MiR-191-5p Attenuates Tau Phosphorylation, Aß Generation, and Neuronal Cell Death by Regulating Death-Associated Protein Kinase 1.

Dysregulation of microRNAs has been implicated in diverse diseases, including Alzheimer's disease (AD). MiR-191-5p in plasma/serum has been identified as a novel and promising noninvasive diagnostic biomarker for AD. However, whether miR-191-5p is involved in AD pathogenesis is largely unknown, and its levels in human AD brains are undetermined. Herein, we demonstrated that miR-191-5p downregulated tau phosphorylation at multiple AD-related sites and promoted neurite outgrowth using immunoblotting, immunofluorescence, and neurite outgrowth assays. Moreover, immunoblotting and enzyme-linked immunosorbent assays indicated that miR-191-5p decreased amyloid precursor protein phosphorylation levels and beta-amyloid (Aß) generation. Furthermore, miR-191-5p reduced ceramide-induced neuronal cell death analyzed by trypan blue staining, the in situ cell death detection kit, and Annexin V-FITC/PI flow cytometry. Next, we verified that death-associated protein kinase 1 (DAPK1) was a direct target of miR-191-5p through the dual luciferase reporter assay and confirmed that the effects of miR-191-5p were antagonized by restoration of DAPK1 expression. Finally, the hippocampal miR-191-5p level was found to be decreased in humans with AD compared with controls and was inversely correlated with the DAPK1 expression level. Collectively, these findings suggest that miR-191-5p might exert inhibitory effects on tau phosphorylation, Aß secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

miR-143-3p Inhibits Aberrant Tau Phosphorylation and Amyloidogenic Processing of APP by Directly Targeting DAPK1 in Alzheimer's Disease.

The neuropathology of Alzheimer's disease (AD) is characterized by intracellular aggregation of hyperphosphorylated tau and extracellular accumulation of beta-amyloid (Aß). Death-associated protein kinase 1 (DAPK1), as a novel therapeutic target, shows promise for the treatment of human AD, but the regulatory mechanisms of DAPK1 expression in AD remain unclear. In this study, we identified miR-143-3p as a promising candidate for targeting DAPK1. miR-143-3p directly bound to the 3' untranslated region of human DAPK1 mRNA and inhibited its translation. miR-143-3p decreased tau phosphorylation and promoted neurite outgrowth and microtubule assembly. Moreover, miR-143-3p attenuated amyloid precursor protein (APP) phosphorylation and reduced the generation of Aß40 and Aß42. Furthermore, restoring DAPK1 expression with miR-143-3p antagonized the effects of miR-143-3p in attenuating tau hyperphosphorylation and Aß production. In addition, the miR-143-3p levels were downregulated and correlated inversely with the expression of DAPK1 in the hippocampus of AD patients. Our results suggest that miR-143-3p might play critical roles in regulating both aberrant tau phosphorylation and amyloidogenic processing of APP by targeting DAPK1 and thus offer a potential novel therapeutic strategy for AD.

Blocking ERK-DAPK1 Axis Attenuates Glutamate Excitotoxicity in Epilepsy.

Glutamate excitotoxicity induces neuronal cell death during epileptic seizures. Death-associated protein kinase 1 (DAPK1) expression is highly increased in the brains of epilepsy patients; however, the underlying mechanisms by which DAPK1 influences neuronal injury and its therapeutic effect on glutamate excitotoxicity have not been determined. We assessed multiple electroencephalograms and seizure grades and performed biochemical and cell death analyses with cellular and animal models. We applied small molecules and peptides and knocked out and mutated genes to evaluate the therapeutic efficacy of kainic acid (KA), an analog of glutamate-induced neuronal damage. KA administration increased DAPK1 activity by promoting its phosphorylation by activated extracellular signal-regulated kinase (ERK). DAPK1 activation increased seizure severity and neuronal cell death in mice. Selective ERK antagonist treatment, DAPK1 gene ablation, and uncoupling of DAPK1 and ERK peptides led to potent anti-seizure and anti-apoptotic effects in vitro and in vivo. Moreover, a DAPK1 phosphorylation-deficient mutant alleviated glutamate-induced neuronal apoptosis. These results provide novel insight into the pathogenesis of epilepsy and indicate that targeting DAPK1 may be a potential therapeutic strategy for treating epilepsy.

Melatonin ameliorates tau-related pathology via the miR-504-3p and CDK5 axis in Alzheimer's disease.

BACKGROUND: Intracellular accumulation of the microtubule-associated protein tau and its hyperphosphorylated forms is a key neuropathological feature of Alzheimer's disease (AD). Melatonin has been shown to prevent tau hyperphosphorylation in cellular and animal models. However, the molecular mechanisms by which melatonin attenuates tau hyperphosphorylation and tau-related pathologies are not fully understood. METHODS: Immunofluorescence, immunoblotting analysis and thioflavin-S staining were employed to examine the effects of early and late treatment of melatonin on tau-related pathology in hTau mice, in which nonmutated human tau is overexpressed on a mouse tau knockout background. High-throughput microRNA (miRNA) sequencing, quantitative RT-PCR, luciferase reporter assay and immunoblotting analysis were performed to determine the molecular mechanism. RESULTS: We found that both early and late treatment of melatonin efficiently decreased the phosphorylation of soluble and insoluble tau at sites related to AD. Moreover, melatonin significantly reduced the number of neurofibrillary tangles (NFTs) and attenuated neuronal loss in the cortex and hippocampus. Furthermore, using miRNA microarray analysis, we found that miR-504-3p expression was upregulated by melatonin in the hTau mice. The administration of miR-504-3p mimics dramatically decreased tau phosphorylation by targeting p39, an activator of the well-known tau kinase cyclin-dependent kinase 5 (CDK5). Compared with miR-504-3p mimics alone, co-treatment with miR-504-3p mimics and p39 failed to reduce tau hyperphosphorylation. CONCLUSIONS: Our results suggest for the first time that melatonin alleviates tau-related pathologies through upregulation of miR-504-3p expression by targeting the p39/CDK5 axis and provide novel insights into AD treatment strategies.

Death-associated protein kinase 1 mediates Aß42 aggregation-induced neuronal apoptosis and tau dysregulation in Alzheimer's disease.

The aggregation of amyloid-ß (Aß) peptides into oligomers and fibrils is a key pathological feature of Alzheimer's disease (AD). An increasing amount of evidence suggests that oligomeric Aß might be the major culprit responsible for various neuropathological changes in AD. Death-associated protein kinase 1 (DAPK1) is abnormally elevated in brains of AD patients and plays an important role in modulating tau homeostasis by regulating prolyl isomerase Pin1 phosphorylation. However, it remains elusive whether and how Aß species influence the function of DAPK1, and whether this may further affect the function and phosphorylation of tau in neurons. Herein, we demonstrated that Aß aggregates (both oligomers and fibrils) prepared from synthetic Aß42 peptides were able to upregulate DAPK1 protein levels and thereby its function through heat shock protein 90 (HSP90)-mediated protein stabilization. DAPK1 activation not only caused neuronal apoptosis, but also phosphorylated Pin1 at the Ser71 residue, leading to tau accumulation and phosphorylation at multiple AD-related sites in primary neurons. Both DAPK1 knockout (KO) and the application of a specific DAPK1 inhibitor could effectively protect primary neurons against Aß aggregate-induced cell death and tau dysregulation, corroborating the critical role of DAPK1 in mediating Aß aggregation-induced neuronal damage. Our study suggests a mechanistic link between Aß oligomerization and tau hyperphosphorylation mediated by DAPK1, and supports the role of DAPK1 as a promising target for early intervention in AD.

Inhibition of Death-associated Protein Kinase 1 protects against Epileptic Seizures in mice.

Epilepsy is a chronic encephalopathy and one of the most common neurological disorders. Death-associated protein kinase 1 (DAPK1) expression has been shown to be upregulated in the brains of human epilepsy patients compared with those of normal subjects. However, little is known about the impact of DAPK1 on epileptic seizure conditions. In this study, we aim to clarify whether and how DAPK1 is regulated in epilepsy and whether targeting DAPK1 expression or activity has a protective effect against epilepsy using seizure animal models. Here, we found that cortical and hippocampal DAPK1 activity but not DAPK1 expression was increased immediately after convulsive pentylenetetrazol (PTZ) exposure in mice. However, DAPK1 overexpression was found after chronic low-dose PTZ insults during the kindling paradigm. The suppression of DAPK1 expression by genetic knockout significantly reduced PTZ-induced seizure phenotypes and the development of kindled seizures. Moreover, pharmacological inhibition of DAPK1 activity exerted rapid antiepileptic effects in both acute and chronic epilepsy mouse models. Mechanistically, PTZ stimulated the phosphorylation of NR2B through DAPK1 activation. Combined together, these results suggest that DAPK1 regulation is a novel mechanism for the control of both acute and chronic epilepsy and provide new therapeutic strategies for the treatment of human epilepsy.

Ube2b-dependent degradation of DNMT3a relieves a transcriptional brake on opiate-induced synaptic and behavioral plasticity.

Compelling evidence suggests that synaptic structural plasticity, driven by remodeling of the actin cytoskeleton, underlies addictive drugs-induced long-lasting behavioral plasticity. However, the signaling mechanisms leading to actin cytoskeleton remodeling remain poorly defined. DNA methylation is a critical mechanism used to control activity-dependent gene expression essential for long-lasting synaptic plasticity. Here, we provide evidence that DNA methyltransferase DNMT3a is degraded by the E2 ubiquitin-conjugating enzyme Ube2b-mediated ubiquitination in dorsal hippocampus (DH) of rats that repeatedly self-administrated heroin. DNMT3a degradation leads to demethylation in CaMKK1 gene promotor, thereby facilitating CaMKK1 expression and consequent activation of its downstream target CaMKIα, an essential regulator of spinogenesis. CaMKK1/CaMKIα signaling regulates actin cytoskeleton remodeling in the DH and behavioral plasticity by activation of Rac1 via acting Rac guanine-nucleotide-exchange factor ßPIX. These data suggest that Ube2b-dependent degradation of DNMT3a relieves a transcriptional brake on CaMKK1 gene and thus activates CaMKK1/CaMKIα/ßPIX/Rac1 cascade, leading to drug use-induced actin polymerization and behavior plasticity.

Programmed Cell Death Protein 1 Blockade Reduces Glycogen Synthase Kinase 3ß Activity and Tau Hyperphosphorylation in Alzheimer's Disease Mouse Models.

Alzheimer's disease (AD) is a central nervous system degenerative disease, with no effective treatment to date. Administration of immune checkpoint inhibitors significantly reduces neuronal damage and tau hyperphosphorylation in AD, but the specific mechanism is unclear. Here, we found that programmed cell death-receptor 1 (PD1) and its ligand PDL1 were induced by an intracerebroventricular injection of amyloid-ß; they were significantly upregulated in the brains of APP/PS1, 5×FAD mice and in SH-SY5Y-APP cell line compared with control. The PD1 and PDL1 levels positively correlated with the glycogen synthase kinase 3 beta (GSK3ß) activity in various AD mouse models, and the PDL1-GSK3ß immune complex was found in the brain. The application of PD1-blocking antibody reduced tau hyperphosphorylation and GSK3ß activity and prevented memory impairments. Mechanistically, we identified PD1 as a critical regulator of GSK3ß activity. These results suggest that the immune regulation of the PD1/PDL1 axis is closely involved in AD.

Melatonin directly binds and inhibits death-associated protein kinase 1 function in Alzheimer's disease.

Death-associated protein kinase 1 (DAPK1) is upregulated in the brains of human Alzheimer's disease (AD) patients compared with normal subjects, and aberrant DAPK1 regulation is implicated in the development of AD. However, little is known about whether and how DAPK1 function is regulated in AD. Here, we identified melatonin as a critical regulator of DAPK1 levels and function. Melatonin significantly decreases DAPK1 expression in a post-transcriptional manner in neuronal cell lines and mouse primary cortical neurons. Moreover, melatonin directly binds to DAPK1 and promotes its ubiquitination, resulting in increased DAPK1 protein degradation through a proteasome-dependent pathway. Furthermore, in tau-overexpressing mouse brain slices, melatonin treatment and the inhibition of DAPK1 kinase activity synergistically decrease tau phosphorylation at multiple sites related to AD. In addition, melatonin and DAPK1 inhibitor dramatically accelerate neurite outgrowth and increase the assembly of microtubules. Mechanistically, melatonin-mediated DAPK1 degradation increases the activity of Pin1, a prolyl isomerase known to play a protective role against tau hyperphosphorylation and tau-related pathologies. Finally, elevated DAPK1 expression shows a strong correlation with the decrease in melatonin levels in human AD brains. Combined, these results suggest that DAPK1 regulation by melatonin is a novel mechanism that controls tau phosphorylation and function and offers new therapeutic options for treating human AD.

The Genetics of Alzheimer's Disease in the Chinese Population.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment. In China, the number of AD patients is growing rapidly, which poses a considerable burden on society and families. In recent years, through the advancement of genome-wide association studies, second-generation gene sequencing technology, and their application in AD genetic research, more genetic loci associated with the risk for AD have been discovered, including KCNJ15, TREM2, and GCH1, which provides new ideas for the etiology and treatment of AD. This review summarizes three early-onset AD causative genes (APP, PSEN1, and PSEN2) and some late-onset AD susceptibility genes and their mutation sites newly discovered in China, and briefly introduces the potential mechanisms of these genetic susceptibilities in the pathogenesis of AD, which would help in understanding the genetic mechanisms underlying this devastating disease.

Dissociative role for dorsal hippocampus in mediating heroin self-administration and relapse through CDK5 and RhoB signaling revealed by proteomic analysis.

Addiction is characterized by drug craving, compulsive drug taking and relapse, which is attributed to aberrant neuroadaptation in brain regions implicated in drug addiction, induced by changes in gene and protein expression in these regions after chronic drug exposure. Accumulating evidence suggests that the dorsal hippocampus (DH) plays an important role in mediating drug-seeking and drug-taking behavior and relapse. However, the molecular mechanisms underlying these effects of the DH are unclear. In the present study, we employed a label-free quantitative proteomic approach to analyze the proteins altered in the DH of heroin self-administering rats. A total of 4015 proteins were quantified with high confidence, and 361 proteins showed significant differences compared with the saline control group. Among them, cyclin-dependent kinase 5 (CDK5) and ras homolog family member B (RhoB) were up-regulated in rats with a history of extended access to heroin. Functionally, inhibition of CDK5 in the DH enhanced heroin self-administration, indicating that CDK5 signaling in the DH acts as a homeostatic compensatory mechanism to limit heroin-taking behavior, whereas blockade of the Rho-Rho kinase (ROCK) pathway attenuated context-induced heroin relapse, indicating that RhoB signaling in the DH is required for the retrieval (recall) of addiction memory. Our findings suggest that manipulation of CDK5 signaling in the DH may be essential in determining vulnerability to opiate taking, whereas manipulation of RhoB signaling in the DH may be essential in determining vulnerability to relapse. Overall, the present study suggests that the DH can exert dissociative effects on heroin addiction through CDK5 and RhoB signaling.