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.

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.

The aggregation of amyloid-β (Aβ) peptides into oligomers and fibrils is a key pathological feature of Alzheimer's disease (AD). An increasing amount of evidence suggests that oligomeric Aβ 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 Aβ species influence the function of DAPK1, and whether this may further affect the function and phosphorylation of tau in neurons. Herein, we demonstrated that Aβ aggregates (both oligomers and fibrils) prepared from synthetic Aβ42 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 Aβ aggregate-induced cell death and tau dysregulation, corroborating the critical role of DAPK1 in mediating Aβ aggregation-induced neuronal damage. Our study suggests a mechanistic link between Aβ oligomerization and tau hyperphosphorylation mediated by DAPK1, and supports the role of DAPK1 as a promising target for early intervention in AD.

Epilepsy is a chronic encephalopathy and one of the most common neurological disorders. Deathassociated 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.

The neuropathology of Alzheimer’s disease (AD) is characterized by intracellular aggregation of hyperphosphorylated tau and extracellular accumulation of beta-amyloid (Aβ). 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 Aβ40 and Aβ42. Furthermore, restoring DAPK1 expression with miR-143-3p antagonized the effects of miR-143-3p in attenuating tau hyperphosphorylation and Aβ 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.

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.

Dysregulation of microRNAs has been implicated in diverse diseases, including Alzheimer's disease (AD). 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 (Aβ) 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, Aβ secretion, and neuronal cell death by directly targeting DAPK1, providing an attractive therapeutic option for AD.

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.