Cdt2-controlled Cyclin-dependent Kinases Activity Regulates The Pathogenesis Of Alzheimer's Disease

Background:Alzheimer's disease(AD),which is characterized histopathologically by extracellular senile plaques made up of p-amyloid(Ap)and intracellular neurofibrillary tangles consisting of hyperphosphorylated tau.Due to a plenty of risk factors involving AD,its underlying mechanism remains unclear.The previous research has shown that,a high level of DTL expression is found in the constructed panoramic genetic network of AD patients and DTL is closely related to the pathological features of AD,supporting that DTL and its encoded protein Cdt2 may play an important role in the pathogenesis of AD.DTL is predicted as the top key driver of cell cycle-enriched module/subnetwork(CCM).DTL encoding protein Cdt2 is an E3-ubiquitination ligase,which can promote the ubiquitination and degradation of P21 and some other substrates.P21 is a cyclin-dependent kinase(CDK)inhibitor,which can inhibit the activity of this kinase by binding to CDK.Previous studies have shown that CDK1 and CDK5 among CDKs,are highly correlated with the pathogenesis of AD.As upstream kinases of tau protein,either CDK1 or CDK5 promotes tau hyperphosphorylation,resulting in neurofibrillary degeneration.The role of CDK5 on AD has been widely reported,which is associated with the dysfunction of neuronal migration and differentiation,and memory consolidation during AD process.In the present study,we try to investigate whether in the central nervous system,E3 ubiquitination ligase Cdt2 encoded by DTL,the key gene of cell cycle,can degrade its substrate P21 by promoting ubiquitmation,and release the inhibition of CDK family activities such as CDK1 or CDK5.In turn,overactivated CDKs promote the phosphorylation of tau and APP,leading to tau hyperphosphorylation and Ap toxicity.Objective:To explore the pathogenesis of Cdt2 involved in AD by regulating CDK activity.To clarify the close relationship between DTL and AD.Methods:RNA-seq profiles were generated from a large-scale of human AD and normal postmortem brain tissues in four brain regions,including parahippocampal gyrus(PHG),inferior frontal gyrus,superior temporal gyrus,and frontal pole(from the Mount Sinai NIH NeuroBioBank,MS-NBTR and the AlzData database)to examine DTL expression in AD.Empty vector or Cdt2 overexpressed plasmid was transfected into HEK293 or HEK293/tau cells instantaneously for 48h.Western blot was employed to detect the protein level of P21.CDK(1-5)was purified by specific antibodies separately and co-incubated with its substrate,Histone-1,and then the alteration of CDKs activity were assessed by the phosphorylation level of Histone-1.The above transfected cell samples were collected and RNA was extracted for link-RNA sequencing.The raw sequencing reads were aligned to human hg19 genome by using the STAR aligner(version 2.5.0b).Following read alignment,feature Counts was used to quantify the gene expression at the gene level based on Ensembl gene model GRCh37.70.CdtZ were instan.Otsuki,L.and A.H.Brand,Cell cycle heterogeneity directs the timing of neural stem cell taneously transfected into HEK293 or HEK293/tau cells for 48h and followed by treatment with CDK inhibitor Roscovitine(10?M)for 4h.The level of tau phosphotylation,soluble Ap42 and CDK1 or CDK5 binding to APP,APP phosphorylation and APP binding to BACE1 were mearsured by Western blot,ELISA and co-IP respectively.Quantitative analysis of gene expression was performed by link-RNA sequencing to determine the role of CDK in Cdt2-mediated AD pathology.The Cdt2-packaged adeno-associated virus was injected into the prefrontal cortex of C57 mice by stereotopic injection for one month.After Roscovitme(10mM)treatment,open field,novel object recognition and water maze tests were conducted to detect behavior changes such as mood,motor,learning and memory functions on animals.LTP was conducted by electrophysiology to detecte the synaptic plasticity of neurons.The morphological and numerical changes of dendritic spines were analyzed by golgi staining.Results:1.Data from four brain regions including parahippocampal gyrus(PHG),inferior frontal gyrus,superior temporal gyrus,and frontal pole were analyzed synthetically and result showed that DTL expression was significantly up-regulated in the brain of AD.2.In cells that overexpress Cdt2,Western Blot results showed that P21 protein level was significantly down-regulated compared with the control.CDK(1-5)activity detection results showed that the phosphorylation level of Histone-1 was increased to different degrees,that is,CdtZ overexpression could lead to the up-regulation of CDK1,CDK2,CDK3,CDK4 and CDK5 activity.The RNA sequencing results showed that compared with the control group,the CdtZ over-expressed group appeared 2713 up-regulated genes and 2760 down-regulated genes.Among the up-regulated differentially expressed genes(DEGs),there are three CDK genes,whereas among the downregulated DEGs,there is one CDK gene and one CDK inhibitor gene.A number of AD GW AS susceptibility genes were also among the DTL OE signatures,such as APOE,CASS4,CELF1,CLU and SORL1.3.After overexpression of Cdt2 in HEK293/tau cells and C57 mice,western blot showed that tau phosphorylation level in Ser396 obviously increased,ELISA result showed that the level of soluble Ap42 was significantly increased,Co-IP showed that the phosphorylation level of APP and the binding level of APP to BACE1,CDK1 and CDK5 were significantly up-regulated,compaired to the control.While after the treatment of Roscovitine,all the above changes were obviously recovered.4.Link-RNA sequencing analysis showed that more than 5400 DEGs regulated by DTL overexpression were restored after Roscovitine treatment to similar expression levels as in the control cells.5.In the C57 mice,open field,novel object recognition and water maze tests showed that CdtZ overexpression did not affect the emotional and motor abilities,and the cognitive function was significantly reduced on mice.Electrophysiological results showed that the synaptic plasticity of neurons in Cdt2 overexpressed mice was significantly reduced,and golgi staining results showed that morphology and number of dendritic spines of neurons in Cdt2 overexpressed mice were significantly reduced.After the treatment of Roscovitine,the above cognitive and nerve injuries were significantly reversed.6.We overlaid the DTL OE signature onto this cell cycle subnetwork and found a significant overlap.Conclusion:1.The up-regulation of DTL in human brain of AD,indicating that DTL was highly correlated with the pathogenesis of AD.2.Cdt2 promotes the ubiquitination degradation of P21,leading to the upregulation of CDKs activity.3.DTL overexpression can not only affect the expression of many CDK family proteins,but also affect the expression of many high-risk genes in AD,such as APOE,CASS4,CELF1,CLU and SORL1.4.Cdt2 upregulated CDKs activity leading to tau hyperphosphorylation and increased toxic A?.CDK Inhibitor reversed the cognitive dysfunction and gene expression changes caused by Cdt2 overexpression significantly.DTL/Cdt2 promotes P21 ubiquitination and degradation,which in turn releases CDKs activity in the AD brain.Especially activated CDK1 and CDK5,on the one hand,cause tau hyperphosphorylation and promote neurofibrillary degeneration,on the other hand,phosphorylate APP and promote Ap toxicity,trigger AD process.Background:Considering the multifactorial nature of Alzheimer's disease(AD),multi-target-directed ligands(MTDLs)might be synergistically beneficial against several etiopathogenic mechanisms of AD.The beneficial effects of the extracts from the medicinal plant Hypericum perforatum in AD animal models have been continuously reported,and these reports have led to the question of whether hyperforin-related PPAPs(Polycyclic Polyprenylated AcylPhloroglucinols)derivatives with unusual architectures,are also bioactive constituents and potential therapeutic agents for AD.Objective:Targeting BACE1 and PP2A activity regulation,to screen and study the anti-alzheimer activity and medicinal value of PPAPs extracts from the medicinal plant Hypericum perforatum.Methods:By using CCK8 kit to test the cell viability of N2a/APP and HEK293/tau cells treated with five PPAPs extracts(compound 1-5)from the medicinal plant Hypericum perforatum,to evaluate their cell toxicity and safety.BACE1 and PP2A activity testing kits were used to test and calculate the IC50 and EC50 of compound 1-5,to screen the effective PPAPs extracts for the further study.ELISA and Western blot were used to evaluate the effects of compounds on toxic A? production and tau phosphorylation in N2a/APP and HEK293/tau cells respectively.Brain stereotactic injection via implanted cannulas were used for the compounds administration,open field,novel object recognition and Morris water maze were used to evaluate the locomotor,learning and memory ability in 3 × Tg AD mice.Golgi staining were used to evaluate the number of dendritic spines.ELISA and Western blot were used to evaluate the effects of compounds on PP2A and BACE1 activity in vivoResults:Cell viability assays showed that compound 1-5 with no cytotoxicities at a concentration up to 8? M both in N2a/APP and HEK293/tau cells,indicating the safety of these compounds.Compounds 2 and 3 simultaneously displayed notable activation of PP2A in HEK293/tau cells(EC50:258.8 and 199 nM,respectively)and inhibition of BACE1 in N2a/APP cells(IC50:136.2 and 98.62 nM,respectively),which showed better activities than positive controls,SCR1693(a PP2A activator,EC50:413.9 nM)and LY2811376(a BACE1 inhibitor of Eli Lilly,IC50:260.2 nM).Compounds 2 and 3 effectively reduced tau phosphorylation level,p-cleavage of amyloid-p precursor protein(APP)and toxic Ap production,while compound 3 exhibited favorable effects.Furthermore,compound 3 shows notable therapeutic effects with respect to the reduction of pathological and cognitive impairments in 3 × Tg AD mice.ConCLUsion:Compound 3 represents the first multitargeted natural product that could activate PPZA and simultaneously inhibit BACE1,which highlights the potential of 3 as a promising lead compound and a versatile scaffold in AD drug development.