Effect Of P75^NTR On Hippocampus Neurogenesis Of Alzhemer's Disease

BackgroundThe aging of the population is one of the greatest challenges for the world in the twenty-first century. This problem in China is more serious in the coming dacades. China has become an aged society since 1999 ,and the aged population(＞60years old) has reached 144 million by the end of last year. According to calculation based on the current prevalence data, there will be 0.45 billion elder people in China by 2050 and the proportion of aged people will exceed 30% by estimation. Alzheimer's disease (AD) is a neurodegenerative disease characterized histopathologically by beta-amyloid (Aβ)-containing senile plaque, neurofibrillary tangle and loss of neuron in certain brain areas, and clinically by progressive dementia. It is the most common cause of senile dementia of later life. Currently the incidence rate of AD among old people over 65 is 0.42 %-0.95% in china and the number of AD patients is about 3-4 million in China. AD is a major cause of disability and death in the elderly and results in heavy burden for the families and the society.Over the past 10 years , it has been demonstrated that Aβis the main pathogenic cause of AD by pathology, genetics and transgenic studies. The senile plaque(SP) mainly copmosed of Aβwhich is currently considered as the key factor in pathogenesis of AD. Aβbegins to accumulate and deposit in the brain long before the clinical symptoms onset of AD .Hippocampus plays an important role in learning and memory ,while hippocampus is the most vulnerable part of the brain affected by AD. The granule cells in the subergranular zone(SGZ) of dentate gyrus is one of the few areas in adult mammalian brain that neurogenesis exists all the life.Local environmental factors play important roles in neurogenesis. Neurogenesis in hippocampus has close relation with memory, and is affected by the local environmental factors.What the role of Aβgenerated in the early stages of AD brain on neurogenesis in hippocampus is unknown, which attracts a lot of attention in the field of neuroscience recent years. It is very important to clarify the neurogenesis changes in hippocampus in the early pathological stage of AD for the prevention and treatment ofAD.p75^NTR, the low affinity receptor of neurotrophins, together with Trk receptors, play critical roles in regulating the survival and death of nerve cells during the neuron development. Recently p75^NTR has identified as an Aβreceptor and it mediates neuronal death induced by Aβ. But whether p75^NTR is involved in neurogenesis in AD brain affected by Aβis not clear.With regard to this purpose, we established a p75^NTR-knockout AD mouse model, and the extracellular domain of p75^NTR (p75^NTR-ECD ) was injected into AD mouse hippocampus and added to neural stem cells (NSCs) cultured with Aβ. Then proliferation of NSCs was observed in vivo and in vitro. By such means we explored the role of p75 NTR in AD hippocampal neurogenesis.Methods1. Generation of animal model and genotypingAPPswe/PS1dE9 and p75^N32TR-/- mice were crossed and their progeny were genotyped. An APPswe/PS1dE9/p75^NTR+/- mouse was repeatedly backcrossed with a p75^NTR-/- mouse for 10 generations to produce APPswe/PS1dE9/p75^NTR-/- mice with nearly pure 129/Sv homozygosity. An APPswe/PS1dE9/p75^NTR-/- mouse was crossed with a 129/Sv mouse to obtain a male APPswe/PS1dE9/p75^NTR+/- mouse, which was next crossed with female 129/Sv mice and p75^NTR-/- mice to breed different kinds of genotype animals for the next experiments. Mice were maintained on ad libitum food and water with a 12-hour light/dark cycle.2. Effect of p75^NTRon neurogenesis of hippocampus in AD micep75 knockout AD mice,AD mice and wild-type mice at age of 3, 6, 9 months were selected for the experiment. Morris Water Maze was used to measure the cognitive function of the mice. Intraperitoneal injection of BrdU (dose 100ug/Kg weight, 2 / day, total of 3 days) and immunohistochemical detection of BrdU were used to observe the proliferation of hippocampal NSCs in different groups of mice.The initial differentiation of hippocampal NSCs was observed by double-labeling immunofluorescence (Brdu + DCXor Brdu + GFAP) and confocal laser scanning microscope . 3. NSCs proliferation in AD mice hippocampus after p75^NTR-ECD injection .Extracellular domain of p75^NTRis the binding site of Aβ. Twelve APPswe PS1dE9 ADmice at the age of 9-month-old were randomly divided into experimental and control groups with 6 mice in each group. The human p75^NTR-ECD was injected into the AD mouse hippocampus to block the endogenous p75^NTR activation,while human IgG was used as control .Intraperitoneal injection of BrdU and immunohistochemistry were used to examine the proliferation of NSCs in hippocampus .4. The effect of p75^NTR-ECD on Aβ1-42cultured NSCs from mouse hippocampus.Adult (2 months old) hippocampal NSCs were isolated and cultured.Aβ42 was addedinto the cultured cells at a concentration of 10um/1 and NSCs proliferation changes was observed.Then p75^NTR-ECD with different concentrations were added into the NSCscultured with Aβ1-42 to examine the role of p75^NTR in NSCs proliferation changes induced by Aβ1-42.Results1. Generation of p75^NTR knocked out AD mouse model.APPswe/PS1dE9 transgenic AD mice carry a C57BL/6 gene back ground, and p75 knockout mice (p75NTR/ExonⅢ-/- mice) has a 129 / sv background .The two kinds of animals were crossed according to the designed step in order to get the same 129/sv back ground. As a result,six genetic types including APP_swe^+/-p75^NTR-/-,APP_swe^+/-p75^NTR+/+,APP_swe^-/-p75^NTR-/-,APP_swe^-/-p75^NTR+/-,APP_swe^-/-p75^NTR+/+,APP_swe^+/-p75^NTR+/- were produced.The different genetype animals were in normal condition. APP_swe^+/-p75^NTR-/-,APP_swe^+/-p75^NTR+/+ , APP_swe^-/-p75^NTR+/+were selected as p75 konock-out AD mice, AD mice and wild mice.2. Neurogenesis changes in AD hippocampus and the role of p75^NTRThe neurogenesis of hippocampus decreased with age increased in each genotype,with more drop in AD brain. The results of double labeling showed that the proportion of NSCs differentiation in p75^NTR knockout AD and AD mice was much different from wild mice-the proportion of BrdU+DCX was lower,and the proportion of BrdU+GFAP was higher than wild mice especially at 6,9 months of age (p＜0.05). There were no significant differences between each groups at 3 months of age. The results of Morris Water Maze test showed that there were no significant differences between AD mice , p75^NTR knockout mice and wild mice in the ability of learning and memory.3. NSCs proliferation changes in AD mice hippocampus after p75^NTR-ECD injectionBrdU-positive cells in AD mouse hippocampus significantly increased afterp75^NTR-ECD hippcampus injection compared with control (p＜0.01).4. The effect of p75^NTR-ECD on mouse hippocampal NSCs cultured with Aβ.In vitro study we demonstrated that Aβ1-42 inhibited the proliferation of hippocampal NSCs, and p75^NTR-ECD blocked the inhibition effect of Aβ1-42 on the proliferation of hippocampal NSCs.Conclusions1. p75^NTR knockout AD mice were successfully constructed .2. p75^NTR plays an inhibitory role in the proliferation of hippocampus NSCs in AD mice.3. The inhibition of proliferation induced by Aβ_1-42 is mediated by p75^NTR.