Journal: PLOS ONE

Article Title: Palmitoyl-L-carnitine induces tau phosphorylation and mitochondrial dysfunction in neuronal cells

doi: 10.1371/journal.pone.0313507

Figure Lengend Snippet: (A) shows changes in protein levels of pGSK-3β (S9), GSK-3β, CDK5, and p25 after treatment with palmitoyl-L-carnitine in SH-SY5Y cells. pGSK-3β (S9) indicates GSK-3β phosphorylated at serine 9. Full blots are provided in . (B) Histogram illustrating changes in protein levels of pGSK-3β (S9), CDK5, and p25 after treatment with palmitoyl-L-carnitine in SH-SY5Y cells, shown as mean ± standard error of the mean (SEM; n = 3). GSK-3β phosphorylation levels were normalized to the total GSK-3β protein. (C) shows changes in protein levels of pTau (T181), pTau (S262), PHF-1, total tau, GSK-3β, CDK5, and p25 after treatment with tau kinase inhibitors in palmitoyl-L-carnitine-treated SH-SY5Y cells. pTau (T181), pTau (S262), and PHF-1 represent tau phosphorylated at threonine 181, serine 262, and serine 396/404, respectively. SB216763, Roscovitine, and PD150606 are GSK-3β inhibitor, CDK5 inhibitor, and calpain inhibitor, respectively. Full blots are provided in . (D) Histogram illustrating changes in protein levels of pTau (T181), pTau (S262), PHF-1, total tau, GSK-3β, CDK5, and p25 after treatment with tau kinase inhibitors in palmitoyl-L-carnitine-treated SH-SY5Y cells, shown as mean ± standard error of the mean (SEM; n = 3). Tau phosphorylation levels were normalized to the total tau protein. In (A) and (D) , BSA and BSA-PC represent bovine serum albumin and BSA-conjugated palmitoyl-L-carnitine, respectively. Statistical significance was determined using an unpaired two-tailed t-test with Welch’s correction and an ordinary two-way ANOVA with Tukey’s multiple comparison test; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: To assess the effects of kinase inhibitors, cells were treated simultaneously with 5 μM GSK-3β inhibitor SB216763 (Tocris), 5 μM CDK5 inhibitor Roscovitine (Tocris), or 5 μM calpain inhibitor PD150606 (Tocris), each combined with 5 μM BSA-PC for 24h.

Techniques: Two Tailed Test, Comparison

Journal: PLOS ONE

Article Title: Palmitoyl-L-carnitine induces tau phosphorylation and mitochondrial dysfunction in neuronal cells

doi: 10.1371/journal.pone.0313507

Figure Lengend Snippet: A schematic illustration showing the mechanism by which palmitoyl-L-carnitine induces tau phosphorylation in SH-SY5Y neurons. Palmitoyl-L-carnitine causes calcium overload by closely interacting with mitochondrial malfunction, including the fission process. This increased calcium overload activates tau kinases (GSK-3β and CDK5/p25), leading to significant tau phosphorylation. Therefore, elevated serum levels of palmitoyl-L-carnitine are likely to contribute significantly to the development of AD pathology with aging.

Article Snippet: To assess the effects of kinase inhibitors, cells were treated simultaneously with 5 μM GSK-3β inhibitor SB216763 (Tocris), 5 μM CDK5 inhibitor Roscovitine (Tocris), or 5 μM calpain inhibitor PD150606 (Tocris), each combined with 5 μM BSA-PC for 24h.

Techniques:

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Expression levels of E2F1 and CDK5 in microglia after CIRI. (A, B) RT‐qPCR experiments assessing the expression levels of E2F1 and CDK5 in the brain tissue of CIRI mice. (C–F) Immunofluorescence experiments detected the expression levels of E2F1 (C, D) and CDK5 (E, F) in the brain tissues of mice 24 h after CIRI. (G, H) RT‐qPCR experiments measuring the expression levels of E2F1 and CDK5 in microglia after OGD/R, scale bars = 25 µm. (I, L) Immunofluorescence experiments investigating the expression levels of E2F1 (I, J) and CDK5 (K, L) in microglia after OGD/R. Scale bars = 25 µm. Each group consisted of six mice, and all cellular experiments were repeated three times. * p < .05, p ** < .01, *** p < 0.001.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Transcriptional regulation of CDK5 by E2F1 in microglia. (A) Schematic diagram of the dual‐luciferase reporter gene experiment (Created with BioRender.com). (B) RT‐qPCR detection of E2F1 expression levels in cells after overexpression or silencing of E2F1. (C) RT‐qPCR detection of CDK5 expression levels in cells after overexpression or silencing of E2F1. (D) Dual‐luciferase assay to detect the effect of E2F1 on CDK5 promoter transcriptional activity: based on lentivirus‐mediated silencing and overexpression of E2F1 in 293T cells (oe‐NC, oe‐E2F1, sh‐NC, sh‐E2F1), co‐transfected with the dual‐luciferase reporter gene vector containing the CDK5 promoter sequence and its binding site mutant, luciferase activity was measured 48 h posttransfection and normalized to Renilla. (E) ChIP experiment to detect the enrichment of E2F1 on the CDK5 promoter. (F, G) TEM image showing mitochondrial morphology in oligodendrocytes. Scale bar = 500 nm. (H, I) Fluorescence images and related quantitative data showing Mito‐ROS MitoSOX fluorescence in oligodendrocytes of each group. Scale bar = 25 µm. (J, K) Immunofluorescence detection of mitochondrial morphology in oligodendrocytes of each group, red marks mitochondria, blue marks nuclei. Scale bar = 15 µm. (L, M) DCFH‐DA staining to detect ROS production in oligodendrocytes of each group. Scale bar = 25 µm. All cellular experiments were conducted thrice, * p < .05, ** p < .01.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Luciferase, Quantitative RT-PCR, Expressing, Over Expression, Activity Assay, Transfection, Plasmid Preparation, Sequencing, Binding Assay, Mutagenesis, Fluorescence, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: CDK5 regulation of DRP1 phosphorylation affects microglia mitochondrial fission and ROS accumulation. (A) TEM image displaying mitochondria in the cerebral cortex post‐CIRI along with quantitative data on mitochondrial length and cristae density per group. Scale bars = 2 µm/500 nm. (B) DCFH‐DA staining assessing ROS production in the cerebral cortex post‐CIRI. Scale bar = 25 µm. (C) Western blot determining the activation status of DRP1, with β‐actin, COX IV, and Tubulin serving as internal references for total, mitochondrial, and cytosolic fractions, respectively. (D, E) Immunofluorescence detecting the co‐localization of DRP1 and mitochondrial marker protein COX IV. Scale bar = 15 µm; (F, G) TEM image featuring mitochondrial morphology in microglia. Scale bar = 500 nm. (H, I) Fluorescence images and corresponding quantitative data showing MitoSOX fluorescence in microglia of each group. Scale bar = 25 µm. Each group comprised six mice, and all cellular experiments were repeated three times. * p < 0.05.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Staining, Western Blot, Activation Assay, Immunofluorescence, Marker, Fluorescence

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Toxic effects of E2F1/CDK5 regulating DRP1 on neurons. (A) RT‐qPCR analysis of E2F1 and CDK5 expression levels in cells. (B, C) Western blot examining the activation status of DRP1, with β‐actin, COX IV, and Tubulin used as internal references for total, mitochondrial, and cytosolic fractions. (D) Morphological changes in neurons in each group. Scale bar = 25 µm. (E) CCK‐8 assay measuring the proliferative activity of neurons in each group. (F, G) Tunel staining assessing apoptosis in neurons of each group. Scale bar = 50 µm. (H) Quantification of LDH release indicating neuronal cell death. All cellular experiments were conducted thrice. * p < .05.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, CCK-8 Assay, Activity Assay, TUNEL Assay, Staining

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Impact of E2F1 silencing in microglia on neurobehavioral functions in mice following CIRI injury. (A) RT‐qPCR analysis of E2F1 and CDK5 expression levels in brain tissue. (B, C) Western Blot analysis of DRP1 activation in brain tissue. (D) Quantitative assessment of TCC staining and infarct area in the mouse brain. (E) H&E staining of the cerebral cortex in mice. Scale bar = 50/25 µm. (F) Evaluation of neurological deficits in mice through the mNSS analysis. (G) Delay in reaching the hidden platform on the 6th day for mice. (H) Time spent in the target quadrant during the probe trial on the 7th day in seconds. (I) Number of crossings over the target platform location by mice on the 7th day during the probe trial. (J) Representative swim paths of mice on the 7th day. (K) Percentage of time spent exploring the novel object in the novel object recognition test phase. Each group comprises six mice, * p < .05.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activation Assay, Staining

Journal: Clinical and Translational Medicine

Article Title: E2F1/CDK5/DRP1 axis mediates microglial mitochondrial division and autophagy in the pathogenesis of cerebral ischemia‐reperfusion injury

doi: 10.1002/ctm2.70197

Figure Lengend Snippet: Schematic representation of the mechanism by E2F1/CDK5/DRP1 mediating microglial mitochondrial division and autophagy impact on CIRI.

Article Snippet: The treatment involved using 15 µmol of the CDK5 inhibitor Roscovitine (product code: HY‐30237, MCE), 10 µmol of the dynamin‐related protein 1 (DRP1) inhibitor Mdivi‐1 (Product Code: HY‐15886, MCE), and the ROS scavenger NAC (product code: HY‐B0215, MCE) to treat the cells separately to establish the OGD/R cellular model. For the OGD/R+sh‐NC and OGD/R+sh‐CDK5 groups, a culture of the cells with the corresponding lentivirus was continued for 48 h, and then the infection efficiency was evaluated following the aforementioned method to construct the OGD/R cellular model.

Techniques:

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Proteins with increased and decreased post-ischemic ubiquitination were assessed for GO enrichment for biological process. Benjamini-corrected P values for the top four terms for the “Ub increased” dataset are depicted. b All proteins with increased ubiquitination after ischemia and enrichment in the four top categories for biological process were investigated for GO enrichment for molecular function and cellular components. Benjamini-corrected P values for the five highest-ranking terms are shown. c Post-ischemic ubiquitination of select PSD-associated kinases and phosphatases was confirmed by immunoprecipitation of proteins of interest from ipsi- and contralateral detergent-insoluble cortical lysates from MCAO/1 h reperfusion-treated mice and detection of ubiquitin by Western Blotting. IgG-isotype antibodies served as controls. Results from n = 3 mice/group were quantified. CaMKIIα: * P = 0.0009; CaMKIIβ: * P = 0.0040; PKCβ: * P = 0.0021; PKCγ: * P = 0.0099; Cdk5: * P = 0.0015; Pyk2: * P = 0.0016; CKβ: * P = 0.0002; Pten: * P = 0.0013. Two-tailed unpaired t -test. Data are expressed as mean ± s.e.m. d Domain structure of prominent PSD-associated kinases and phosphatases found ubiquitinated after ischemia. Numbers represent amino acid positions, and arrows indicate ubiquitinated residues identified by MS analysis. BP biological process, c contralateral, C carboxy-terminus, C1a, and C1b diacylglycerol-binding domain, C2 calcium-binding domain, CaM calmodulin, FAT focal adhesion kinase-targeting domain, FERM, 4.1 protein, Ezrin radixin, and moesin domain, GO gene ontology, i ipsilateral, LTP long-term potentiation, N amino-terminus, nc not called, P proline-rich region, PDZ-b PSD95, Dlg1 Zo-1-containing domain-binding domain, POI protein of interest, Ub ubiquitination.

Article Snippet: The specificity of substrate phosphorylation by Cdk5 was verified by adding 10 μM Cdk5 inhibitor ((R)-CR8, Tocris Bioscience, Bristol, UK) to companion reactions.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Two Tailed Test, Binding Assay

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Phosphorylation levels of Cdk5 downstream targets were determined in PSD lysates of sham and MCAO-treated animals. Tau: * P = 0.0137 from s, # P = 0.0055 from s; Crmp2: * P = 0.0496 from s and P = 0.0430 from c; one-way ANOVA with Bonferroni test; n = 4–5 animals/group. b Cortical PSD-lysates from sham and MCAO-treated mice were assessed for Cdk5 activity (* P < 0.0001, two-tailed unpaired t -test; n = 6 mice/group). c Cdk5 activity was measured in post-ischemic PSD lysates untreated and treated with recombinant deubiquitinase USP2 (* P = 0.0057, two-tailed paired t -test; n = 6 animals). c contralateral, i ipsilateral, S serine, s sham. Data are expressed as mean ± s.e.m.

Article Snippet: The specificity of substrate phosphorylation by Cdk5 was verified by adding 10 μM Cdk5 inhibitor ((R)-CR8, Tocris Bioscience, Bristol, UK) to companion reactions.

Techniques: Phospho-proteomics, Activity Assay, Two Tailed Test, Recombinant

Journal: Communications Biology

Article Title: Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases

doi: 10.1038/s42003-024-06009-8

Figure Lengend Snippet: a Post-ischemic detergent-resistant ubiquitination is elevated in neurons, particularly at the postsynaptic density (PSD) of glutamatergic neurons, while it is reduced in all other brain cell types. EC endothelial cell, glut glutamatergic, Tx Triton X100, Ub ubiquitination. b Postsynaptic proteins with increased ubiquitination after ischemic stroke include receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), Densin-180, G protein-coupled receptor (GPCR), tropomyosin receptor kinase (TrkB)), scaffolding proteins and their adapters (brain-enriched guanylate kinase-associated protein (Begain), Disks large-associated protein 2–4 (Dlgap2-4), guanylate kinase-associated protein (GKAP), postsynaptic density protein 93/95 (PSD93/95), synapse-associated protein 97/102 (SAP97/102), SH3 and multiple ankyrin repeat domain (Shank)), G proteins (guanine nucleotide-binding protein G(i) subunit alpha (Gnai), guanine nucleotide-binding protein G(o) subunit alpha (Gnao), guanine nucleotide-binding protein G(s) subunit alpha isoforms short (Gnas), guanine nucleotide-binding protein G(t) subunit alpha (Gnat)), and signaling proteins (calcium-calmodulin-dependent protein kinase II (CaMKII), cyclin-dependent kinase 5 (Cdk5), creatine kinase b (CKb), Kalirin-7, protein kinase C (PKC), protein phosphatase 2 (PP2), phosphatase and TENsin homolog (Pten), proline-rich tyrosine kinase (Pyk2), synaptic Ras GTPase-activating protein 1 (SynGAP1)). c PSD-kinases, such as CaMKII, PKC, Cdk5, and Pyk2, are prominent post-ischemic ubiquitination targets. While CaMKII, PKC, and Cdk5 activities at the PSD are decreased after stroke, leading to reduced target phosphorylation, Pyk2 exhibits increased activity, thereby accelerating the phosphorylation of target proteins. In all cases, kinase activity regulation after stroke was dependent on ubiquitination, whose removal normalized activity. P phosphorylation. This figure was created with BioRender.com .

Article Snippet: The specificity of substrate phosphorylation by Cdk5 was verified by adding 10 μM Cdk5 inhibitor ((R)-CR8, Tocris Bioscience, Bristol, UK) to companion reactions.

Techniques: Ubiquitin Proteomics, Scaffolding, Binding Assay, Phospho-proteomics, Activity Assay