Base-promoted Lewis acid catalyzed synthesis of quinazoline derivatives.

A one-pot protocol has been developed for the synthesis of quinazolinones from amide-oxazolines with TsCl via a cyclic 1,3-azaoxonium intermediate and 6π electron cyclization in the presence of a Lewis acid and base. The process is operationally simple and has a broad substrate scope. This method provides a unique strategy for the construction of quinazolinones.

CD200-CD200R dysfunction exacerbates microglial activation and dopaminergic neurodegeneration in a rat model of Parkinson's disease.

BACKGROUND: Increasing evidence suggests that microglial activation may participate in the aetiology and pathogenesis of Parkinson's disease (PD). CD200-CD200R signalling has been shown to be critical for restraining microglial activation. We have previously shown that expression of CD200R in monocyte-derived macrophages, induced by various stimuli, is impaired in PD patients, implying an intrinsic abnormality of CD200-CD200R signalling in PD brain. Thus, further in vivo evidence is needed to elucidate the role of malfunction of CD200-CD200R signalling in the pathogenesis of PD. METHODS: 6-hydroxydopamine (6-OHDA)-lesioned rats were used as an animal model of PD. CD200R-blocking antibody (BAb) was injected into striatum to block the engagement of CD200 and CD200R. The animals were divided into three groups, which were treated with 6-OHDA/Veh (PBS), 6-OHDA/CAb (isotype control antibody) or 6-OHDA/BAb, respectively. Rotational tests and immunohistochemistry were employed to evaluate motor deficits and dopaminergic neurodegeneration in animals from each group. HPLC analysis was used to measure monoamine levels in striatum. Morphological analysis and quantification of CD11b- (or MHC II-) immunoreactive cells were performed to investigate microglial activation and possible neuroinflammation in the substantia nigra (SN). Finally, ELISA was employed to assay protein levels of proinflammatory cytokines. RESULTS: Compared with 6-OHDA/CAb or 6-OHDA/Veh groups, rats treated with 6-OHDA/BAb showed a significant increase in counts of contralateral rotation and a significant decrease in TH-immunoreactive (TH-ir) neurons in SN. A marked decrease in monoamine levels was also detected in 6-OHDA/BAb-treated rats, in comparison to 6-OHDA/Veh-treated ones. Furthermore, remarkably increased activation of microglia as well as up-regulation of proinflammatory cytokines was found concomitant with dopaminergic neurodegeneration in 6-OHDA/BAb-treated rats. CONCLUSIONS: This study shows that deficits in the CD200-CD200R system exacerbate microglial activation and dopaminergic neurodegeneration in a 6-OHDA-induced rat model of PD. Our results suggest that dysfunction of CD200-CD200R signalling may be involved in the aetiopathogenesis of PD.

Proteasome inhibitor lactacystin induces cholinergic degeneration.

OBJECTIVE: Ubiquitin proteasome system dysfunction is believed to play an important role in the development of Parkinson's disease (PD), and almost all studies till now have mainly focused on the susceptibility of dopaminergic neurons to proteasome inhibition. However, in fact, there are many other types of neurons such as cholinergic ones involved in PD. In our present study, we attempt to figure out what effect the failure of ubiquitin proteasome function would execute on cholinergic cells in culture. METHODS: We treated cholinergic cells in culture with various doses of lactacystin. Then MTT assay was used to evaluate the cellular viability and the AnnexinV-PI method was used to detect apoptosis. Both cellular soluble and insoluble polyubiquitinated proteins were detected by western blot. Furthermore, the mitochondrial membrane potential was analyzed using JC-1 and the intracellular production of reactive oxygen species (ROS) was determined using the fluorescent probe CM-H2DCFDA. RESULTS: We found that low doses of lactacystin were enough to induce significant apoptotic cell death, disturb the mitochondrial membrane potential, and cause oxidative stress. We also found that the amounts of polyubiquitinated proteins dramatically increased with high doses, although the loss of cells did not increase accordingly. CONCLUSIONS: Our results suggest that cholinergic cells are sensitive to ubiquitin proteasome system dysfunction, which exerts its toxic effect by causing mitochondrial dysfunction and subsequent oxidative stress, not through polyubiquitinated proteins accumulation.

PMS777, a new cholinesterase inhibitor with anti-platelet activated factor activity, regulates amyloid precursor protein processing in vitro.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by progressive impairment of memory and cognition. Previous data have shown that beta-amyloid (Abeta) cascade plays a central role in AD pathophysiology and thus drugs regulate amyloid precursor protein (APP) metabolism may have therapeutic potential. Here the effects of PMS777, a new cholinesterase inhibitor with anti-platelet activated factor activity, on APP processing were investigated. Using SH-SY5Y(APP695) cells, it showed that PMS777 treatment caused significant decreased secretion of sAPPalpha into the conditioned media without affecting cellular holoAPP synthesis. When PC12 cells were incubated with PMS777, the same effect was observed. The data also indicated that 10 muM PMS777 incubation decreased the release of Abeta42 into the cell media as compared with vehicle group in SH-SY5Y(APP695) cells. Pretreatment of cells with M-receptor scopolamine antagonized the decreased secretion of sAPPalpha induced by PMS777, but N-receptor alpha-bungarotoxin pretreatment did not have such an effect. These results indicated that PMS777 could modulate APP processing in vitro and that decreasing Abeta generation might demonstrate its therapeutic potential in AD.

Parkinson disease IgG and C5a-induced synergistic dopaminergic neurotoxicity: role of microglia.

Increasing evidence suggests the involvement of immune/inflammatory system in Parkinson's disease (PD). Many immune/inflammatory factors may synergistically participate in PD. In this study, we demonstrated that immunoglobulin G from the serum of 4/11 PD patients (PDIgG, 60microg/ml) and recombinant human C5a (0.1nM) synergistically induced selective dopaminergic neurodegeneration in rat mesencephalic neuron-glia cultures, while that PDIgG alone or C5a alone was minimally toxic or nontoxic. IgG from 17 disease controls and from 7 normal controls did not significantly induce dopaminergic neurotoxicity in the cultures even in the presence of C5a. Using mesencephalic neuron-enriched cultures, we found that the synergistic dopaminergic neurotoxicity was mediated by glia. The results from microglia-supplemented neuronal cultures, astroglia-supplemented neuronal cultures and neuron-astroglia cocultures indicated that microglia, not astroglia, played a pivotal role in the neurotoxicity. Through immunocytochemistry analysis and assay of proinflammatory factors, we observed that each of the four PDIgGs (60microg/ml) and C5a (0.1nM) synergistically induced microglia activation and production of superoxide and nitric oxide (NO) in neuron-glia cultures. Further investigations indicated that superoxide and NO were both responsible for the synergistic neurotoxicity. Finally, using F(ab')(2) fragments of PDIgG, we demonstrated that microglial Fc receptors may play an important role in the neurotoxicity. Our work provides new evidence for the involvement of the immune/inflammatory system in PD and helpful clues for studying the combined effect of antibody and complement on microglia.

Mitogen activated protein kinase and protein kinase C activation mediate promotion of sAPPalpha secretion by deprenyl.

The beta amyloid cascade plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Therefore, drugs that regulate amyloid precursor protein (APP) processing toward the nonamyloidgenic pathway may have therapeutic potential. Many anti-dementia drugs can regulate APP processing in addition to their pharmacological properties. Deprenyl is a neuroprotective agent used to treat some neurodegenerative diseases, including AD. In the present study, the effects of deprenyl on APP processing were investigated. Using SK-N-SH and PC12 cells, it was demonstrated that deprenyl stimulated the release of the nonamyloidogenic alpha-secretase form of soluble APP (sAPPalpha) in a dose-dependent manner without affecting cellular APP expression. The increase of sAPPalpha secretion by deprenyl was blocked by the mitogen activated protein (MAP) kinase inhibitor U0126 and PD98059, and by the protein kinase C (PKC) inhibitor GF109203X and staurosporine, suggesting the involvement of these signal transduction pathways. Deprenyl induced phosphorylation of p42/44 MAP kinase, which was abolished by specific inhibitors of MAP kinase and PKC. Deprenyl also phosphorylated PKC and its major substrate, and myristoylated alanine-rich C kinase (MARCKS) at specific amino acid residues. The data also indicated that 10microM deprenyl successfully induced two PKC isoforms involved in the pathogenesis of AD, PKCalpha and PKCepsilon, to translocate from the cytosolic to the membrane fraction. This phenomenon was substantiated by immunocytochemistry staining. These data suggest a novel pharmacological mechanism in which deprenyl regulates the processing of APP via activation of the MAP kinase and PKC pathways, and that this mechanism may underlie the clinical efficacy of the drug in some AD patients.

Transplantation of bone marrow stromal cells containing the neurturin gene in rat model of Parkinson's disease.

The experiment was to evaluate the therapeutic benefit of transplanted bone marrow stromal cells (BMSCs) transfected with a kind of neurotrophic factor gene, neurturin (NTN) gene, in treating the rat model of Parkinson's disease (PD). The 6-OHDA-lesioned rats were assigned to one of three groups, those receiving BMSCs transfected with NTN gene, those receiving untransfected BMSCs containing a void plasmid and those receiving phosphate buffer solution (PBS). Treatments were injected into the right striatum (6-OHDA-lesioned side). One to six months post-transplantation, apomorphine-induced rotational behavior was observed. One month after transplantation, green fluorescent protein (GFP)/NTN, GFP/glial fibrillary acidic protein (GFAP), GFP/neuron specific enolase (NSE) and GFP/tyrosine hydroxylase (TH) fluorescence determinations of brain sections were carried out. One to six months after transplantation, brain sections containing striatum and substantia nigra were stained for TH. In situ hybridization and Western blots were used to determine NTN mRNA and protein concentration, respectively, in affected brain regions. High performance liquid chromatography (HPLC) was used to measure the dopamine (DA) content in the lesioned striatum 1 and 3 month(s) post-transplantation. The results were shown that: in the first 3 months after transplantation, the number of rotations was lower in NTN-transplant group than the void vector group, and during 1-6 months post-transplantation, the number of rotations was lower in both transplant groups than that in the PBS group (P<0.05). One month after transplantation, we detected GFP/NTN-, GFP/GFAP- and GFP/NSE-labeled cells in the transplantation area of the NTN-transplanted group, but no obvious GFP/TH labeled cells were found. Quantitative analysis of TH-positive cells 1 to 6 months after transplantation indicated that there were no significant differences between groups in survival rates of TH-positive neurons in the lesioned substantia nigra (P>0.05). In situ hybridization and Western blot identified NTN mRNA and protein expression in the transplantation area of the NTN-transplanted group. After transplantation of NTN-expressing cells, DA content in the lesioned striatum was significantly higher in the transgenic group than that in the void vector group or the PBS group (P<0.05). The overall therapeutic effects of the NTN-transplanted group were superior to those of the void plasmid group and the PBS group. The mechanisms by which transgenic therapy treats PD might involve functional enhancement of residual dopaminergic neurons by NTN, which significantly reduces the number of rotations in animals, but not increase the numbers of existing dopaminergic neurons.

Effect of transplantation of c17.2 cells transfected with interleukin-10 gene on intracerebral immune response in rat model of Parkinson's disease.

Currently, regulation of immune response after grafting has become a hot topic in Parkinson's disease (PD) transplantation research. Interleukin-10 (IL-10) is an important regulator of immune system. Presently, we transplanted c17.2 neural stem cells transfected with pcDNA3.1-Hygro-IL-10 vector (IL-10-c17.2 cells) or Mock-c17.2 cells (c17.2 cells transfected with pcDNA3.1-Hygro vector) into the brains of 6-hydroxydopamine-lesioned PD model rats. From days 10 to 60 after grafting, double immunohistochemistry showed that IL-10 expression was detected in IL-10-c17.2 cells in vivo. Further immunohistochemistry analyses revealed that intracerebral cellular (ED1 and CD8) and humoral (C3 and IgM) immune responses were down-regulated in the rats treated with IL-10-c17.2 cells compared with controls treated with Mock-c17.2 cells. The reduction in ED1 immunostaining in the rats treated with IL-10-c17.2 cells remained significant until day 60 after transplantation. Our results suggest the potential application value of IL-10 in the transplantation treatment of PD.

Dopaminergic neuroprotection by neurturin-expressing c17.2 neural stem cells in a rat model of Parkinson's disease.

Genetically engineered neural stem cell (NSC) lines are promising vectors for the treatment of regenerative diseases, especially Parkinson's disease (PD). Neurturin (NTN), a member of the glial cell line-derived neurotrophic factor-family, has been demonstrated to act specifically on mesencephalic dopaminergic neurons, suggesting its therapeutic potential for PD. Here, we have generated a NTN-secreting c17.2 NSC line and investigated the protective effect of NTN-c17.2 on PD rat models. These NTN-releasing NSCs engrafted and integrated in the host striatum with good success, gave rise to neurons, astrocytes and oligodendrocytes, and maintained stable, high-level NTN expression. In addition, inverse transfer of NTN protein into the substantia nigra (SN) was able to protect dopaminergic neurons from 6-OHDA toxicity. Observation of rotational behavior showed that the NTN group performed significantly better than the Mock group, and the protective effect of NTN lasted for at least 4 months. HPLC tests indicated that the contents of neurotransmitters (e.g. dopamine) in the corpus striatum area of the NTN-c17.2 group and the Mock-c17.2 group were significantly higher than in the PBS group, but there was no significant difference between expression in the NTN-c17.2 and Mock-c17.2 groups. Taken together, our results suggest that transplantation of NTN-secreting NSCs exerted protective on PD rat models.

Therapeutic effects of differentiated bone marrow stromal cell transplantation on rat models of Parkinson's disease.

This study was to explore curative effect of bone marrow stromal cells (BMSCs) differentiated into nestin-positive cells transplantation on rat Parkinson's disease (PD) model. The PD rats were selected and allocated randomly into three groups. BMSCs with differentiation, BMSCs without differentiation and physiological saline were injected into right striatum of PD rat. The rotation test and immunofluorescence double staining were done. Frequency of rotation was significantly less in differentiated or non-differentiated BMSCs groups than that in normal saline group. Brdu/GFAP- and Brdu/NSE-positive cells appeared except BrdU/TH-positive cells. BMSCs differentiated had better effect than that of BMSCs without differentiated and physiological saline group.

[Effects of different doses of levodopa on paraquat-induced neurotoxicity: an experiment with mice].

OBJECTIVE: To investigate the effects and mechanisms of different doses of levodopa on paraquat-induced neuro-toxicity. METHODS: 72 C57BL mice were divided into 2 equal groups: acute experiment group and chronic experiment groups. The acute experiment group was re-divided into 2 subgroup: subgroup A to be injected with levodopa of the doses of 0 (distilled water instead), 10 mg/kg, or 100 mg/kg and then paraquat 30 mg/kg (levodopa + paraquat), and then killed 90 minutes after; and subgroup B, to be injected with paraquat 30 mg/kg and then levodopa 0, 10 mg/kg, or 100 mg/kg (paraquat + levodopa), and then killed 2 hours after. The chronic experiment group was re-divided into 2 subgroups to be injected with levodopa + paraquat or paraquat + levodopa once a week for 3 weeks, and then killed 24 hours after the injection. Fluorescent microscopy was used to observe the fluorescent staining of paraquat in the substantia nigra in the acute experiment group and the fluorescent staining of tyrosine hydroxylase (TH) in the substantia nigra in the chronic experiment group. in the chronic experiment group Western blotting was used to examine the protein expression of TH; thioflavine double labeling was used to observe the alpha-synuclein aggregation by immunofluorescence staining and Western blotting. The slices of substantia nigra of the mice in the chronic experiment group treated with distilled water + paraquat were inoculated with or without 250 micromol/L levodopa and then underwent thioflavine staining to observe the alpha-Syn aggregation. RESULTS: The paraquat staining was strongly positive in the substantia nigra of the mice in Group A-1, and was decreased gradually in the group A-2 and A-3. The paraquat staining was strongly positive in the substantia nigra of Group B-1 without a significant difference between Group A-1 and Group B-1, and was not remarkable in Group B-2 and B-3. The TH staining and protein expression in the substantia nigra of Group A-2 were significantly stronger than that of Group A-1 (P < 0.05), and the TH staining was remarkably weaker in Group A-3 (P < 0.05), as shown by immunofluorescence staining and Western blotting. There was no significant difference in TH staining and protein expression in the substantia nigra among Group A-1, Group B-1, and Group B-2 (all P > 0.05). However, the TH staining was remarkably weaker in Group B-3 (P < 0.05). The thioflavine and alpha-Syn double staining was significantly weaker in Groups A-2 and A-3 in comparison with Group A-1. There was no significant difference in the double staining among Group A-1, Group B-1, and Group B-2 (all P > 0.05). However, the double staining was remarkably weaker in Group B-3 (P < 0.05). The thioflavine positive staining in the tissue slices inoculated with levodopa was significantly weaker in comparison with those un-inoculated. CONCLUSION: Pre-treatment with lower dose L-dopa before the paraquat administration is neuroprotective by preventing paraquat from access into central nervous system through a blood-brain barrier competitive uptake mechanism, while higher dose L-dopa shows neurotoxicity through disaggregating alpha-synuclein deposits in Parkinsonian mice.

[Proteasomal inhibitor lactacystin induces cell apoptosis and caspase 3 activation in PC12 cells].

OBJECTIVE: To explore whether proteasome dysfunction cam induce dopaminergic cell apoptosis and investigate the probable molecular mechanism. METHODS: MTT assay was applied to measure the cell vitality of rat pheochrom-ocytoma cells of the Line PC12 exposed to highly specific proteasomal inhibitor lactacystin (0, 1 micromol/L, 5 micromol/L or 10 micromol/L) for 24 hours. After the PC12 cells were treated with 10 micromol/L lactacystin for 24 hours, apoptosis was estimated by Hoechst fluorescence staining and flow cytometry. When the PC12 cells were treated with 10 micromol/L lactacystin for 0, 24 or 48 hours, the level of caspase 3 cleaved fragments were analyzed by Western blotting. RESULTS: The PC12 cells exposed to 5 micromol/L or 10 micromol/L lactacystin for 24 hours showed a significant decrease in cell vitality (P < 0.05). Following treated with 10 micromol/L lactacystin for 24 hours, PC12 cells were seen to be nuclear condensation and fragmentation consistent with an apoptotic nuclear morphology by were seen in the Hoechst fluorescence staining and confirmed to have a significant increase of apoptotic cells (about 31.4%) by flow cytometry. Western blotting showed that there was a very low level of caspase 3 cleaved fragments (17,000) in control cells. But, after PC12 cells were exposed to 10 micromol/L lactacystin for 48 hours, the protein level of caspase 3 cleaved fragments (17,000) increased obviously. CONCLUSION: Proteasomal inhibitor lactacystin leads to dopaminergic cell apoptosis. The activation of caspase 3 protease may contribute to the mechanism of lactacystin-induced apoptosis in PC12 cells. Proteasome dysfunction may play an important role in the pathogenesis of Parkinson's disease.

[Surgical treatment of acetabular type C1 acetabular fracture by posteroproximal-posteroanterior sequential reduction and internal fixation].

OBJECTIVE: To investigate the operative reduction techniques and clinical results of surgical treatment of type C1 (AO/ASIF) acetabular fracture by posteroproximal-posteroanterior sequential reduction and internal fixation. METHODS: From August 2004 to January 2012, 13 patients with type C1 (AO/ASIF) acetabular fracture were treated by posteroproximal-posteroanterior sequential reduction and internal fixation. Of them, 8 cases were male and 5 cases were female with an average age of 42 years years old (ranged, 18 to 64). Pelvis 3-dimentional CT reconstruction were used to confirmed the classification of fracture, and the operation were performed during from 5 to 20 days with an average of 9.5 days. Operation time, blood loss, complications and reduction were recorded and evaluated. The function of hip joint were accessed at the final follow-up. RESULTS: The operation time ranged from 190 to 290 min with an average of 240 min. The mean blood loss was 1 800 ml (ranged, 1 300 to 3 000 ml). One case had superficial infection and healed after 3 weeks. According to Matta reduction criteria, 8 cases obtained anatomical reduction, 4 cases got satisfied results and 1 cases got unsatisfied results. Eleven cases were followed up with an average of (24.0 +/- 8.0) months, and 2 cases were lost to follow-up. According to revised Mede d'Aubingne and Postel evaluation system, 7 cases got excellent results, 2 good, 1 moderate and 1 poor. CONCLUSION: Posteroproximal-posteroanterior sequential reduction and internal fixation for the treatment of type C1 (AO/ASIF) acetabular fracture can achieve satisfied surgical proces and operation quality.

[Treatment of acromion base fractures with double plates internal fixation].

OBJECTIVE: To study clinical effects of double plates fixation for the treatment of acromion base fracutres. METHODS: From January 2010 to May 2012, 7 patients with acromion base fractures were treated with double plates ORIF surgical treatment. There were 5 males and 2 females, with an average age of 36.3 years old (ranged, 24 to 62 years old). All fractures were acuted and closed injuries. The duration from injury to surgery was 4.6 days (ranged, 2 to 10 days). Hardegger functional criterion, Visual Analogue Scale (VAS) and complications of the patients were documented analysis. RESULTS: All the patients were followed up,and the duration ranged from 4 to 13 months (averaged 8.9 months). The healing duration of fractures ranged from 8 to 14 weeks without any infection, shoulder instability, subacromial impingement syndrome, nonunion and failure of internal fixation. At the latest follow-up, the VAS ranged from 0 to 5. According to Hardegger criterion, 2 patients got an excellent result, 4 good and 1 poor. CONCLUSION: Double plates ORIF plays a positive role in the treatment of acromion base fractures, which reduces complications and maximally restore the function of shoulder.

Impaired CD200-CD200R-mediated microglia silencing enhances midbrain dopaminergic neurodegeneration: roles of aging, superoxide, NADPH oxidase, and p38 MAPK.

CD200-CD200R signaling holds microglia in a quiescent state. Parkinson disease (PD) neurodegeneration may be associated with impairment of CD200-CD200R-mediated microglia silencing in the substantia nigra (SN). In this study, an anti-CD200R blocking antibody (ACDR) selectively and significantly enhanced the susceptibility of dopaminergic neurons to neurotoxicity induced by rotenone (Rot) and iron (Ir) in mesencephalic neuron/glia cultures. Microglia were shown to mediate dopaminergic neurotoxicity induced by ACDR/Rot (combination of ACDR and Rot) and ACDR/Ir (combination of ACDR and Ir). ACDR significantly enhanced the microglial activation induced by Rot and Ir in neuron/glia cultures. NADPH oxidase-mediated superoxide generation was a key contributor to dopaminergic neurotoxicity induced by ACDR/Rot and ACDR/Ir. p38 MAPK contributed to NADPH oxidase activation induced by ACDR/Rot and ACDR/Ir. Interestingly, there were a decrease in CD200 expression (mRNA and protein) and an enhancement of microglial response (MHCII mRNA and ICAM-1 protein) in the rat SN with aging. ICAM-1 expression was significantly inversely correlated with CD200 expression. These results strongly indicate the participation of SN CD200-CD200R dysfunction in the etiopathogenesis of PD and provide a new insight into the molecular mechanisms underlying the involvement of aging in PD and help to elucidate the mechanisms of the combined involvement of immune/inflammatory factors, environmental substances, and aging in PD.

[Curative effect of the fibrinogen gel for sacral canal arachnoid cyst].

OBJECTIVE: To evaluate curative effect of the fibrinogen gel for treating sacral canal arachnoid cyst. METHODS: Nineteen patients with sacral canal arachnoid cysts included 7 males and 12 females; The average age was 48.4 years ranging from 19 to 68 years. The course was from 2 weeks to 7 months. Of all the patients, 9 were in level of S1, 4 were in level of S1 to S2, 5 were in level of S2, 1 was in level of S1 to S3. Cystis wall greater partial excision adopted in 11 cases, partial resection in 8, then all patients were treated by spray painting fibrinogen gel. RESULTS: Nineteen patients were followed-up for 13 to 30 months (mean 21.3 months). The clinical symptom disappeared completely in 18 patients, and only one patient urinated incapably, but after 2 weeks returned to normal. No one found recurrence by MRI after 12 months. CONCLUSION: This method of fibrinogen gel for treating sacral canal arachnoid cyst has advantages of easy performing, safety, achieve good results, less neck symptoms and early commencing of mobilization.

Dopaminergic regeneration by neurturin-overexpressing c17.2 neural stem cells in a rat model of Parkinson's disease.

BACKGROUND: Genetically engineered neural stem cell (NSC) lines are promising vectors for the treatment of neurodegenerative diseases, particularly Parkinson's disease (PD). Neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family, has been demonstrated to act specifically on mesencephalic dopaminergic neurons, suggesting its therapeutic potential for PD. In our previous work, we demonstrated that NTN-overexpressing c17.2 NSCs exerted dopaminergic neuroprotection in a rat model of PD. In this study, we transplanted NTN-c17.2 into the striatum of the 6-hydroxydopamine (6-OHDA) PD model to further determine the regenerative effect of NTN-c17.2 on the rat models of PD. RESULTS: After intrastriatal grafting, NTN-c17.2 cells differentiated and gradually downregulated nestin expression, while the grafts stably overexpressed NTN. Further, an observation of rotational behavior and the contents of neurotransmitters tested by high-performance liquid chromatography showed that the regenerative effect of the NTN-c17.2 group was significantly better than that of the Mock-c17.2 group, and the regenerative effect of the Mock-c17.2 group was better than that of the PBS group. Further research through reverse-transcriptase polymerase chain reaction assays and in vivo histology revealed that the regenerative effect of Mock-c17.2 and NTN-c17.2 cell grafts may be attributed to the ability of NSCs to produce neurotrophic factors and differentiate into tyrosine hydroxylase-positive cells. CONCLUSION: The transplantation of NTN-c17.2 can exert neuroregenerative effects in the rat model of PD, and the delivery of NTN by NSCs may constitute a very useful strategy in the treatment of PD.

New protein kinase C activator regulates amyloid precursor protein processing in vitro by increasing alpha-secretase activity.

The beta amyloid (Abeta) cascade has been at the forefront of the hypothesis used to describe the pathogenesis of Alzheimer's disease (AD). It is generally accepted that drugs that can regulate the processing of the amyloid precursor protein (APP) toward the non-amyloidogenic pathway may have a therapeutic potential. Previous studies have shown that protein kinase C (PKC) hypofunction has an important role in AD pathophysiology. Therefore, the effects of a new PKC activator, alpha-APP modulator [(2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam (TPPB)], on APP processing were investigated. Using PC12 cells and SH-SY5Y(APP695) cells, it was found that TPPB promoted the secretion of sAPPalpha without affecting full-length expression of APP. The increase in sAPPalpha by TPPB was blocked by inhibitors of PKC, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and tyrosine kinase, suggesting the involvement of these signal transduction pathways. TPPB increased alpha-secretase activity [a disintegrin and metalloproteinase (ADAM)10 and 17], as shown by direct fluorescence activity detection and Western blot analysis. TPPB-induced sAPPalpha release was blocked by the metalloproteinase inhibitor TAPI-2, furin inhibitor CMK and by the protein-trafficking inhibitor brefeldin. The results also showed that TPPB decreased beta-secretase activity, Abeta40 release and beta site APP-cleaving enzyme 1 (BACE1) expression, but did not significantly affect neprilysin (NEP) and insulin-degrading enzyme (IDE) expression. Our data indicate that TPPB could direct APP processing towards the non-amyloidogenic pathway by increasing alpha-secretase activity, and suggest its therapeutic potential in AD.

Neuroprotection by pergolide against levodopa-induced cytotoxicity of neural stem cells.

Neural stem cells (NSCs) are currently considered very hopeful candidates for cell replacement therapy in neurodegenerative pathologies such as Parkinson's disease (PD), but like embryonic neural tissue transplantation, levodopa medication may still be required to improve symptoms even after cell transplantation. The issues of whether levodopa induces cytotoxicity and apoptosis of NSCs following transplantation, as well as the means to prevent these processes from occurring remain to be elucidated. In this study, the possible cytotoxicity of levodopa at different doses on C17.2 neural stem cells and subsequent neuroprotection by pergolide were investigated. The cell viability was determined by the MTT assay. Cell proliferation was assayed by BrdU labeling, while apoptosis was detected by Annexin-V-FLUOS staining and flow cytometry. Levels of p53, Bax, Bcl-2, NFkB, cytochrome c, caspase-3 as well as cleavage of caspase-3 were measured by western blotting. We found levodopa induced a concentration- and time-dependent decrease in cell viability and proliferation. Apoptotic cells were observed at different stages, specifically 12 and 24 h following exposure to levodopa (200 microM). Elevated p53, Bax, cytochrome c, caspase-3 and active fragments of caspase-3 protein were observed in the cells exposed to levodopa. These alterations were partly inhibited by pergolide, a dopamine receptor agonist, while Bcl-2 and NFkB p65 levels remained constant at the various time-points in all the groups examined. These observations indicate that levodopa at high concentrations (> or = 200 microM) was neurotoxic to C17.2 neural stem cells via inhibition of DNA synthesis and cell proliferation. Activation of the mitochondria-dependent pathway and caspase-3 protease may contribute to the mechanism by which levodopa induces apoptosis. Pergolide, an anti-Parkinson drug, has a neuroprotective effect and partly blocks levodopa-induced cytotoxicity.