ILK Participate In The Enhancement Of Memory Caused By Environmental Enrichment And Rescue Memory Deficit In APP/PS1Mice

BackgroundIntegrin-linked kinase (ILK) was discovered in1996and it was a widely expressed serine/threonine protein kinase located in focal adhesions (FAs). ILK has been known playing a central role in transducing many of the biochemical signals such as growth, proliferation, survival, differentiation, migration, invasion and angiogenesis. And in cancer cells ILK expressed abnormal, which meant ILK maybe have a tight connection with the start and cure of the cancer. It has been known that ILK had a huge expression in many regions of the central nervous system(CNS), which means ILK may play an important role in CNS. The deletion of ILK in mouse brain resulted in cortical-lamination defects resembling cobblestone lissencephay. In neuronal-cellmlgration abnormalities. And the mutant hippocampus showed scalloping of the granule cell layer in the lower portion of the dentate gyrus with displacement of granule cells extending to the pial surface. Although it has been known ILK played an essential role in neurite outgrowth and dendrite initiation and growth as well as neural polarityf﹁we are still unknown about its function in high order functions such as learning and memory. Our research used various technologies and researched the molecular and behavior lays and found ILK participate in learning and memory in mouse and its mechanism and found its function in Alzheimer’s Disease. This research gave a theoretical basis to clinical treat.Objective1. The function of ILK in the enhancement of neurogenesis and memory caused by environmental enrichment.2. The mechanism of ILK which took part in environmental enrichment, neurogenesis and memory.3. In APP/PS1transgene mousef﹁increasing ILK expression could rescue its neurogenesis and memory impaired in APP/PS1transgene mouse.Method1. Environmental enrichment training and ILK protein, mRNA test1.1Environmental enrichment training Put mouse into the box having running wheel, ladder, tunnel, grounder etc. checking ILK protein level with time.1.2ILK protein and mRNA test Using western blot and RT-PCR technology to research ILK protein and mRNA level in mouse hippocampus.2. Lentivirus intervention affects neurogenesis and learning and memory.2.1ILK siRNA lentivirus and overexpression lentivirus Using PGW and PCCL transform plasmids make ILK siRNA lentivirus and overexpression lentivirus. Using microinjection inject lentivirus to DG and one month later, checking neurogenesis and memory.2.2the memory model Using contextual fear conditioning evaluate the fear memory and using water maze model evaluate the special memory.2.3Detection of neurogenesis BrdU staining, NeuN staining were used to analyze number of new born cells and survival of new born cells.3. Alzheimer’s Disease transgene model APP/PS1knocked in transgene mouse were bought to imitate human disease for experiment test.these mouse would appear many age pigments which include mainly A β and appear physiology phenomenon like human Alzheimer’s Disease.Results1. ILK improved significantly after environmental enrichment We took mouse hippocampus tissue once a week for four weeks after environmental enrichment. What we found was ILK protein and mRNA level began improved from the second week and getting the max level when third and fourth week.2. ILK played role in the enhancement of neurogenesis and memory caused by environmental enrichment2.1ILK affected memory enhanced by environmental enrichment One month after environmental enrichment, contextual fear memory and spatial memory was detected and found both of them improved. When test phase of water maze training, we found the latent time decreased and its time in quadrantal of testing phase increased, which means its spatial memory enhanced. At the same time, when testing the memory of contextual fear conditioning, we found its freezing time increased significantly. But after ILK interruption, this improvement was inhibited.2.2ILK affected neurogenesis enhanced by environmental enrichment One month after environmental enrichment, neurogenesis of DG in mouse improved obviously, which counted up the number of BrdU positive cells and BrdU. When we gave ILK siRNA lentivirus, detecting proliferation and survival and found the neurogenesis enhancement caused by environmental enrichment was reduced because of ILK interruption.3. The mechanism of ILK participating in neurogenesis and memory3.1Change of GSK3β in DG after environmental enrichment GSK3β, which is the downstream molecular of ILK, had a reduced activity when given environmental enrichment. After giving ILK siRNA lentivirus, the activity of GSK3β of the control mouse improved and it could not be reversed by environmental enrichment.3.2the GSK3β inhibitor SB216763could rescue the damaged neurogenesis and memory.We gave mouse SB216763for two weeks, detecting Morris water maze found decreasing ILK caused the latent time increased and its time in quadrantal of testing phase’decreased and in contextual fear conditioning we found its freezing time decreased significantly. When given SB216763, these impairment was rescued. We tested neurogenesis in DG and found out that reducing ILK could impair BrdU positive cells and BrdU NeuN positive cells in DG and given SB216763could rescue these impairment.4. Enhance ILK could rescue memory deficits in APP/PSltransgene mouse4.1ILK expression and memory reduced in APP/PS1transgene mouse We found the expression of ILK reduced in APP/PS1mouse and their BrdU NeuN positive cells reduced, which means its neurogenesis impairs in APP/PS1transgene mouse. We tested Morris water maze and found transgene mouse had a increased latent time in training phase and a decreased time in quadrantal of testing phase. At the same time, when we gave contextual fear conditioning, we found its freezing time reduced significantly.4.2ILK overexpression rescued neurogenesis and memory deficits in APP/PSltransgene mouseAfter we overexpressed ILK in APP/PS1mouse, there was a remission about their impaired neurogenesis and memory. We found ILK overexpression increased BrdU NeuN positive cells number in DG and found the latent time in the training phase decreased and the time in quadrantal of testing phase increased in water maze. When testing contextual fear conditioning, we found the freezing time increased after enhanced ILK.4.3Fluoxetine affected Alzheimer’s Disease After we gave fluoxetine to APP/PS1transgene mouse for two weeks, We found fluoxetine increased BrdU NeuN positive cells number in DG and found the latent time in the training phase decreased and the time in quadrantal of testing phase increased in water maze. When testing contextual fear conditioning; we found the freezing time increased after given fluoxetine.Conclusion1. ILK improved after environmental enrichment and it could impair the enhancement of neurogenesis and memory caused by environmental enrichment. These results told us ILK participated in environmental enrichment.2. ILK-GSK3(?) Asignal pathway participated in environmental enrichment and gave mouse SB216763could rescue the impairment caused by ILK interruption. 3. Overexpression ILK could remitt the impaired neurogenesis and memory in APP/PS1mouse, revealed neurogenesis had a tight connection with Alzheimer’s Disease. We gave fluoxetine which could stimulated neurogenesis could rescue the impairment in APP/PS1mouse, which offer a new way to treat Alzheimer’s Disease.Innovation1. We found out ILK could affected neurogenesis and memory enhanced by environmental enrichment.2. We found the ILK-GSK3(?) Apathway played an important role in neurogenesis and memory.3. We found ILK overexpression could resuce the damage in Alzheimer’s Disease.