Maohong Cao

Up-regulation of NFATc4 Involves in Neuronal Apoptosis Following Intracerebral Hemorrhage

Nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4), a transcriptional factor, is involved in the control about the flow of genetic information and the modulation of diverse cellular activities. Accumulating evidence has demonstrated that NFATc4 exerted a pro-apoptotic effect in multiple diseases. Here, we explored the NFATc4’s roles during the pathophysiological processes of intracerebral hemorrhage (ICH). An ICH rat model was built and evaluated according to behavioral testing. Using Western blot, immunohistochemistry, and immunofluorescence, significant up-regulation of NFATc4 was found in neurons in brain areas surrounding the hematoma following ICH. Increasing NFATc4 expression was found to be accompanied by the up-regulation of Fas ligand (FasL), active caspase-8, and active caspase-3, respectively. Besides, NFATc4 co-localized with active caspase-3 in neurons, indicating its role in neuronal apoptosis. Our in vitro study, using NFATc4 RNA interference in PC12 cells, further confirmed that NFATc4 might exert its pro-apoptotic function in neuronal apoptosis through extrinsic pathway. Thus, NFATc4 may play a role in promoting the brain secondary damage following ICH.

Increased Expression of Small Heat Shock Protein αB-crystallin After Intracerebral Hemorrhage in Adult Rats

AbstractαB-crystallin (αBC) is involved in diverse cellular activities. Previous studies demonstrated that αBC had anti-apoptotic and proliferation-promoting effects in multiple diseases. Here, we explored the αBC’s roles in the pathophysiology of intracerebral hemorrhage (ICH). An ICH rat model was established and assessed by behavioral tests. Using Western blot and immunohistochemistry, significant up-regulation of αBC was found in neurons and astrocytes in brain areas surrounding the hematoma following ICH. Increase of αBC expression was found to be accompanied by the increased expression of proliferating cell nuclear antigen (PCNA), p53, Bax, and active-caspase-3. αBC was co-localized with PCNA in astrocytes or active-caspase-3 in neurons, suggesting its role in astrocyte proliferation and neuronal apoptosis. Our in vitro study, using αBC RNA interference in PC12 cells, indicated that αBC might exert its anti-apoptotic function in neuronal apoptosis. Thus, αBC may play a role in protecting the brain from secondary damage following ICH.

Upregulation of Ras homolog enriched in the brain (Rheb) in lipopolysaccharide-induced neuroinflammation.

Ras homolog enriched in the brain (Rheb) is a homolog of Ras GTPase that regulates cell growth, proliferation, and cell cycle via mammalian target of rapamycin (mTOR). Recently, it has been confirmed that Rheb activation not only promotes cellular proliferation and differentiation but also enhances cellular apoptosis in response to diverse toxic stimuli. However, the function of Rheb in the central nervous system (CNS) is still with limited understanding. To elaborate whether Rheb was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats. Upregulation of Rheb was observed in the brain cortex by performing western blotting and immunohistochemistry. Double immunofluorescent staining demonstrated that Rheb was mainly in active astrocytes and neurons. PCNA and active caspase-3 were upregulated, and co-labeling with Rheb, which indicated that Rheb might be relevant to astrocytic proliferation and neuronal apoptosis following the inflammatory response by LPS-induced. Furthermore, we also found that the expression profiles of cyclinD1 and CDK4 were parallel with that of Rheb in a time-space dependent manner. Finally, knocking down Rheb by siRNA and treatment with rapamycin or lovastatin showed that not only astrocytic proliferation decreased but also neuronal protection. Based on our data, we suggested that Rheb might play an important role in physiological and pathological functions following neuroinflammation caused by LPS, which might provide a potential target to the treatment of neuroinflammation.

CDK5 Contributes to Neuronal Apoptosis via Promoting MEF2D Phosphorylation in Rat Model of Intracerebral Hemorrhage

Cyclin-dependent kinase-5 (CDK5), a serine/threonine kinase which can be activated by its neuron-specific activator p35, or its truncated form p25, plays an important role in a variety of neuronal events, including neuronal migration, synaptic transmission, and neuronal death. Accumulating evidence has shown that abnormal activation of CDK5 was a critical neuronal pro-death signal in central nervous system (CNS) diseases. However, it remains unclear how CDK5 functions upon neuronal apoptosis following intracerebral hemorrhage (ICH). In the present study, we established ICH models by injecting autologous whole blood into the right basal ganglia of adult rats and assessed their neurological deficits by behavioral tests. CDK5 protein levels and kinase activities were upregulated adjacent to the hematoma following ICH. Immunofluorescent staining showed CDK5 was mainly localized in neurons, rather than in astrocytes or oligodendrocytes. Furthermore, active caspase-3, an apoptotic marker, showed a temporally parallel expression with the protein levels/kinase activities of CDK5 following ICH. Meantime, myocyte enhancer factor 2D (MEF2D), a pro-survival transcription factor which could be phosphorylated inactivation by CDK5, also exhibited high phosphorylation levels following ICH. In vitro, we obtained a consistent upregulation of CDK5 kinase activity in primary cortical neurons after thrombin treatment. Knocking down CDK5 kinase activity suppressed neuronal apoptosis and coupled with reduced MEF2D phosphorylation at ser444 residues. Thus, we speculated that CDK5 might exert an important function in the regulation of neuronal apoptosis following ICH.

Upregulation of SYF2 is associated with neuronal apoptosis caused by reactive astrogliosis to neuroinflammation

SYF2, known as CCNDBP1‐interactor or p29, is likely involved in pre‐mRNA splicing and cell cycle progression. The present study was designed to elucidate dynamic changes in SYF2 expression and distribution in the cerebral cortex in a lipopolysaccharide (LPS)‐induced neuroinflammation rat model. It was found that SYF2 expression was induced strongly in active astrocytes after LPS injection. In vitro studies showed that the upregulation of SYF2 might be involved in the activation of C6 cells after LPS challenge and the neuronal apoptosis after conditioned media challenge. In addition, with silencing of SYF2 in C6 and PC12 cells by siRNA, the results indicated that SYF2 was required for astrocyte activation and neuronal apoptosis induced by LPS. Our findings on the cellular signaling pathway may provide a new therapeutic strategy against neuroinflammation in the CNS.

Increased expression of BAG-1 in rat brain cortex after traumatic brain injury

BAG-1 protein was initially identified as a Bcl-2-binding protein. It was reported to enhance Bcl-2 protection from cell death, suggesting that BAG-1 represents a new type of anti-cell death gene. Moreover, recent study has shown that BAG-1 can enhance the proliferation of neuronal precursor cells, attenuate the growth inhibition induced by siah1. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of BAG-1 expression in the brain cortex. Double immunofluorescence staining revealed that BAG-1 was co-expressed with NEURON and glial fibrillary acidic protein (GFAP). In addition, we detected that proliferating cell nuclear antigen had the co-localization with GFAP, and BAG-1. All our findings suggested that BAG-1 might involve in the pathophysiology of brain after TBI.

Up-Regulation of Podoplanin Involves in Neuronal Apoptosis in LPS-Induced Neuroinflammation

Podoplanin (PDPN) is a mucin-type transmembrane sialoglycoprotein expressed in multiple tissues in adult animals, including the brain, lungs, kidney, and lymphoid organs. Studies of this molecule have demonstrated its great importance in tumor metastasis, platelet aggregation, and lymphatic vessel formation. However, information regarding its regulation and possible function in the central nervous system is still limited. In this study, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats and detected increased expression of PDPN in the brain cortex. Immunofluorescence indicated that PDPN was located in the neurons, but not astrocytes. Moreover, there was a concomitant up-regulation of active caspase-3, cyclin D1, and CDK4 in vivo and vitro studies. In addition, the expression of these three proteins in cortical primary neurons was decreased after knocking down PDPN by siRNA. Collectively, all these results suggested that the up-regulation of PDPN might be involved in neuronal apoptosis in neuroinflammation after LPS injection.

Wip1 phosphatase involved in lipopolysaccharide-induced neuroinflammation.

Wild type p53-induced phosphatase 1 (Wip1) is a phosphatase which belongs to protein phosphatase type 2C family, which have been predominantly linked to cell growth and to cellular stress signaling. Numerous downstream targets of Wip1 have been identified, and genetic studies confirm that some play a part in tumorigenesis. Recent evidence highlights a new role for Wip1 in the regulation of NF-κB p65, which indicated that it might play a critical role in immune system. However, its regulation role in central nervous system (CNS) remains poorly understood. To elaborate whether Wip1 was involved in CNS injury, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral–ventral injection in adult rats. Wip1 expression was strongly upregulated in active astrocytes in inflamed brain cortex. In vitro studies indicated that the upregulation of Wip1 may be involved in the subsequent astrocytic activation following LPS exposure, and knockdown of Wip1 in primary astrocytes by siRNA showed that Wip1 inhibited the synthesis of TNF-α. Collectively, these results suggested that Wip1 may be important in host defense in CNS immune response, which might provide a potent therapeutic target of neuroinflammation.

Investigation of Long Non-coding RNA Expression Profiles in the Substantia Nigra of Parkinson’s Disease

Genetics is considered as an important risk factor in the pathological changes of Parkinson’s disease (PD). Substantia nigra (SN) is thought to be the most vulnerable area in this process. In recent decades, however, few related long non-coding RNAs (lncRNAs) in the SN of PD patients had been identified and the functions of those lncRNAs had been studied even less. In this study, we sought to investigate the lncRNA expression profiles and their potential functions in the SN of PD patients. We screened lncRNA expression profiles in the SN of PD patients using the lncRNA mining approach from the ArrayExpress database, which included GSE20295. The samples were from 11 of PD and 14 of normal tissue samples. We identified 87 lncRNAs that were altered significantly in the SN during the occurrence of PD. Among these lncRNAs, lncRNA AL049437 and lncRNA AK021630 varied most dramatically. AL049437 was up-regulated in the PD samples, while AK021630 was down-regulated. Based on the results, we focused on the potential roles of the two lncRNAs in the pathogenesis of PD by the knockdown of the expression of AL049437 or AK021630 in human neuroblastoma SH-SY5Y cell line. Results indicated that the reduction in AL049437 level increased cell viability, mitochondrial transmembrane potential (Δψm), mitochondrial mass, and tyrosine hydroxylase (TyrH) secretion. By contrast, the knockdown of AK021630 resulted in the opposite effect. Based on these results, we speculated that lncRNA AL049437 likely contributed to the risk of PD, while lncRNA AK021630 likely inhibited the occurrence of PD.

Up-regulation of Glis2 involves in neuronal apoptosis after intracerebral hemorrhage in adult rats.

The novel Krüppel-like zinc finger protein Gli-similar 2 (Glis2), one member of the transcription factors, is involved in controlling the flow of genetic information and the modulation of diverse cellular activities. Accumulating evidence has demonstrated its important roles in adult development and several diseases. However, information regarding the regulation and possible function of Glis2 in the central nervous system is still limited. In this study, we explored the roles of Glis2 during the pathophysiological process of intracerebral hemorrhage (ICH). An ICH rat model was established and assessed by behavioral tests. Expression of Glis2 was significantly up-regulated in brain areas surrounding the hematoma following ICH. Immunofluorescence showed that Glis2 was strikingly increased in neurons, but not astrocytes or microglia. Up-regulation of Glis2 was found to be accompanied by the increased expression of active caspase-3 and Bax and decreased expression of Bcl-2 in vivo and vitro studies. Moreover, knocking down Glis2 by RNA-interference in PC12 cells reduced active caspase-3 and Bax expression while increased Bcl-2. Collectively, we speculated that Glis2 might exert pro-apoptotic function in neurons following ICH.