The Molecular Mechanism Of Learning And Memory Impairment Via DAPK1 During Aging

BackgroundAging is the phenomenon that the organism’s physiological and psychological adaptability to the environment is progressively reduced.There are two types of aging:physiological aging and pathological aging.Physiological senescence refers to the physiological degenerative process that occurs after the maturation period,and pathological senescence is due to senile changes caused by various external factors（including various diseases）.In the process of physiological aging,age-associated cognitive decline is an inevitable phenomenon that predisposes individuals for neurological and psychiatric disorders eventually affecting the quality of life.The decline of learning and memory is one of the manifestations of cognitive impairment in the aging brain,and scientists try to identify the key molecular switches that drive cognitive decline with advancing age.But the mechanism of aging-related deficit in learning and memory is not clear.In our early studies,DAPK1 was found to be involved in the impairment of spatial learning and memory in Alzheimer’s disease,but its molecular mechanism remains unclear.Caytaxin is known to be highly abundant in the hippocampus and is expressed in presynaptic area and involved in the synthesis and transport of neurotransmitters such as glutamate.During aging of the brain,whether DAPK1 is involved in the expression of Caytaxin in the hippocampus and whether Caytaxin is involved in the regulation of learning and memory is still unclear.Aim（1）To confirm whether DAPK1 is involved in learning and memory impairment during aging;（2）Molecular mechanism of DAPK1 involved in learning and memory impairment during aging.（3）Selectively knocking out the kinase domain of DAPK1 in excitatory neurons by means of genetic means to explore an effective strategy for anti-aging.MethodsWe selected mice with 4 months old（young group）and 18 months old（old group）according to the age-specific correspondence between mice and humans.We used C57BL/6J mice,DAPK1-KD^loxp/loxp and CaMK2α-cre mice as study subjects respectively.Through a series of behavioral experiments to detect spatial learning memory and fear memory of mice;Western blot to detect senescence of hippocampus in aging mouse（p16/p53 as the aging related indicators）and the activity of DAPK1（pMLC）,the activity of caspase 3（cleaved caspase 3）and the expression of Caytaxin;senescence model of cultured neurons in vitro was established by D-galactose,Caytaxin expression was detected by immunoblotting,and the distribution of Caytaxin in hippocampus of the aging mouse was observed by immunofluorescence.Using D-galactose to induce senescence in cultured primary neurons,adding the inhibitor of DAPK1--HS38 and the inhibitor of caspase3--z-VAD-fmk,expression of Caytaxin was detected by immunoblotting.Expression system was constructed in HEK293T cells and transfected with DAPK1 mutants（DAPK1K42A and DAPK1ΔCaM）,In addition,caspase3 inhibitor z-VAD-fmk was added,then detect the expression of Caytaxin by immunoblotting.The lentivirus was used to inhibit the expression of Caytaxin in the hippocampus.A series of behavioral experiments were used to detect spatial learning memory and fear memory in mice.Results1.A reduction of Caytaxin protein correlates with DAPK1 activation in aging processThe behaviors of 4/18-month-old mice were detected and we found that the spatial learning and memory of old mice was decreased,and the fear-related memory of hippocampus was decreased too.After detection of 4,8,12,18 months of C57 mouse hippocampal tissue,WB showed up-regulation of the activity of DAPK1 and Caspase3,Caytaxin expression was down-regulated,and senescence-related genes such as p53/p16 were up-regulated.While the change of DAPK1 expression was not obviously;The activity of DAPK1 and Caspase3 were up-regulated,Caytaxin expression was down-regulated,and expression of aging-related genes such as p53/p16 were up-regulated in the senescent cell model（D-gal）.2.Silencing Caytaxin reduces neuronal excitability and impairs learning and memoryOver-express or inhibit the expression of Caytaxin in primary neurons,detect mEPSC,synaptic dysfunction was found in group of inhibit the expression of Caytaxin,and silence the expression of Caytaxin in hippocampus,electrophysiological tests and behavioral tests showed that CA3-CA1 in long-term enhanced lesions in the group of inhibit the expression of Caytaxin,spatial learning and memory and hippocampal related fear memory were deficits.3.Activation of DAPK1 degrades Caytaxin via caspase-3In D-galactose-induced senescent cultured primary neurons,the addition of HS38or z-VAD-fmk can prevent the degradation of Caytaxin.In HEK293T cells,the expression of Caytaxin in activation of the DAPK1 group was recovered by addition of z-VAD-fmk.4.Inactivation of DAPK1 inhibits Caytaxin degradation in agingImmunoblot detection of hippocampal tissue in DAPK1-KD^-/-old mice showed that the activity of DAPK1 and Caspase3 were down-regulated,Caytaxin expression was up-regulated,and expression of aging-related genes such as p53/p16 were down-regulated compared to DAPK1-KD^loxp/loxp old mice.Immunohistochemistry showed that the projected fibers which expressed Caytaxin from DG to CA3 was significantly down-regulated in aged mice,and in DAPK1-KD^-/-old mice,the fibers which expressed Caytaxin in DG was significantly upregulated compared to DAPK1-KD^loxp/loxp old mice.5.Inhibition of Caytaxin degradation rescues learning and memory deficits in agingWe divided the animals into four groups,4-month-old DAPK1-KD^-/-mice and DAPK1-KD^loxp/loxp mice,18-month-old DAPK1-KD^-/-mice,and DAPK1-KD^loxp/loxpoxp/loxp mice.The water maze results showed that the spatial learning and memory of DAPK1-KD^-/-mice at 18 months was significant improved and recovered to a level similar to that of DAPK1-KD^loxp/loxp mice at 4 months.The results of contextual fear condition showed that knock out the kinase domain of DAPK1 can recsue the loss of fear memory.ConclusionIn aged mice,the hippocampus undergoes senescence,the activity of DAPK1 was increased,while the expression of Caytaxin was decreased,which results in impaired spatial learning and memory.Genetic deletion the kinase domain of DAPK1 in excitatory neurons can improve spatial learning and memory.