Ablation of Death-Associated Protein Kinase 1 Changes the Transcriptomic Profile and Alters Neural-Related Pathways in the Brain.

Death-associated protein kinase 1 (DAPK1), a Ca2+/calmodulin-dependent serine/threonine kinase, mediates various neuronal functions, including cell death. Abnormal upregulation of DAPK1 is observed in human patients with neurological diseases, such as Alzheimer's disease (AD) and epilepsy. Ablation of DAPK1 expression and suppression of DAPK1 activity attenuates neuropathology and behavior impairments. However, whether DAPK1 regulates gene expression in the brain, and whether its gene profile is implicated in neuronal disorders, remains elusive. To reveal the function and pathogenic role of DAPK1 in neurological diseases in the brain, differential transcriptional profiling was performed in the brains of DAPK1 knockout (DAPK1-KO) mice compared with those of wild-type (WT) mice by RNA sequencing. We showed significantly altered genes in the cerebral cortex, hippocampus, brain stem, and cerebellum of both male and female DAPK1-KO mice compared to those in WT mice, respectively. The genes are implicated in multiple neural-related pathways, including: AD, Parkinson's disease (PD), Huntington's disease (HD), neurodegeneration, glutamatergic synapse, and GABAergic synapse pathways. Moreover, our findings imply that the potassium voltage-gated channel subfamily A member 1 (Kcna1) may be involved in the modulation of DAPK1 in epilepsy. Our study provides insight into the pathological role of DAPK1 in the regulatory networks in the brain and new therapeutic strategies for the treatment of neurological diseases.

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.

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.

Blockade of Fgfr1 with PD166866 Protects Cartilage from the Catabolic Effects Induced by Interleukin-1ß: A Genome-Wide Expression Profiles Analysis.

OBJECTIVE: Previously we showed that genetic deletion of Fgfr1 in chondrocytes protected mice from progression of osteoarthritis (OA). The aim of this study is to evaluate the effect of PD166866, a potent selective inhibitor of Fgfr1, on cartilage degeneration induced by interleukin-1ß (IL-1ß) and to clarify underlying global gene expression pattern. DESIGN: Cartilage explants and primary rat chondrocytes were stimulated with IL-1ß to establish an inflammatory OA in vitro model. The effects of PD166866 were determined by measuring the release of glycosaminoglycans (GAG) in cartilage explants and primary rat chondrocytes, and the underlying molecular mechanism was analyzed by microarray and RT-PCR analysis in primary chondrocytes. RESULTS: In cartilage explants, PD166866 significantly counteracts IL-ß stimulated GAG release. In addition, PD166866 impede IL-1ß-stimulated nuclear translocation of p65 in rat chondrocytes. Based on microarray analysis, a total of 67 and 132 genes with more than 1.5-fold changes were identified in IL-1ß-treated versus control and PD166866 cotreatment versus IL-1ß treatment alone, respectively. Only 19 thereof were coregulated by IL-1ß and PD166866 simultaneously. GO and KEGG pathway analysis showed that some pathways, including "cytokine-cytokine receptor interaction," "chemokine signaling pathway," and "complement and coagulation cascades," as well as some key genes like chemokines, complement, and matrix metalloproteinases may relevant for therapeutic application of Fgfr1 blockade in IL-1ß-stimulated chondrocytes. CONCLUSION: Our results clearly demonstrated that blockade of Fgfr1 with PD166866 could effectively suppress the catabolic effects induced by IL-1ß, and elucidated whole genomic targets of Fgfr1 inhibition responsible for the therapeutic effects of Fgfr1 blockade against inflammatory OA.

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 root canal morphology in mandibular first premolars: a comparative evaluation of cone-beam computed tomography and micro-computed tomography.

OBJECTIVES: This study evaluated the accuracy of cone-beam computed tomography (CBCT) in detecting the root canal morphology of mandibular first premolars using micro-computed tomography (micro-CT) as a reference standard. MATERIALS AND METHODS: In total, 143 extracted human mandibular first premolars were selected and scanned using micro-CT and CBCT. The acquired images were used to evaluate the root canal morphology in each tooth, and evaluations were repeated after 2 weeks. The root canal configurations observed on the three-dimensional images were recorded, and the findings from both modalities were compared using chi-square tests. The actual agreement between the two modalities was assessed using kappa statistics. RESULTS: In total, the root morphologies in 136 mandibular first premolars were consistently identified by both CBCT and micro-CT: type I in 104, type III in five, type V in 20, and type IX in seven. Of the remaining seven teeth, the morphology in two, one, and four teeth was identified as type I, type VII, and type IX (type 1-3 in two and type 1-2-3 in two), respectively, by micro-CT and misdiagnosed as type III, type V, and type V, respectively, by CBCT. There were no significant differences between the two modalities with regard to the accurate detection of root canal configurations, with a kappa value of 0.886 for the actual agreement. CONCLUSIONS: Although CBCT may be accurate in detecting the root canal configuration in mandibular first premolars, it produces poorer image details compared with micro-CT. CLINICAL RELEVANCE: CBCT is a reliable radiological technique, but its accuracy in detecting details of the root canal morphology in mandibular first premolars, especially in some complex root canal configurations, needs to be improved.