Ablation of aberrant neurogenesis fails to attenuate cognitive deficit of chronically epileptic mice.

Pilocarpine-induced acute seizures strongly induce aberrant hippocampal neurogenesis, characterized by increased proliferation of neural progenitors and abnormal integrations of newly generated granule cells - hilar ectopic granule cells (EGCs), mossy fibre sprouting (MFS), and hilar basal dendrites (HBDs), which may disturb hippocampal neuronal circuits and thus contribute to cognitive impairment in temporal lobe epilepsy (TLE) patients and animal models. Previous studies via ablating hippocampal neurogenesis after acute seizures produced inconsistent results regarding the development of long-term cognitive impairment. Furthermore, a sufficient decrease of subsequent abnormal integrations in chronically epileptic hippocampus was not well-established in these studies. Therefore, the link between seizure-induced aberrant hippocampal neurogenesis and cognitive decline associated with epilepsy is still in need to be clarified. In this study, the mice were injected with methylazoxymethanol acetate (MAM) both before and after pilocarpine-induced status epilepticus (SE) to achieve an overall ablation of newborn cells contributing to the pathological recruitment. In addition, a protracted time point was chosen for behavioral testing considering it takes a fairly long time for newborn granule cells to adequately develop abnormal integrations, especially MFS. Although an overall reduction of abnormal integrations, including EGCs, MFS and HBDs was confirmed following the ablation regime, the performance of ablated and non-ablated mice in the Morris Water Maze (MWM) task did not differ. The current findings therefore provide novel evidences that ablation of neurogenesis with an overall decrease of abnormal integrations cannot attenuate subsequent cognitive impairment at least in the model used in this study.

Reduced abnormal integration of adult-generated granule cells does not attenuate spontaneous recurrent seizures in mice.

Epileptic seizures lead to aberrant hippocampal neurogenesis, including increased proliferation of neural progenitors and abnormal integrations of newly generated granule cells - hilar ectopic granule cells (EGCs), mossy fiber sprouting (MFS), and hilar basal dendrites (HBDs). Previous results from ablating hippocampal neurogenesis after acute seizures have been controversial with regards to the development of spontaneous recurrent seizures (SRSs). While ablation of hippocampal newborn cells was effective, a sufficient decrease of subsequent abnormal integrations in chronically epileptic hippocampus was not well-established in these studies. Evaluations of the role of aberrant neurogenesis in epileptogenesis were therefore inconclusive. In this study, we ablated the hippocampal neurogenesis by methylazoxymethanol acetate (MAM) treatment both before and after pilocarpine induced status epilepticus (SE). We found that an overall ablation of newborn granule cells and a protracted delay after the cell ablation are required to eliminate subsequent abnormal integrations, including EGCs, MSF and HBDs. However, there were no alterations in frequency, duration and severity of chronic seizures were demonstrated following this regime. The current findings provide novel evidences that an overall decrease of abnormal integrations via cell ablation cannot exert significant effects on the development of SRSs at least in the model used in this study.

Immature neurons born one week before pilocarpine-induced status epileptic exhibit aberrant integration at chronic stage after SE / 西安交通大学学报(医学版)

Objective To determine whether the integration of immature neurons born before status epilepticus (SE)can be disrupted by an epileptogenic insult.Methods Pilocarpine was used to induce SE in mice. At week 1 before induction,BrdU or retroviral vector expressing green fluorescent protein (RV-GFP)was used to label the newly born cells in the dentate gyrus (DG).At week 8 after SE,BrdU+Map2 or BrdU+NeuN double-labeling staining was carried out to visualize hilar basal dendrite or hilar ectopic migration.Virus-transduced GFP signals were used to identify the mossy fiber sprouting from the newly generated neurons.The number of cells with aberrant integrations was compared using unpaired Student's t-test.Results The percentage of newborn neurons with aberrant dendritic morphology was (20.8±8.4)% at week 8 after SE.The percentage of BrdU+NeuN double labeled cells ectopically migrated into the hilus was (15.9 ± 7.4)%.At week 8 after SE,the chronically epileptic mice showed many GFP+ processes in the IML with the same axonal appearance and small mossy fiber bouton-like structures as those seen in the hilus.The number of newborn neurons with aberrant integrations in SE mice wassignificantly increased when compared with the control mice (P <0.05).Conclusion These data demonstrate the existence of aberrant integrations-hilar basal dendrites,hilar ectopic migration and mossy fiber sprouting in the DG-generated cells born 1 week before an SE insult.

Immature neurons born one week before pilocarpine-induced status epileptic exhibit aberrant integration at chronic stage after SE / 西安交通大学学报(医学版)

Objective To determine whether the integration of immature neurons born before status epilepticus (SE)can be disrupted by an epileptogenic insult.Methods Pilocarpine was used to induce SE in mice. At week 1 before induction,BrdU or retroviral vector expressing green fluorescent protein (RV-GFP)was used to label the newly born cells in the dentate gyrus (DG).At week 8 after SE,BrdU+Map2 or BrdU+NeuN double-labeling staining was carried out to visualize hilar basal dendrite or hilar ectopic migration.Virus-transduced GFP signals were used to identify the mossy fiber sprouting from the newly generated neurons.The number of cells with aberrant integrations was compared using unpaired Student's t-test.Results The percentage of newborn neurons with aberrant dendritic morphology was (20.8±8.4)% at week 8 after SE.The percentage of BrdU+NeuN double labeled cells ectopically migrated into the hilus was (15.9 ± 7.4)%.At week 8 after SE,the chronically epileptic mice showed many GFP+ processes in the IML with the same axonal appearance and small mossy fiber bouton-like structures as those seen in the hilus.The number of newborn neurons with aberrant integrations in SE mice wassignificantly increased when compared with the control mice (P <0.05).Conclusion These data demonstrate the existence of aberrant integrations-hilar basal dendrites,hilar ectopic migration and mossy fiber sprouting in the DG-generated cells born 1 week before an SE insult.

Puerarin attenuates learning and memory impairments and inhibits oxidative stress in STZ-induced SAD mice.

Puerarin (PUE), an isoflavone purified from the root of Pueraria lobata (Chinese herb), has been reported to attenuate learning and memory impairments in the transgenic mouse model of Alzheimer's disease (AD). In the present study, we tested PUE in a sporadic AD (SAD) mouse model which was induced by the intracerebroventricular injection of streptozotocin (STZ). The mice were administrated PUE (25, 50, or 100mg/kg/d) for 28 days. Learning and memory abilities were assessed by the Morris water maze test. After behavioral test, the biochemical parameters of oxidative stress (glutathione peroxidase (GSH-Px), superoxide dismutases (SOD), and malondialdehyde (MDA)) were measured in the cerebral cortex and hippocampus. The SAD mice exhibited significantly decreased learning and memory ability, while PUE attenuated these impairments. The activities of GSH-Px and SOD were decreased while MDA was increased in the SAD animals. After PUE treatment, the activities of GSH-Px and SOD were elevated, and the level of MDA was decreased. The middle dose PUE was more effective than others. These results indicate that PUE attenuates learning and memory impairments and inhibits oxidative stress in STZ-induced SAD mice. PUE may be a promising therapeutic agent for SAD.

Vasoactive intestinal peptide administration after stroke in rats enhances neurogenesis and improves neurological function.

The aim of this study was to investigate the effects of vasoactive intestinal peptide (VIP) on neurogenesis and neurological function after cerebral ischemia. Rats were intracerebroventricular administered with VIP after a 2h middle cerebral artery occlusion (MCAO) and sacrificed at 7, 14 and 28 days after MCAO. Functional outcome was studied with the modified neurological severity score. The infarct volume was evaluated via histology. Neurogenesis, angiogenesis and the protein expression of vascular endothelial growth factor (VEGF) were measured by immunohistochemistry and Western blotting analysis, respectively. The treatment with VIP significantly reduced the neurological severity score and the infarc volume, and increased the numbers of bromodeoxyuridine (BrdU) immunoreactive cells and doublecortin immunoreactive area in the subventricular zone (SVZ) at 7, 14 and 28 days after ischemia. The cerebral protein levels of VEGF and VEGF expression in the SVZ were also enhanced in VIP-treated rats at 7 days after stroke. VIP treatment obviously increased the number of BrdU positive endothelial cells in the SVZ and density of cerebral microvessels in the ischemic boundary at 28 days after ischemia. Our study suggests that in the ischemic rat brain VIP reduces brain damage and promotes neurogenesis by increasing VEGF. VIP-enhanced neurogenesis is associated with angiogenesis. These changes may contribute to improvement in functional outcome.

Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aß). Reuptake of extracellular Aß is believed to contribute significantly to the intraneuronal Aß pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aß1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aß internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aß1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aß internalization in neurons. We found that extracellular Aß1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aß1-42 and LRP1 were also found co-localized in neurons during Aß1-42 internalization, and they could form Aß1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aß1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aß1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aß1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aß levels and served a potential therapeutic target for AD.

[Optimization of trypsin digestion intensity to obtain high-purity in vitro cultured astrocytes].

The aim of the present study was to observe the effect of trypsin digestion on the purity of in vitro cultured astrocytes and optimize the culture methods. The cerebral cortical tissue from newborn Sprague Dawley (SD) rats was isolated and digested with 0.25% trypsin for 20, 30, or 40 min. The obtained single cell suspension was then cultured. Once reaching confluence, the cells were shaken at a constant temperature. Then, each of 20 and 30 min groups was subdivided into two groups, the control group with normal digestion and two-time-digestion group, and the cells were passaged and purified. Through inverted phase contrast microscope and MTT assay, cell growth and proliferation were observed, respectively. Immunofluorescence for glial fibrillary acidic protein (GFAP) was used to observe the morphology of astrocytes and to assess their purity in different stages. Flow cytometric analysis was used to detect the apoptotic rates of purified astrocytes. The results showed that, the cells being digested for 20 min usually reached confluence at 9 d after seeding. When the digestion time was extended to 30 min, the cells grew faster and reached confluence at 7 d after seeding, meanwhile the morphology of astrocytes was normal, GFAP positive rate (70.2 ± 4.0)% being much higher than that of the 20 min group (P < 0.05). Compared with 20 min group, 40 min group showed higher GFAP positive rate, whereas the cell proliferation was slower, and cell injury was more obvious. After shaking at constant temperature, two times of trypsin digestion could decrease the number of contaminated cells after passage. The GFAP positive rates of two-time-digestion groups in passage 1 (P1) were higher than those of corresponding control groups, and the GFAP positive rate of 30 min + two-time-digestion group in P1 reached (98.1 ± 1.7)%, which was equivalent to that of the 20 min + control group in P3. However, the apoptotic rate showed no significant difference between these two groups. Based on above mentioned results, we conclude that 30 min + two-time of trypsin digestion effectively improves the purity of astrocytes and shortens the time of primary culture and purification, suggesting that it is a rapid and effective method to obtain astrocytes with high purity in vitro.

The effects of valsartan on cognitive deficits induced by aluminum trichloride and d-galactose in mice.

OBJECTIVES: Valsartan has been reported to reduce brain beta-amyloid protein levels and improve spatial learning in the Tg2576 transgenic mouse model of Alzheimer's disease (AD). However, the exact mechanism of neuroprotective effects of valsartan has not been properly studied especially in cholinergic function and oxidative damage, the essential factors that undergo impairment in AD. Therefore, the present study examined the effects of valsartan on memory impairment, cholinergic dysfunction, and oxidative stress in aluminum trichloride (AlCl3) and d-galactose (d-gal)-induced experimental sporadic dementia of Alzheimer's type. METHODS: Valsartan was administered intragastrically (i.g.) (20 mg/kg/day) for 60 days after mice were given AlCl3 (10 mg/kg/day) and d-gal (150 mg/kg/day) intraperitoneally (i.p.) once daily for 90 days. Then, memory function was evaluated by Morris water maze test. Acetylcholinesterase (AChE), superoxide dismutases (SOD) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) level in cortex and hippocampus were also assessed with biochemical technique. RESULTS: Chronic administration of valsartan not only improved learning and memory but also restored the elevation of AChE activity induced by AlCl3 and d-gal in cortex and hippocampus. In addition, valsartan significantly restored SOD and GSH-Px activities and reduced MDA level in cortex and hippocampus indicating attenuation of oxidative stress. DISCUSSION: Our results indicate that valsartan prevents AlCl3- and d-gal-induced cognitive decline partly to restore cholinergic function and attenuate oxidative damage. These findings further support the potential of valsartan to be used in AD treatment.

The effects of perindopril on cognitive impairment induced by d-galactose and aluminum trichloride via inhibition of acetylcholinesterase activity and oxidative stress.

Preclinical and clinical studies indicate involvement of renin angiotensin system (RAS) in memory functions. However, exact role of RAS in cognition is still ambiguous. The present study investigated the effects of perindopril on dementia of Alzheimer's type induced by d-galactose (d-gal) and aluminum trichloride (AlCl3). Perindopril, an angiotensin converting enzyme inhibitor, was administered intragastrically (0.5mg/kg/day) for 60days after mice were given d-gal (150mg/kg/day) and AlCl3 (10mg/kg/day) intraperitoneally for 90days. Then, memory function was evaluated by Morris water maze test. The biochemical studies were conducted in cerebral cortex and hippocampus of mouse brain after the behavioral studies. d-Gal and AlCl3 caused significant memory impairment along with significant elevation of acetylcholinesterase (AChE) activity in cerebral cortex and hippocampus. Further, a significant reduction of superoxide dismutases (SOD) and glutathione peroxidase (GSH-Px) activities, and elevation of malondialdehyde (MDA) level in cerebral cortex and hippocampus were observed. Perindopril not only improved cognitive impairment but also restored the elevation of AChE activity induced by d-gal and AlCl3. In addition, perindopril significantly increased SOD and GSH-Px activities, reduced MDA level in cerebral cortex and hippocampus. Taken together, the above findings indicate that perindopril improves learning and memorizing probably by restoring cholinergic function and attenuating oxidative damage.

Vasoactive intestinal peptide increases VEGF expression to promote proliferation of brain vascular endothelial cells via the cAMP/PKA pathway after ischemic insult in vitro.

Vasoactive intestinal peptide (VIP) enhances angiogenesis in rats with focal cerebral ischemia. In the present study, we investigated the molecular mechanism of the proangiogenic action of VIP using an in vitro ischemic model, in which rat brain microvascular endothelial cells (RBMECs) are subjected to oxygen and glucose deprivation (OGD). Western blotting and immunocytochemistry were carried out to examine the expression of VIP receptors and vascular endothelial growth factor (VEGF) in cultured RBMECs. The cell proliferation was assessed by the MTT assay. Cyclic adenosine monophosphate (cAMP) and VEGF levels were measured by using the enzyme-linked immunosorbent assay. The cultured RBMECs expressed VPAC1, VPAC2 and PAC1 receptors. Treatment with VIP significantly promoted the proliferation of RBMECs and increased OGD-induced expression of VEGF, and this effect was antagonized by the VPAC receptor antagonist VIP6-28 and VEGF antibody. VIP significantly increased contents of cAMP in RBMECs and VEGF in the culture medium. The VIP-induced VEGF production was blocked by H89, a protein kinase A (PKA) inhibitor. These data suggest that treatment with VIP promotes VEGF-mediated endothelial cell proliferation after ischemic insult in vitro, and this effect appears to be initiated by the VPAC receptors leading to activation of the cAMP/PKA pathway.

[Prokaryotic expression and immunogenicity analysis of the chimeric HBcAg containing APP beta cleavage site peptide and Aß(1-15);].

AIM: To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aß(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli. METHODS: The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aß(1-15); gene(ABCSP-Aß(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aß(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aß(15-c);. c-ABCSP-Aß(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA. RESULTS: The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aßantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable. CONCLUSION: Recombinant c-ABCSP-Aß(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.

[Immunization with recombinant HBcAg and ß-amyloid peptide fusion protein promotes clearance of intrahippocampally injected ß-amyloid peptide in rats].

OBJECTIVE: To study the effects of immunization with the fusion protein CAC (a product of prokaryotic expression of recombinant HBcAg and ß-amyloid peptide fusion gene) against the toxicity induced by intrahippocampal injection of aggregated ß-amyloid peptide (Aß) in rats. METHODS: SD rats were immunized intraperitoneally with the fusion protein CAC, and the titer of anti-Aß antibody was evaluated by ELISA. When the titers of the anti-Aß antibody reached 1:3 000, aggregated Aß was injected into the CA1 region of the rat hippocampus. Two weeks after Aß injection, the rats underwent morris water maze test before sacrificed to prepare the brain slices with Congo red and haematoxylin staining. RESULTS: The titer of anti-Aß antibody reached 1:3 000 after 5 immunizations with the fusion protein. After Aß injection, the saline-immunized rats showed a reduced cognitive behavior in the Morris water maze test compared to the CAC-immunized rats. In the saline-immunized rats, the neurons around the site of Aß injection exhibited obvious cell damages with Aß deposits and glial infiltration, whereas in CAC-immunized rats, Aß deposits were significantly reduced or even absent. CONCLUSION: Immunization with the fusion protein CAC can inhibit the toxicity induced by intrahippocampal aggregated Aß injection.

[Effect of [Gly14]-Humanin on Abeta(25-35)-induced PC12 cell apoptosis].

AIM: To investigate the effect of [Gly14]-Humanin overexpression on Abeta(25-35);-induced PC12 cell apoptosis. METHODS: Recombinant plasmid pcDNA3.1(-)/HNG-FLAG was transfected into PC12 cells by liposome method. The subclone cell lines were obtained by persistent G418 selection. The HNG gene expression of PC12 cells was detected by immunocytochemistry. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability was determined by MTT assay, and apoptosis rate was detected by using flow cytometric analysis. Hochest33258 staining was used to observe the morphological changes of cellular nuclei. RESULTS: PC12 cell lines stably expressing HNG gene was successfully selected. After being treated with 25 micromol/L Abeta(25-35); for 24 h, cell viability of PC12 cells overexpression HNG was significant elevated compared with empty plasmid transfected cells (P<0.05), and the apoptosis rate was lower significantly (P<0.05). By Hoechst 33258 staining, the nuclei of empty plasmid transfected PC12 cells exhibited highly condensed and fragmented nuclei morphology which was the typical characteristics of apoptosis, and the nuclei of PC12 cells overexpression HNG were round and homogeneously stained. CONCLUSION: Overexpression of HNG prevented the cell apoptosis induced by Abeta(25-35); in PC12 cells.

[Effect of puerarin on PC12 cells apoptosis induced by Abeta25-35 in vitro].

OBJECTIVE: To establish a nerve cell injury model by incubating PC12 cell line in the presence of Abeta25-35 to study the effect of puerarin on apoptosis of nerve cells. METHODS: PC12 cells were incubated with Abeta25-35 and puerarin. Cell viability was detected by MTT. The cellular morphology was observed with electron microscopy. FITC-labeled Annexin V and propidium iodide were adopted to evaluate the rate of cell apoptosis in different groups by means of flow cytometry. RESULTS: Following incubation Abeta25-35, the cells were induced to undergo apoptosis. The viability of PC12 cells decreased in a time-dependent manner. Morphological evidences for apoptosis nuclear condensation were observed in PC12 cells. Cells incubated in the presence of Abeta25-35 showed increasing apoptotic rates, but cells treated with puerarin and Abeta25-35 revealed decreasing apoptotic rates, it demonstrated that puerarin had a significant protective action against Abeta25-35 evoked apoptosis. CONCLUSION: Puerarin can resist the adverse effects of Abeta25-35 on increasing apoptotic rates, and it has the protective function towards nerve cells.

[Biological effects of immune serum from fusion protein of beta-amyloid peptide and HbcAg/MIR on AD transgeneic cells].

OBJECTIVE: To investigate the expression of fusion protein c-Abeta-c, a fusion protein from the beta amyloid peptide (Abeta) and the Hepatitis B core antigen/ Major immunodominant region (HbcAg/MIR), in the BL21/pET28 prokaryotic expression system and to immunize mice with the expressed fusion protein and evaluate the immunogenicity and biological effects of the serum in vitro. METHODS: The recombinant prokaryotic expression plasmid PET-28a /c-Abeta-c was constructed by molecular cloning technique and the c-Abeta-c fusion gene expression was induced in E. coli BL21 by isopropyl-1-thio-b-Dgalactopyranoside (IPTG). The expressed fusion protein was analyzed by SDS-PAGE. Intraperitoneal injection (i.p.) of the purified c-Abeta-c fusion protein was given to the BALB/c mice. The anti-Abeta effect of the immune serum was detected by indirect ELISA. The biological effect of the immune serum on Alzheimer's disease (AD) transgeneic cells was assessed by MTT assay and flow cytometer. RESULTS: The c-Abeta-c fusion protein was found in the sediment of the isolated bacteria. The expressed protein comprised more than 30% of the total proteins in the bacteria sediment. The anti-Abeta antibody in the serum of the immunized mice reached 1:16000. The antiserum reduced the cytotoxicity of Abeta peptide in the AD transgeneic cells and significantly decreased the apoptosis of cells. CONCLUSION: The c-Abeta-c fusion protein has good Abeta immunogenicity and the animal immune serum efficiently inhibits the cytotoxicity of Abeta peptide.

[Specific suppression of beta-secretase gene expression by short interfering RNA in mammalian cells].

OBJECTIVE: To investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR, respectively. The PCR products were inserted into plasmid pLXSN. The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE. The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method. RESULT: The interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively, and the production of A beta was reduced. CONCLUSION: BACE siRNA can inhibit the expression of BACE gene of mammalian cells.

Specific suppression of beta-secretase gene expression by short interfering RNA in SK-N-SH cells.

OBJECTIVE: To test the effect of short interfering RNAs (siRNAs) of beta-site APP cleaving enzyme (BACE) on inhibiting the expression of BACE in mammalian cells. METHODS: The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR. The PCR products were inserted into the retrovirus plasmid pLXSN. The interfering vector was identified as pLXSN/ EGFP-U6-siBACE. The SK-N-SH cell line was produced, which can highly expressed BACE. The inhibitive effect of BACE siRNA on BACE expression was examined by fluoroscopy and immunohistochemistry tests. RESULTS: The interfering vector, pLXSN/EGFP-U6-siBACE, was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cell and reduced the production of Abeta. CONCLUSION: BACE siRNA inhibits the expression of BACE gene of mammalian, which has implications for RNA interference of Alzheimer's disease.

[Construction and prokaryotic expression of recombinant gene c-Abeta-c and the immunogenicity analysis of the fusion protein].

AIM: To construct the recombinant prokaryotic expression plasmid pHGhis/c-Abeta-c and evaluate the immunogenicity of the fusion protein expressed in E.coli DH5 alpha. METHODS: The gene fragments of HBc1-71, HBc88-144 and Abeta(1-42) were amplified by PCR. Then the Abeta(1-42) gene was inserted between HBc1-71 and HBc88-144, yielding the recombinant gene c-Abeta-c. c-Abeta-c gene was cloned into pGEMEX and then subcloned into pHGhis plasmids. c-Abeta-c fusion protein expression in transformed E.coli DH5alpha was induced at 42degrees Celsius. The expressed fusion protein was analyzed by SDS-PAGE. Six BALB/c mice recieved intraperitoneal injection (i.p.) of c-Abeta-c fusion protein purified by saturated ammonium sulfate. The anti-Abeta antibody was detected by indirect ELISA. RESULTS: The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After temperature induction, fusion protein was expressed and existed in the sediment of the bacterial lysate. The expression level was 16% of all the proteins in the sediment. After 3 times of immunization, the titer of anti-Abeta antibody in sera of BALB/c mice reached up to 1:16,000, while the anti-HBc antibody was undetectable. CONCLUSION: Recombinant c-Abeta-c gene can be expressed in E.coli DH5 alpha. The expressed protein exists in the sediment of the bacterial lysate and has a strong immunogenicity. This study lays the foundation for the experimental animal study of Alzheimer's disease genetic engineering vaccine.

[Protective effects of gastrodin on the cellular model of Alzheimer's disease induced by Abeta25-35].

OBJECTIVE: To assess the effects of gastrodin (GAS) on the induced neurons of Alzheimer's disease (AD). METHODS: The cellular model of AD was established with primary cultured cerebral cortical and hippocampal cells induced by Abeta325-35. The total saponin ginseng was taken as positive control. The effects of gastrodin on the cellular model of AD were studied by morphological observation and biochemical methods. RESULTS: Compared with the control, the cellular model induced by Abeta25-35 showed remarkable morphological changes, significant increase in LDH release, and significant decrease in cell survival. When different doses of gastrodin, total saponin of ginseng were added beforehand, significant decrease in LDH release and significant increase in cell survival were noted by comparison with the experimental control, which indicated that the two medicines could protect the cells from the damage induced by Abeta25-35 to some extent. CONCLUSION: The results suggested that gastrodin has potential effects on the prevention and therapy of AD.