BACKGROUND: To date, no drugs are able to halt the progression of Alzheimer's disease (AD). Neural stem cells (NSCs) transplantation has been widely used to treat AD, but the mechanism of AD treatment remains un...BACKGROUND: To date, no drugs are able to halt the progression of Alzheimer's disease (AD). Neural stem cells (NSCs) transplantation has been widely used to treat AD, but the mechanism of AD treatment remains unclear. OBJECTIVE: To observe changes in protein and factors in the hippocampus and frontal lobe of AD rats following NSCs transplantation, and to understand mechanism of action of NSCs transplantation in AD treatment. DESIGN, TIME AND SETTING: A randomized, controlled animal study was conducted at the First Clinical Hospital, Jilin University, China from July 2007 to March 2009. MATERIALS: NSCs were harvested from the hippocampus of 10 E16 Wistar rats. METHODS: A total of 57 male adult Wistar rats were equally and randomly divided into normal control, AD model and NSCs groups. AD models were established in the AD model and NSCs groups by bilateral removal of hippocampus. At 2 weeks postsurgery, NSCs were transplanted into the hippocampus of rats from the NSCs group. MAIN OUTCOME MEASURES: Protein levels were measured in the hippocampus of rats from normal control, NSCs and AD model groups using proteomics. Expression of choline acetyl transferase mRNA, glial fibrillary acidic protein and S100β was measured in the hippocampus and frontal lobe of rats using in situ hybridization and immunohistochemistry. RESULTS: Expression of choline acetyl transferase mRNA, heat shock protein 70, heat shock protein 90, F-actin and actin was significantly higher in the NSCs group compared with AD model group. Glial fibrillary acidic protein and S100β expression was less in the NSCs group compared with AD model group. CONCLUSlOIN: NSCs implanted into the brain may generate new neural cells, which can relieve damage to the cholinergic system and resist apoptosis. NSCs transplantation plays a protective role in the cholinergic system in the AD rats to some extent.展开更多
BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal dege...BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear. OBJECTIVE: To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING: A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS: PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS: PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES: Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used. RESULTS: In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P 〈 0.01). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P 〈 0.01), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition (P 〈 0.01). By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION: γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.展开更多
Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by its progressive course. The current therapies are aimed at alleviating symptoms by rescuing the unbalanced physi...Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by its progressive course. The current therapies are aimed at alleviating symptoms by rescuing the unbalanced physiological dopamine metabolism and recovery of damaged neuronal circuits. However, these strategies result in insufficient clinical benefits for many patients and fail to halt disease progression. Therefore, new therapeutic targets could serve as the gateway against PD degeneration. One pathological hallmark of PD is the formation of intracytoplasmic protein inclusions or Lewy bodies, in neurons. Recent studies have suggested that Lewy bodies are formed similarly to aggresomes, and results have supported the concept that the novel cellular organelle, the aggresome, is a cytoprotective response that sequesters and facilitates clearance of potentially toxic protein aggregates. In addition, a-tubulin deacetylase has been shown to regulate aggresome formation and rescue neural cell viability in response to misfolded protein. Therefore, the regulation of aggresome formation to trigger cellular self-protection system could arrest PD progression. The present study discusses research progress related to Lewy bodies, aggresomes, and histone deacetylases, with an emphasis on histone deacetylase 6 and sirtuin type 2.展开更多
Dopamine (DA) exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid (RA,10 pmol/L,72 hours) followed by phorbol ester 12...Dopamine (DA) exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid (RA,10 pmol/L,72 hours) followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA,80 nmol/L,72 hours). However,it remains unclear whether the alteration of phenotype observed in response to oxidative stress is associated with protein regulation in this cellular model for Parkinson's disease. The present study detected protein regulation affected by oxidative stress at a proteomic level:selection of differentially altered proteins using two dimensional difference in-gel electrophoresis and identification of these proteins using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated significant alterations in expression of six proteins in SH-SY5Y cells following the differentiation and fourteen proteins in the differentiated cells following the exposure,exemplified by an increase of tubulin alpha1 in the former but a decrease of tubulin alpha-ubiquitous chain in the latter. These results suggest that two potentially specific but relevant patterns of proteomic change may be produced in SH-SY5Y cells with the induction of differentiation by RA followed by TPA,and in the differentiated cells after DA exposure.展开更多
Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic prot...Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic proteasome inhibitor-induced inclusions in PC12 cells contained six subunits in the 26S proteasome. In the present study, mass spectrometry analysis of single protein spots resolved by two-dimensional gel electrophoresis and identified by bioinformatic analysis of peptide mass fingerprint (PMF) data were performed to comprehensively characterize the proteomic profile of the proteasome subunits. Results showed that six subunits in the 26S proteasome were characterized through accurate assignment by PMF data-specific protein identification in protein databases. Additionally, identification of one of the proteasome subunits was further confirmed using a subunit-specific antibody against non-adenosine triphosphatase subunit 11 of the 19S regulatory particle. Results suggest that the potential proteomic profile of six subunits in the 26S proteasome could be established from proteasome inhibitor-induced inclusions in PC12 cells.展开更多
Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson's disease patients. In the present study, proteasome inhibitor Z-Ile-Glu(O...Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson's disease patients. In the present study, proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was used to inhibit the function of the ubiquitin-proteasome system in PC12 cells to simulate Parkinson's disease. Oxidatively modified proteins were identified to determine pathogenesis of Parkinson's disease. Results demonstrated that 24 hours of 10 IJmol/L PSI-treatment in PC12 cells simulated pathological characteristics of Parkinson's disease: neuronal degeneration and eosinophilic inclusion formation in neurons. In PSI-treated PC12 cells, three oxidative proteins and a molecular chaperone family member were detected: chaperonin containing t-complex polypeptide 1 subunit 3, glucose-regulated protein 58, and heat shock protein 70. This is the first study to demonstrate oxidative modification of a molecule family in a cell model of Parkinson's disease induced with PSI.展开更多
To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass...To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify proteins differentially-expressed in SH-SY5Y cells treated with resveratrol. Compared with the control group, resveratrol treatment significantly affected the expression of four proteins: endoplasmic reticulum oxidoreductin 1-like protein alpha, p21-activated kinase 1, Archain 1, and T cell receptor beta chain. The former three were downregulated and the latter was upregulated. These proteins are primarily associated with endoplasmic reticulum stress, intracellular trafficking, and immune function.展开更多
基金the Jilin Pro-vincial Technology Devel-opment Foundation, No. 200505204, 200705129the Postdoctorate Founda-tion from Northeast Normal University, No. 111258000
文摘BACKGROUND: To date, no drugs are able to halt the progression of Alzheimer's disease (AD). Neural stem cells (NSCs) transplantation has been widely used to treat AD, but the mechanism of AD treatment remains unclear. OBJECTIVE: To observe changes in protein and factors in the hippocampus and frontal lobe of AD rats following NSCs transplantation, and to understand mechanism of action of NSCs transplantation in AD treatment. DESIGN, TIME AND SETTING: A randomized, controlled animal study was conducted at the First Clinical Hospital, Jilin University, China from July 2007 to March 2009. MATERIALS: NSCs were harvested from the hippocampus of 10 E16 Wistar rats. METHODS: A total of 57 male adult Wistar rats were equally and randomly divided into normal control, AD model and NSCs groups. AD models were established in the AD model and NSCs groups by bilateral removal of hippocampus. At 2 weeks postsurgery, NSCs were transplanted into the hippocampus of rats from the NSCs group. MAIN OUTCOME MEASURES: Protein levels were measured in the hippocampus of rats from normal control, NSCs and AD model groups using proteomics. Expression of choline acetyl transferase mRNA, glial fibrillary acidic protein and S100β was measured in the hippocampus and frontal lobe of rats using in situ hybridization and immunohistochemistry. RESULTS: Expression of choline acetyl transferase mRNA, heat shock protein 70, heat shock protein 90, F-actin and actin was significantly higher in the NSCs group compared with AD model group. Glial fibrillary acidic protein and S100β expression was less in the NSCs group compared with AD model group. CONCLUSlOIN: NSCs implanted into the brain may generate new neural cells, which can relieve damage to the cholinergic system and resist apoptosis. NSCs transplantation plays a protective role in the cholinergic system in the AD rats to some extent.
文摘BACKGROUND: Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear. OBJECTIVE: To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level. DESIGN, TIME AND SETTING: A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007. MATERIALS: PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA. METHODS: PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μmol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively. MAIN OUTCOME MEASURES: Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used. RESULTS: In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P 〈 0.01). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P 〈 0.01), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition (P 〈 0.01). By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury. CONCLUSION: γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.
文摘Parkinson's disease (PD) is the second most common neurodegenerative disorder and is characterized by its progressive course. The current therapies are aimed at alleviating symptoms by rescuing the unbalanced physiological dopamine metabolism and recovery of damaged neuronal circuits. However, these strategies result in insufficient clinical benefits for many patients and fail to halt disease progression. Therefore, new therapeutic targets could serve as the gateway against PD degeneration. One pathological hallmark of PD is the formation of intracytoplasmic protein inclusions or Lewy bodies, in neurons. Recent studies have suggested that Lewy bodies are formed similarly to aggresomes, and results have supported the concept that the novel cellular organelle, the aggresome, is a cytoprotective response that sequesters and facilitates clearance of potentially toxic protein aggregates. In addition, a-tubulin deacetylase has been shown to regulate aggresome formation and rescue neural cell viability in response to misfolded protein. Therefore, the regulation of aggresome formation to trigger cellular self-protection system could arrest PD progression. The present study discusses research progress related to Lewy bodies, aggresomes, and histone deacetylases, with an emphasis on histone deacetylase 6 and sirtuin type 2.
基金the Science and Technology Development Program of Jilin Province, No. 200505200the Distinguished Professor Foundation of Jilin University, No. 450011011204
文摘Dopamine (DA) exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid (RA,10 pmol/L,72 hours) followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA,80 nmol/L,72 hours). However,it remains unclear whether the alteration of phenotype observed in response to oxidative stress is associated with protein regulation in this cellular model for Parkinson's disease. The present study detected protein regulation affected by oxidative stress at a proteomic level:selection of differentially altered proteins using two dimensional difference in-gel electrophoresis and identification of these proteins using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated significant alterations in expression of six proteins in SH-SY5Y cells following the differentiation and fourteen proteins in the differentiated cells following the exposure,exemplified by an increase of tubulin alpha1 in the former but a decrease of tubulin alpha-ubiquitous chain in the latter. These results suggest that two potentially specific but relevant patterns of proteomic change may be produced in SH-SY5Y cells with the induction of differentiation by RA followed by TPA,and in the differentiated cells after DA exposure.
基金the Science and Technology Commission Foundation of Jilin Province,No.200505200the Distinguished Professor Foundation of Jilin University,No.450011011204
文摘Proteasome dysfunction during dopaminergic degeneration induces proteolytic stress, and is a contributing factor for the onset and formation of Lewy bodies. Results from our previous studies showed that synthetic proteasome inhibitor-induced inclusions in PC12 cells contained six subunits in the 26S proteasome. In the present study, mass spectrometry analysis of single protein spots resolved by two-dimensional gel electrophoresis and identified by bioinformatic analysis of peptide mass fingerprint (PMF) data were performed to comprehensively characterize the proteomic profile of the proteasome subunits. Results showed that six subunits in the 26S proteasome were characterized through accurate assignment by PMF data-specific protein identification in protein databases. Additionally, identification of one of the proteasome subunits was further confirmed using a subunit-specific antibody against non-adenosine triphosphatase subunit 11 of the 19S regulatory particle. Results suggest that the potential proteomic profile of six subunits in the 26S proteasome could be established from proteasome inhibitor-induced inclusions in PC12 cells.
基金the Scientific Research Foundation of Department of Science and Technology of Jilin Province,No. 200505200
文摘Previous studies have demonstrated that ubiquitin-proteasome system function is significantly decreased in the substantia nigra of Parkinson's disease patients. In the present study, proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was used to inhibit the function of the ubiquitin-proteasome system in PC12 cells to simulate Parkinson's disease. Oxidatively modified proteins were identified to determine pathogenesis of Parkinson's disease. Results demonstrated that 24 hours of 10 IJmol/L PSI-treatment in PC12 cells simulated pathological characteristics of Parkinson's disease: neuronal degeneration and eosinophilic inclusion formation in neurons. In PSI-treated PC12 cells, three oxidative proteins and a molecular chaperone family member were detected: chaperonin containing t-complex polypeptide 1 subunit 3, glucose-regulated protein 58, and heat shock protein 70. This is the first study to demonstrate oxidative modification of a molecule family in a cell model of Parkinson's disease induced with PSI.
文摘To gain insight into the molecular mechanisms of resveratrol-mediated neuroprotection, two-dimensional difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify proteins differentially-expressed in SH-SY5Y cells treated with resveratrol. Compared with the control group, resveratrol treatment significantly affected the expression of four proteins: endoplasmic reticulum oxidoreductin 1-like protein alpha, p21-activated kinase 1, Archain 1, and T cell receptor beta chain. The former three were downregulated and the latter was upregulated. These proteins are primarily associated with endoplasmic reticulum stress, intracellular trafficking, and immune function.