular function and increases neuronal apoptosis in the cell model of the ICH. Our findings demonstrated a new role for O-glycosylation in neuronal apoptosis and provided a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identified a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development, and apoptosis.

Up-Regulation of TAB3 Is Involved in Neuronal Apoptosis After Intracerebral Hemorrhage.

Human transforming growth factor ß-activated kinase (TAK1)-binding protein 3 (TAB3) is a regulator of NF-κB which has been mainly found in a variety of cancers. While TAB3 is highly expressed in brain tissue, little is known about the function of TAB3 in central nervous system. Our group established an animal ICH model with autologous whole blood injected into brain, and also a cell ICH model with hemin stimulation. Our Western blot result showed up-regulation of TAB3 during neuronal apoptosis in the model of intracerebral hemorrhage (ICH), which was also approved by immunofluorescence and immunohistochemistry result. Besides, increasing TAB3 level was accompanied by the increased expression of active-caspase-3, active-caspase-8, and decreased expression of Bcl-2. Furthermore, in in vitro study, the level of neuronal apoptosis was decreased by applying TAB3- RNA interference in PC12 cells. All the results above suggested that TAB3 probably participates in the process of neuronal apoptosis following ICH.

Increased expression of HERPUD1 involves in neuronal apoptosis after intracerebral hemorrhage.

Homocysteine-inducible endoplasmic reticulum stress-inducible ubiquitin-like domain member 1 protein (HERPUD1) is involved in endoplasmic reticulum stress response. Immense amounts of research showed HERPUD1 plays multiple roles in various models. In this work, we explored the role of HERPUD1 during the pathophysiological processes of intracerebral hemorrhage (ICH). Rat ICH model was established and verified by behavioral test. Western blot and immunohistochemistry revealed a significant up-regulation of HERPUD1 expression around the hematoma after ICH. Besides, the expression of cytochrome c (cyt c) and active caspase-3 increased accompanied to HERPUD1 expression. Double-labeled immunofluorescence indicated HERPUD1 mainly colocalized with neurons. Further study showed HERPUD1 silence brought about up-regulation of apoptosis markers including cyt c and active caspase-3 coupled with increased cell apoptosis in vitro model. All these findings suggested that HERPUD1 might play a protective role in ICH-induced neuronal apoptosis in rat models.

OTUB1 attenuates neuronal apoptosis after intracerebral hemorrhage.

OTUB1 is a member of deubiquitinating enzymes, which was shown as a proteasome-associated DUB to be involved in the proteins Ub-dependent degradation. Previous studies have indicated that OTUB1 was expressed in brain. But its distribution and function in the brain remain unclear. In this study, we explored the roles of OTUB1 protein in the pathophysiology of intracerebral hemorrhage (ICH). From the results of Western blot, immunohistochemistry, and immunofluorescence, we found an obvious up-regulation of OTUB1 in neurons adjacent to the hematoma after ICH. Furthermore, we also found that the increase of OTUB1 expression was accompanied by the enhanced expression of Bax and active caspase-3, and decreased expression of Bcl-2 in the pathological process of rat ICH. What's more, our in vitro study, using OTUB1 RNA interference in PC12 cells, suggested that OTUB1 might exert its anti-apoptotic function in neuronal apoptosis. Therefore, OTUB1 may play a role in protecting the brain from secondary damage following ICH.

MEKK1 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage.

The JNKs have been implicated in a variety of biological functions in mammalian cells, including apoptosis and the responses to stress. However, the physiological role of these pathways in the intracerebral hemorrhage (ICH) has not been fully elucidated. In this study, we identified a MAPK kinase kinase (MAPKKK), MEKK1, may be involved in neuronal apoptosis in the processes of ICH through the activation of JNKs. From the results of western blot, immunohistochemistry and immunofluorescence, we obtained a significant up-regulation of MEKK1 in neurons adjacent to the hematoma following ICH. Increasing MEKK1 level was found to be accompanied with the up-regulation of p-JNK 3, p53, and c-jun. Besides, MEKK1 co-localized well with p-JNK in neurons, indicating its potential role in neuronal apoptosis. What's more, our in vitro study, using MEKK1 siRNA interference in PC12 cells, further confirmed that MEKK1 might exert its pro-apoptotic function on neuronal apoptosis through extrinsic pathway. Thus, MEKK1 may play a role in promoting the brain damage following ICH.

EP3, Prostaglandin E2 Receptor Subtype 3, Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage.

EP3 is prostaglandin E2 receptor subtype 3 and mediates the activation of several signaling pathways, changing in cAMP levels, calcium mobilization, and activation of phospholipase C. Previous studies demonstrated a direct role for EP3 in various neurodegenerative disorders, such as stroke and Alzheimer disease. However, the distribution and function of EP3 in ICH diseases remain unknown. Here, we demonstrate that EP3 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). From the results of Western blot and immunohistochemistry, we obtained a significant up-regulation of EP3 in neurons adjacent to the hematoma following ICH. Up-regulation of EP3 was found to be accompanied by the increased expression of active caspase-3 and pro-apoptotic Bcl-2-associated X protein (Bax) and decreased expression of anti-apoptotic protein B cell lymphoma-2 (Bcl-2) in vivo and vitro studies. Furthermore, the expression of these three proteins reduced active caspase-3 and Bax expression, while increased Bcl-2 were changed after knocking down EP3 by RNA interference in PC12 cells, further confirmed that EP3 might exert its pro-apoptotic function on neuronal apoptosis. Thus, EP3 may play a role in promoting the neuronal apoptosis following ICH.

A New Role Discovered for IGTP: The Protective Effect of IGTP in ICH-Induced Neuronal Apoptosis.

Interferon gamma-induced GTPase (IGTP), which is also named Irgm3, has been widely described in regulating host resistance against intracellular pathogens. Previous researches have demonstrated that IGTP exerts beneficial function during coxsackievirus B3 (CVB3) infection. However, little information is available regarding the role of IGTP in central nervous system. Here, our study revealed that IGTP may have an essential role during ICH-induced neuronal apoptosis. We found the expression level of IGTP adjacent to hematoma was strongly increased after ICH, accompanied with the up-regulation of proliferating cell nuclear antigen (PCNA), active-caspase-3, p-GSK-3ß, and Bax. IGTP was also observed to be co-localized with PCNA in astrocytes and active-caspase-3 in neurons, indicating its association with astrocyte proliferation and neuronal apoptosis after ICH. Finally, in vitro study, knocking down IGTP with IGTP-specific siRNA promoted active-caspase-3, p-GSK-3ß, and Bax expression, and led to more severe neuronal apoptosis after ICH. All these results above suggested that IGTP might play a critical role in protecting neurons from apoptosis after ICH.