| BackgroundAlzheimer’s disease(AD) is a neurodegenerative disease with features of hidden onset and progressive course, which is also the main cause of dementia. As recent clinical trials of immunotherapy that targeting at amyloid-beta(Aβ) has turned out to be failure, the effective therapy for AD becomes an urgent mission, and researchers are aiming back at the AD pathogenesis again to search for a new therapeutic strategy. AD is pathologically characterized by intracellular neurofibrillary tangles(NFT), extracellular senile plaques(SP) and neuron loss, as well as synaptic dysfunction. There were many factors involved in the pathogenesis of AD, among which brain-derived neurotrophic factor(BDNF) might play a crucial role. Several studies had shown an abnormal level of BDNF in the brain and serum of patients with AD, which had been proposed to be the biomarker, however, this hypothesis needed to be confirmed. The proprotein, precursor of BDNF(brain-derived neurotrophic factor precursor, pro BDNF), has been recently found to exert important bioactivities in central neural system(CNS). Studies in vivo and in vitro have provided evidences that pro BDNF has differing or opposing effects against its mature form(m BDNF), and exerts p75 neurotrophin receptor(p75NTR)-dependent neurotoxic effects to elicit synapse dysfunction and neuronal death. Clinically, it is also proposed that the elevated ratio of pro BDNF/m BDNF caused by proteolytic disorder and other factors may make the toxic effects of pro BDNF prevail.Aβ aggregates and deposits and then forms the senile plaques around neural cells, which is the important feature of the AD pathology. It is believed that upregulated production and abnormal deposition of Aβ that responses to enhanced neurotoxicity could in turn exacerbate the imbalance of Aβ production and clearance, and finally form a vicious cycle. As Aβ deposits and aboundant plaques and tangles forms specifically in cortex and hippocampus, neurons lose and lead to cognitive impairment. Thus, how to maintain neuronal normal function or slow down the Aβ deposition remains to be a crucial point for the prevention and treatment of AD. It is unclear at present whether pro BDNF plays a role in Aβ metabolism and the progress of cognitive impairment. Therefore, we in the current study: 1. investigate the effect of pro BDNF on neuronal viability and axonal growth, as well as the possible mediating pathway of p75 NTR. 2. examine the effect of pro BDNF on Aβ metabolism in the AD mice brain, and on the progress of their cognitive impairment.Materials and Methods1. Newborn cortical neurons primarily cultured from eighteen-day transgenic fetal mice(p75NTR+/+ and p75NTR-/-, 129sv) were applied in the present study. The concentration of cleavage-resistant recombinant pro BDNF was set as 0, 10, 30, 100ng/ml, respectively. The viability of the newborn neurons was measured after 24 h by an approach of MTT method. Then the same measurement was conducted when co-cultured with p75/Fc at a variety of different concentrations(0, 10, 30, 100μg/ml) and pro BDNF(30ng/ml). Neuroblastoma cell line(SH-SY5Y) and primary cultured neurons(p75NTR+/+ and p75NTR-/-) under 30ng/ml pro BDNF were used to examine the axonal length by crystal violet staining and immunofluorescent staining at 72 h.2. Genetically modified mice of Alzheimer’s disease model(AD mice, APPswe PS1 d E9, 5-month old, female) were stereotaxically delivered into right lateral ventricle with recombinant adeno-associated virus-pro BDNF(AAV-pro BDNF), which was expected to express cleavage-resistant protein of pro BDNF. Enzyme-linked immunosorbent assay(ELISA) was conducted to test the pro BDNF level in the brain at week 4 and week 8 postoperatively, respectively.3. AD mice(5-month old, female) were employed to receive stereotaxically microinjection into right lateral ventricle, with normal saline(NS), recombinant adeno-associated virus-pro BDNF(AAV-pro BDNF) and mouse-derived monoclonal antibody to p75NTR(Anti-p75Ab), respectively. After a period of eight weeks, Morris water maze(MWM) was conducted to examine the memorial and learning function of the AD mice.4. ELISA was performed to quantify the Aβ level(including Aβ40 and Aβ42) in the mice brain. To visualize the Aβ depositon and SPs, immunohistochemistry(6E10) and Congo red staining was performed, after which the number of SPs in mice brains from each group was calculated.Result1. pro BDNF exerted dose-dependent suppressive effects on newborn neuronal viability with significance at 30ng/ml. While co-cultured with 30ng/ml pro BDNF, neurons revealed a dose-dependent attenuated death when treated with gradient-based p75/Fc, with significance at 100μg/ml. For axonal growth of SH-SY5 Y and primary cells, pro BDNF(30ng/ml) showed a suppressive effect, which, however, was neutralized by p75/Fc(100μg/ml). No effect was observed in p75NTR-/- neurons both on neural viability and axonal growth.2. AAV-pro BDNF was successfully delivered into the lateral ventricle confirmed by an approach of bromophenol blue microinjection which permeated in the ventricular system in AD mice brain. ELISA assay showed that pro BDNF was of a higher level at week 4 and week 8 after ventricular delivery of AAV-pro BDNF, compared with that as at week 0 as at the same period of the control group,indicating that ventricular delivery of AAV-pro BDNF could lead to a high expression in the brain.3. Results of MWM showed that AD mice from AAV-pro BDNF group had longer latency to hidden platform than those from control group with a significant difference(P﹤0.05), but not differed from those out of AAV-pro BDNF+Antip75 group(P﹥0.05). Similar result was revealed in spatial probe test about the time in the targeting quadrant.4. Aβ in the AD mice brain from AAV-pro BDNF group turned out a higher level than that from control group, with a significant difference(P ﹤ 0.05). Aβ level from AAV-pro BDNF+Antip75 group was higher than that from control, and less than AAV-pro BDNF group(P﹤0.05). The same results were found with Aβ plaque number among the three groups.Conclusion1. pro BDNF has suppressive effects on neuronal viability and axonal growth, which may mediated via p75 NTR pathway.2. pro BDNF may exacerbate Aβ production and depositon in AD mice brain, which is mediated by p75 NTR and responsible for the memory and learning impairment. |
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