Quantitative Proteomic and Phosphoproteomic Analyses Reveal a Role of Death-Associated Protein Kinase 1 in Regulating Hippocampal Synapse.

Death-associated protein kinase 1 (DAPK1) is a stress-responsive calcium/calmodulin (CaM)-regulated serine/threonine protein kinase that is actively involved in stress-induced cell death. The dysregulation of DAPK1 has been established in various neurological disorders such as epilepsy, Alzheimer's disease (AD), and Parkinson's disease (PD). Recent research indicates a synaptic localization of DAPK1 in neurons, suggesting a potential role of DAPK1 in modulating synaptic structure and function. However, the key molecules and pathways underlying the influence of DAPK1 on synapses remain elusive. We utilized quantitative proteomic and phosphoproteomic analyses to compare the differences in protein expression and phosphorylation in hippocampal tissues of wild-type (WT) and DAPK1-knockout (KO) mice. Bioinformatic analysis of differentially expressed proteins and phosphoproteins revealed a preferential enrichment of proteins involved in regulating synaptic function, cytoskeletal structure, and neurotransmission. Gene set enrichment analysis (GESA) highlighted altered presynaptic functions including synaptic vesicle priming and glutamate secretion in KO mice. Besides, we observed that proteins with potential phosphorylation motifs of ERK and DAPK1 were overrepresented among the differential phosphoproteins and were highly enriched in neuronal function-related pathways. Furthermore, Western blot analysis validated differences in the expression of several proteins closely associated with presynaptic organization, dendrites and calcium transmembrane transport between KO and WT mice, further corroborating the potential involvement of DAPK1 in the regulation of synaptic functions. Overall, our data provide molecular evidence to elucidate the physiological links between DAPK1 and neuronal functions and help clarify the role of DAPK1 in the pathogenesis of neurodevelopmental and neurodegenerative diseases.

Activation of transient receptor potential vanilloid 1 ameliorates tau accumulation-induced synaptic damage and cognitive dysfunction via autophagy enhancement.

AIMS: The autophagy-lysosomal pathway is important for maintaining cellular proteostasis, while dysfunction of this pathway has been suggested to drive the aberrant intraneuronal accumulation of tau protein, leading to synaptic damage and cognitive impairment. Previous studies have demonstrated that the activation of transient receptor potential vanilloid 1 (TRPV1) by capsaicin has a positive impact on cognition and AD-related biomarkers. However, the effect and mechanism of TPRV1 activation on neuronal tau homeostasis remain elusive. METHODS: A mouse model of tauopathy was established by overexpressing full-length human tau in the CA3 area. Mice were fed capsaicin diet (0.0125%) or normal diet for 9 weeks. The cognitive ability, synaptic function, tau phosphorylation levels, and autophagy markers were detected. In vitro, capsaicin-induced alterations in cellular autophagy and tau degradation were characterized using two cell models. Besides, various inhibitors were applied to validate the role of TRPV1-mediated autophagy enhancement in tau clearance. RESULTS: We observed that TRPV1 activation by capsaicin effectively mitigates hippocampal tau accumulation-induced synaptic damages, gliosis, and cognitive impairment in vivo. Capsaicin promotes the degradation of abnormally accumulated tau through enhancing autophagic function in neurons, which is dependent on TRPV1-mediated activation of AMP-activated protein kinase (AMPK) and subsequent inhibition of the mammalian target of rapamycin (mTOR). Blocking AMPK activation abolishes capsaicin-induced autophagy enhancement and tau degradation in neurons. CONCLUSION: Our findings reveal that capsaicin-induced TRPV1 activation confers neuroprotection by restoring neuronal tau homeostasis via modulating cellular autophagy and provides additional evidence to support the potential of TRPV1 as a therapeutic target for tauopathies.

Potential Implications of miRNAs in the Pathogenesis, Diagnosis, and Therapeutics of Alzheimer's Disease.

Alzheimer's disease (AD) is a complex multifactorial disorder that poses a substantial burden on patients, caregivers, and society. Considering the increased aging population and life expectancy, the incidence of AD will continue to rise in the following decades. However, the molecular pathogenesis of AD remains controversial, superior blood-based biomarker candidates for early diagnosis are still lacking, and effective therapeutics to halt or slow disease progression are urgently needed. As powerful genetic regulators, microRNAs (miRNAs) are receiving increasing attention due to their implications in the initiation, development, and theranostics of various diseases, including AD. In this review, we summarize miRNAs that directly target microtubule-associated protein tau (MAPT), amyloid precursor protein (APP), and ß-site APP-cleaving enzyme 1 (BACE1) transcripts and regulate the alternative splicing of tau and APP. We also discuss related kinases, such as glycogen synthase kinase (GSK)-3ß, cyclin-dependent kinase 5 (CDK5), and death-associated protein kinase 1 (DAPK1), as well as apolipoprotein E, that are directly targeted by miRNAs to control tau phosphorylation and amyloidogenic APP processing leading to Aß pathologies. Moreover, there is evidence of miRNA-mediated modulation of inflammation. Furthermore, circulating miRNAs in the serum or plasma of AD patients as noninvasive biomarkers with diagnostic potential are reviewed. In addition, miRNA-based therapeutics optimized with nanocarriers or exosomes as potential options for AD treatment are discussed.

Death-associated protein kinase 1 phosphorylates MDM2 and inhibits its protein stability and function.

Breast cancer is one of the major malignancies in women, and most related deaths are due to recurrence, drug resistance, and metastasis. The expression of the mouse double minute 2 (MDM2) oncogene is upregulated in breast cancer; however, its regulatory mechanism has yet to be fully elucidated. Herein, we identified the tumor suppressor death-associated protein kinase 1 (DAPK1) as a novel MDM2 regulator by unbiased peptide library screening. DAPK1 is directly bound to MDM2 and phosphorylates it at Thr419. DAPK1-mediated MDM2 phosphorylation promoted its protein degradation via the ubiquitin-proteasome pathway, resulting in upregulated p53 expression. DAPK1 overexpression, but not its kinase activity-deficient form, decreased colony formation and increased doxorubicin-induced cell death; however, DAPK1 knockdown produced the opposite effects in human breast cancer cells. In a xenograft tumorigenesis assay, DAPK1 overexpression significantly reduced tumor formation, whereas inhibition of DAPK1 kinase activity reduced its antitumorigenic effect. Finally, DAPK1 expression was negatively correlated with MDM2 levels in human breast cancer tissues. Thus, these results suggest that DAPK1-mediated MDM2 phosphorylation and its protein degradation may contribute to its antitumorigenic function in breast cancer.

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.

NIX protein enhances antioxidant capacity of and reduces the apoptosis induced by HSP90 inhibitor luminespib/NVP-AUY922 in PC12 cells.

Pheochromocytomas and paragangliomas (PCPGs) are catecholamine-producing neuroendocrine tumors. Accumulating evidences indicate that the blockade of antioxidative pathways might be a novel therapeutic approach to the treatment of PCPG. NIX has been confirmed to play a key role in maintaining redox homeostasis in tumors, while the function of NIX in PCPG remains unclear. In this study, the analyses of the disease-free survival (DFS) showed that high NIX protein level is related to poor prognosis in patients of PCPG. Consistent with this, high level of NIX protein upregulates the level of p-NF-κB and promotes the migration of PC12 cells. In NIX-over-expressing PC12 cells, the level of reactive oxygen species (ROS) is decreased while trolox-equivalent antioxidant capacity (TEAC) increased. But in NIX-silencing cells, ROS level is increased, while TEAC reversely reduced, consequently antioxidase and phase II enzymes of NRF2 signaling were activated, and elevated endoplasmic reticulum (ER) stress was observed. Additionally, the apoptosis induced by luminespib/NVP-AUY922, an inhibitor of heat shock protein 90 (HSP90, a cellular stress response factor), was enhanced in NIX-silencing cells but reduced in the NIX-over-expressing cells. All of these results indicated that high NIX protein level enhances antioxidant capacity of PC12 cells and reduces the apoptosis caused by cell stress, such as induced by luminespib/NVP-AUY922. Therefore, luminespib/NVP-AUY922 might be effective only for PCPG with low NIX level, while targeting NIX could be a further supplement to the therapeutic treatment strategy for PCPG patients with high NIX protein level.