The Strategy Of Secreting Neuroprotective Peptide In Vivo And Providing Neuroprotection

The use of nerve growth factor proteins as neuroprotective therapeutics is limited by their hindered mobility through the blood-brain barrier. Recent researches suggested peptides are an attractive alternative. ADNP (activity-dependent neuroprotective protein), one of the factors within the neurotrophic milier produced by VIP (vasoactive intestinal peptide) stimulated astroglia, provide neuroprotection at subfemotomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), theβ-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. An 8-amino acid peptide derived from ADNP (NAP, NAPVSIPQ) has been reported protects cultured neurons from multiple neurotoxins. NAP has greater potency and broader effective concentration range (10-15 to 10-13 M) than ADNP in preventing neuronal death associated with tetrodotoxin treatment. The small molecular weight (<1000) of NAP enables it to cross the blood-brain barrier (BBB), however researches suggested that the halftime (or halflife period, half-life and so on) of NAP in the cortex is 15 min. This results in the need for multiple applications and high synthesis costs including solid-phase technology and HPLC purificaton, all of which restrict NAP application in clinical therapy. The current study focused on the molecular cloning and sequencing of NT4-NAP fusion gene, construction of the corresponding lentiviral vector and evaluation the bioactivity of NAP. We hope this study would further the research on the gene therapy of Alzheimer's disease intensively. Construction Lentiviral vector secreting NAP The cDNA encoding NT4-NAP was cloned by PCR technique. The NT4-NAP cDNA was subcloned into pLenti6/V5 TOPO by using DNA recombinant technique. It was introduced into 293FT cells by transient calcium phosphate transfection using four plasmids as well as the report virus rLent/EGFP. The NT4-NAP cDNA cloning and pLent/NT4-NAP construction were confirmed by DNA sequence analysis and corresponding restriction enzymes digestion. The transfected NIH 3T3 cells were found containing strong expression of EGFP and the titer of the rLent/EGFP was 2×106. After 72 h of transfection, the higher fluorescent intensity of EGFP was found in the primary neocorticall cells under confocal microscopy.APPsw transgeneic cellular model In order to provide the experimental basis in vitro,the transgeneic cellular model of AD was established with neuroblastoma cell strain(SK-N-SH). First, we constructed recombinant recombinant retrovirus (RV) carrier APP695.sw (HuAPP695SWE). The APP695.sw is the human amyloid precursor protein (APP) gene with Swedish mutation with the double mutation Lys670-Asn and Met671-Leu (K670N, M671L), which was found in a large Swedish family with early onset of AD. APP immunocytochemistry staining showed that mAPP expression significantly increase comparing with the control group(P<0.01). It exhibited cytotoxicity effects and induced cells apoptosis pathway in AD cellular model.rLent/NAP protected SK-N-SH cells against APPsw-induced death There were 5 groups of the neuroblastoma cells (SK-N-SH), the negative (or normal) control, the APPsw, the TOP (positive control), the NAP supernatant and the rLent (NAP protective group?). The recombinant retroviruses were transfected into SK-N-SH respectivly. The results were evaluated by morphological observation, MTT assay and flow cytometer methods. APPsw exhibited cytotoxicity effects and induced cells to die as apoptosis and necrosis manners in AD cellular model(compare with the control, P<0.01). The Lent/NT4-NAP significantly decreased neurotoxicity of Aβand the rate of apoptosis and necrosis in vitro(P<0.01).The overall mortality in the CHI (what's this?) and rLent-treated (is the NAP protective group?) groups was significantly reduced.rLent/NAP ameliorated injury response in closed head injury in mice The model of closed head injury was made by a metal rod hated the left hemispheres. Facilitated neurobehavioral recovery by NSS and brain edema was calculated as pertentage of H2O. Brain cortex histology changes in brain injury region of groups stained by HE stain. The overall mortality in the CHI and rLent-treated groups (the same problem as above, restate the group clearly) was significantly reduced. The NSS of CHI groups are lower than those of controls and the percentage of H2O also decreased in the rLent-treated mice. After 24 h of injury, cellular edema was observed. After 72 h, glial cells hyperplasia, satellite phenomena and inflammatory infiltration were observed by HE stain. There was significantly GFP fluorescence on the nasal epithelium, but no fluorescence on olfactory nerve and cereal.Conclusion: The recombinant rLent/NT4-NAP was successfully constructed. The results showed that the cellular model of AD was established as supposed. The rLent/NAP could infect and protect SK-N-SH cells in vitro against APPsw-induced death high performance. More importantly, the rLent/NT4-NAP could also efficietnly infect nasal epithelium in vivo. NAP provides significant neuroprotective faction from the complex array of injuries elicited by head trauma.In summary, we demonstrated that the strategy, peptide secretive expression by lentiviral vector, is feasible at both in vitro and in vivo studys. To further our study, more investigation in other animal model, i.e. transgenic mouse, should be carried out in the future. The achievements could assist us to use other neuroprotective peptides in chronic CNS diseases.