Study On The Role Of TAG1-APP Signalling Pathway In Adult Neurogenesis

Objective(s):Alzheimer disease,commonly known as Dementia,was first discovered by German surgon Alzheimer.It’s a progressive and fatal neurodegenerative disease.Since the discovery of the disease in 1906,its precise pathogenesis has not been completely defined,there is also no an accurate method for early diagnosis and effective treatment.In recent years,a number of studies have confirmed that neurogenesis occurs throughout the central nervous system of adult mammals,which provides hope for the treatment of brain injuries and neurodegenerative diseases such as AD.In addition,increasing studies on aging and neurodegenerative diseases such as AD have found that neurogenesis is reduced and that key molecules and signaling pathways associated with pathological processes of AD also affect neurogenesis.It is thus clear that the decrease of neurogenesis might be closely related to the pathogenesis of AD.Our previous studies have demonstrated that the TAG1-APP signaling pathway negatively regulates neurogenesis of embryonic neurogenic ventricular zone by Fe65,but its respective roles in adult neurogenesis and neurodegenerative diseases such as AD are not clear.Hereby,this study will further investigate the role of TAG1-APP signaling pathway on adult neurogenesis,in order to provide an important basis for understanding the pathogenesis of AD and exploring new targets for AD therapy.Extensive papers report that there is persistent neurogenesis mainly in the SVZ-RMS-OB system and the hippocampal DG system of the adult mammalian brain.Two places of expressing neural stem cells in the two systems are described:the subventricular zone(SVZ)of the lateral ventricles and the subgranular zone(SGZ)of the dentate gyrus of the hippocampus.In the SVZ and SGZ,the neural stem cells divide into either a directional progenitor cells or neural precursor cells by asymmetric division.These new neural precursor cells can gradually migrate into the functional region,where they differentiate into mature neurons,and establish synaptic connections with other neurons,and integrated into the existing neural networks to play a role.These process is called neurogenesis.Due to anatomical differences the NSCs of the SVZ neeed long distance migration to the functional areas in OB granule cell layer,and the NSCs located in the SGZ area can be migrated to the function area in the hippocampal granule cell layer by short distance,where they differentiate into mature granular neurons.According to reports by some papers,the SVZ region can fastly produce more neurons than the SGZ region,and the new neurons were produced in the SVZ and SGZ regions play an important role in learning and memory associate with olfactory bulb and hippocampus dependent.The extent of neurogenesis is closely related to the proliferation of NSCs and NPCs,migration and differentiation of NPCs and the survival of new neurons in the critical brain regions where is related to adult neurogenesis.In order to extend the previous research work of this research group,This project focuses on the study of adult neurogenesis in SVZ region to investigate the role of TAG1-APP signaling pathway in the differentiation of adult neurogenic SVZ cells into neurons.While neurogenesis in another important system,will be performed in subsequent studies.Here,this research is divided into two parts to solve the following problems:Part 1 The study of the expression of TAG1 and APP in two critical brain regions of adult neurogenesis1.Western Blot were used to detect whether TAG1 and APP were expressed in SVZ and DG regions of adult mice?2.Double-staining by Immunofluorescence was used to detect whether TAG1 and APP were expressed in SVZ and DG regions of adult mice and whether they could be co-localized with NSCs and NPCs markers involved in neurogenesis?Part 2 The study of TAG1-APP signaling pathway in the differentiation of neonatal cells derived from the subventricular zone1.Does TAG1 affect neurogenesis in the SVZ region of adult mice?2.Does APP affect neurogenesis in the SVZ region of adult mice?3.Does the interaction of TAG 1 and APP affect neurogenesis in the SVZ region of adult mice?Methods:(一)8-10 week-old C57BL/6J male mice were used to study.1.Mice were decapitated and the brains were quickly separated.And placing the brain tissue in a 4℃ HBSS solution.Then,coronal cutting the brains using a vibrating microtome and the brain slices were collected at 4℃ HBSS.Next,the brain tissue of the SVZ region and the DG region was isolated by using micro forceps and observing under an anatomical microscope.Western Blot were used to detect the expression of TAG1 and APP in these two brain regions of adult neurogenesis;2.Mice were deeply anesthetized and perfused.The brains were removed and embedded with OCT.Serial coronal frozen sections of SVZ and hippocampus DG were obtained on a confocal microscope to carry on immunofluorescence staining of two groups that the one is TAG1 and NSCs and NPCs maker and the other is APP and NSCs and NPCs maker,such as Nestin,GFAP,SOX2,EGFR,PSA-NCAM and DCX.(二)Male mice of APP knockout and TAG1 knockout,APP and TAG1 double knockout and wild type with litter born at the age of 8-10 weeks were used to study.1.Doing genotyping by using PCR method to get APP-KO,TAG1-KO,APP and TAG1 double KO and littermate WT mice.2.Brdu(100mg/kg body mass)was injected intraperitoneally three times daily for consecutive 2 days.Mice were then kept for 4 weeks before perfusion.3.Mice were deeply anesthetized and perfused.The brains were removed and embedded with OCT.Non serial coronal frozen sections of 16-um sections with 240-um interval for OB、SVZ and hippocampus DG were collected.These sections(approximately three sections)were attached to a glass slide for staining.For OB sections were double stained with antibodies against Brdu and NeuN.Positive cells on all sections on the same slide co-immunostaining of Brdu and NeuN were observed under a confocal microscope.The number of Brdu+ and NeuN+ were counted.Results:1.Western Blot showed that TAG1 and APP expressed the two Brain regions related to adult neurogenesis.2.Immunofluorescence double staining showed that TAG1 and APP were coexpressed in the two brain regions of adult neurogenesis,and TAG1 and APP could be colocalized with NSCs and NPCs markers in SVZ and DG regions,respectively;3.Results of neurogenesis in adult SVZ-RMS-OB systems are presented as follows:In the OB region,most of BrdU positive cells were distributed in granular cell layer and most of them were colocalized with NeuN.Both BrdU and NeuN positive cells were considered as new neurons migrated from SVZ in OB.Compared with TAG1 wild type mice,new neurons increased in TAG1 knockout mice.According to the counting of new neurons in OB,the number of new neurons in TAG1 KO was 152.17 ± 23.48 and more than that of WT(109.50±23.46);Compared with littermate WT,TAG1 KO increased significantly(P=0.003).Compared with APP wild type mice,new neurons increased in APP knockout mice.According to the counting of new neurons in OB,the number of new neurons in APP KO was 208.50±57.98 and more than littermate WT which number was 175.42±31.45(P=0.011);Compared with wild type mice,new neurons increased in TAG1/APP double knockout mice.According to the counting of new neurons in OB,the number of new neurons in TAG 1/APP double KO was 210.14±37.45 and more than age paired WT which number was 100.59±24.95(P=0.028).Conclusion(s):1.TAG1 and APP can coexpress in two critical brain regions of adult neurogenesis.2.Both TAG1 and APP can negatively regulate adult neurogenesis in the SVZ region;The TAG 1-APP signaling pathway inhibits neurogenesis by reducing the number of the differentiation of neonatal cells derived from the subventricular zone.