Neural Differentiation Of Bone Marrow Mesenchymal Stem Cells From An Alzheimer’s Disease Mouse Model

Background&ObjectiveAlzheimer’s disease (AD) is the leading cause of dementia and is characterizedby the presence of large numbers of neuritic plaques, neurofibrillary tangles，degeneration of hippocampal pyramidal cells and neuron loss. The main clinicalfeature is a progressive decline in cognitive function and generally accompanied bypersonality changes, moreover, the incidence rates increased gradually with age. Thestatistics from Alzheimer’s Association show that:there are about5.2million patientswith Alzheimer’s disease in the United States, among these patients about200,000areyounger than65years and the remaining5million are over65years. With the adventof the baby boom in the next few decades will be about10million Americans developinto new AD. Currently, someone in America develops AD every68seconds. By2050, one new case of AD is expected to develop every33seconds In the UnitedStates, AD is the sixth leading cause of death, and fifth leading cause of death in theage over65years(including65years old). In the past decade, the proportion of deathsattributing to heart disease, stroke and prostate cancer, respectively, decreased by16%,23%and8%, while the proportion of deaths caused by AD has increased byabout68%. In addition to the patient himself,AD has brought serious health problems, decreased quality of life and ability to work, but also brought a severe Challenge totheir families, Apart from carefully taking care of the AD patients, the economic costsare too expensive to bear for many families. However the cause and the specificmechanisms of AD are not very clear, what‘s more there is no effective treatment canreverse or prevent the development of AD. Even given the treatment, the disease willgradually progress. So the new effective treatment methods are needed. Studies haveshown that bone marrow mesenchymal stem cells (MSCs) play an important role inthe treatment of some neurological diseases, especially neurodegenerative diseases.autologous MSCs, easily accessible and without immunological rejection, may beexcellent alternatives for cell transplantation. Notch signaling is a highly conservedsignaling pathway which directly controls stem cell survival, proliferation, anddifferentiation. Notch1signaling plays an important role in the maintenance of neuralstem cells in the undifferentiated state by inhibiting neuronal differentiation. APP andNotch are both hydrolyed by γ-secretase. However, it is still unknown whether thereis cross-talk between the APP and Notch1signaling pathways in the process of MSCneural differentiation. Autophagy plays a vital role in the development of AD, is anon-selective degradation process in which long-lived proteins and organelles aresequestered in autophagosomes and are degraded upon fusing with lysosomalcomponents. The possible therapeutic use of autologous MSCs in AD raises thequestion of whether their neuronally differentiated MSCs have deficiencies inautophagy, which would offset the benefits of autologous MSC transplantation.Therefore,it’s still unknown whether it’s feasible and effective for AD bytransplanting autologous MSCs.MSCs are spindle-shaped cells discovered by Friedenste in cell culture andpresent in bone marrow tissue. MSCs are not very many, but plays an important rolein the survival and maintenance of hematopoietic stem cell. MSCs have a highcapacity in self-renewal and proliferation, and have the potential to differentiate intovarious cell types such as osteoblasts, chondrocytes,cardiocyes, adipocytes,neurons.Because of these characteristics，MSCs can be widely used in the diagnosis andtreatment of many diseases, such as neurodegenerative diseases. However, there aremany questions in the process of differentiation,remain to be explored. In this study, we estimate the neural differentiation efficiency of bone marrowmesenchymal stem cells (MSCs) derived from amyloid precursor protein (APP)transgenic mice and investigate their correlation with Notch1signaling and theautophagy activity during the differentiation.Materials and MethodologyThe MSCs were divided into2groups: the APP group (MSCs from APPtransgenic mice) and the WT group (MSCs from wild-type mice). MSCs were treatedwith β-mercaptoethanol (β-ME) as an inducer to differentiate into neurons. The levelsof Aβ40and Aβ42were measured using human amyloid β peptide Aβ40and Aβ42enzyme-linked immunosorbent assay kits. The expression of neural cell specificmarkers, neuron-specific enolase (NSE) and microtubulin-associated protein2(MAP-2) were measured by immunocytochemistry and western blot. the expressionlevels of Notch-1, Notch-1intracellular domain (NICD),Hes5,LC3and P62(aselective substrate of autophagy)were detected by western blot.Results1. our data showed that there were higer expressions of the neuron-specificmarkers,NSE and MAP-2, in the APP group than in the WT group（P <0.05）.2. The results showed that after induction, the level of Aβ40in the inductionmedium of APP group was (23.2±3.5) pg/mL and the level of Aβ42in the inductionmedium of APP group was (3.3±0.6) pg/mL. Aβ40and Aβ42were not detected inthe induction medium of WT group. there were higher expression of Aβin MSCs fromAPP group3. The results showed that the levels of Notch-1, NICD and Hes5in the APPMSCs were lower than WT MSCs before induction (P<0.05), the expression levels ofNotch-1, NICD and Hes5significantly decreased after5d induction in both groups,and of which the expression levels of Notch-1, NICD and Hes5were still lower in thedifferentiated APP MSCs than differentiated WT MSCs（P <0.05）. These resultsindicated that Notch signaling was inhibited during MSC differentiation. 4. Our results showed an accumulation of fluorescent LC3-positive dots in thedifferentiated APP MSCs and higher expression of LC3Ⅱ in the differentiated APPMSCs than differentiated WT MSCs. we further detected the level of P62, found P62didn’t decrease in the differentiated APP MSCs during the autophagy induction, andhigher than WT MSCs, indicating partial block of autophagosomal maturation in thedifferentiated APP MSCs.ConclusionOverexpression of APP might contribute to the high neural differentiationcapacity of MSCs by inhibiting Notch1signaling pathway in vitro,however,autophagy is impaired in the differentiated MSCs from APP transgenic mice.