Journal: The Journal of Neuroscience

Article Title: Soluble β-Amyloid 1-40 Induces NMDA-Dependent Degradation of Postsynaptic Density-95 at Glutamatergic Synapses

doi: 10.1523/JNEUROSCI.3034-05.2005

Figure Lengend Snippet: PSD-95 levels are reduced after Aβ treatment in rat primary cortical neurons. A, Aβ selectively downregulates PSD-95 levels in a time-dependent manner, without altering the expression of other synaptic proteins. Cells were exposed to 10 μm soluble Aβ1-40 (see Materials and Methods) for between 15 and 120 min before analysis by Western blot. As shown in the histogram, semiquantitative evaluation of PSD-95 levels, normalized against tubulin, shows that Aβ treatment led to a significant reduction of PSD-95 expression (53.3 ± 6.5% of control levels; p < 0.05) within 60 min; despite a trend to recover by 120 min after treatment, PSD-95 expression remained significantly lower than in controls (76 ± 10.9%; p < 0.05). Analysis of levels of the synaptic proteins GluR2, GAP-43, synapsin I, cdk5, and CaMKII revealed no significant effect of Aβ over the treatment duration. Actin and tubulin levels were not influenced by the experimental manipulations. B, Dose-dependent effects of Aβ on PSD-95 levels. Neurons were exposed to Aβ (0.1-10 μm) for 1 h before they were analyzed for levels of PSD-95 expression in Western blot assays. One representative Western blot is shown, and the semiquantitative data from three independent experiments are shown in the histogram. Levels of PSD-95, normalized with respect to tubulin levels, are shown as means ± SD. Asterisks indicate significant changes from untreated control cells (p < 0.05). The dose-response curve had r = 0.879 (p = 0.007). C, Aβ effect on synaptic PSD-95. Primary rat cortical neurons were treated with 10 μm Aβ for 1 h, fixed, and immunostained for synapsin I and PSD-95. Synaptic sites were identified as synapsin I-positive puncta. Puncta density in vehicle-treated control cultures was 36 ± 2.4 puncta/100 μm; after Aβ treatment, the density of PSD-95 immunoreactive puncta was 24.6 ± 2.1/100 μm (p < 0.001). Numerous synaptic sites (identified by synapsin staining) showed prominent decreases in PSD-95 fluorescence after Aβ treatment; fluorescence intensity of synaptic PSD-95 was reduced after Aβ treatment (49.4 ± 2.4% compared with 100 ± 6.7% in controls; p < 0.01, p < 0.05; n = 200). Scale bars, 5 μm. All numerical data represent mean ± SD.

Article Snippet: Membranes were blocked in PBS containing 5% nonfat milk powder and 0.2% Tween-20 and incubated with the following antibodies: anti-PSD-95 (1:6000; Upstate Biotechnology, Lake Placid, NY), anti-synapsin I (1:500; Chemicon, Temecula, CA), anti-cdk5 (1:1000; Biomol, Plymouth Meeting, PA), anti-GAP-43 (1:5000; Chemicon), and anti-actin (1:10,000; Chemicon) or anti-β-tubulin (1: 2000; Oncogene Sciences, Uniondale, NY).

Techniques: Expressing, Western Blot, Control, Staining, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Soluble β-Amyloid 1-40 Induces NMDA-Dependent Degradation of Postsynaptic Density-95 at Glutamatergic Synapses

doi: 10.1523/JNEUROSCI.3034-05.2005

Figure Lengend Snippet: Aβ-induced downregulation of synaptic PSD-95 requires cdk5 activity. A, cdk5 activity is necessary for Aβ effects in cortical neurons. Primary rat cortical neurons were pretreated (1 h) with roscovitine (15 μm) or PNU 112455A (10 μm), or vehicle (DMSO) before exposure to Aβ (10 μm; 1 h). As shown by Western blotting, both inhibitors abrogated the ability of Aβ to reduce PSD-95 levels. C, Control. B, Aβ cannot downregulate PSD-95 levels when SK-N-MC cells are transiently transfected with the T19A, S25A, S35A phosphorylation mutant of PSD-95. Cells transfected with wild-type PSD-95 and expressing p35/cdk5 responded to Aβ with the expected decrease in PSD-95 levels (57.9 ± 5.6% of controls; p < 0.05). C, The Aβ effects on synaptic PSD-95 are blocked by roscovitine, an inhibitor of cdk5 activity. Primary rat cortical neurons were pretreated (1 h) with roscovitine (15 μm) or vehicle (DMSO) before exposure to Aβ (10 μm; 1 h). After fixation, neurons were immunostained for synapsin I and PSD-95. Roscovitine alone did not show a significant effect on the density of PSD-95 puncta or their fluorescence intensity but inhibited the effects of Aβ on these parameters. Scale bar, 5 μm. Asterisks indicate significant changes from untreated control cells (p < 0.05).

Article Snippet: Membranes were blocked in PBS containing 5% nonfat milk powder and 0.2% Tween-20 and incubated with the following antibodies: anti-PSD-95 (1:6000; Upstate Biotechnology, Lake Placid, NY), anti-synapsin I (1:500; Chemicon, Temecula, CA), anti-cdk5 (1:1000; Biomol, Plymouth Meeting, PA), anti-GAP-43 (1:5000; Chemicon), and anti-actin (1:10,000; Chemicon) or anti-β-tubulin (1: 2000; Oncogene Sciences, Uniondale, NY).

Techniques: Activity Assay, Western Blot, Control, Transfection, Phospho-proteomics, Mutagenesis, Expressing, Fluorescence

Journal: International Journal of Cancer

Article Title: NOTCH , ASCL1 , p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas

doi: 10.1002/ijc.29835

Figure Lengend Snippet: ASCL1 mediated phosphorylation of RB triggered by CDK5. ( a ) RB and CDK protein levels determined by Western blot. ( b+c ) Mitotic cell number (pHistoneH3 – pH3) and induction of apoptosis (cleaved Caspase 3 – cCaspase3) determined by flow cytometry upon CDK5 inhibition by Roscovitine for 48 hrs. ( d ) RB and CDK5 protein levels measured by flow cytometry. Mean fluorescence intensity was normalized on secondary antibody control (Index). Data are presented as mean ± SEM of analysis in triplicates. Statistical significance was calculated using the Student's t test, two‐sided, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CDK5 inhibitor Roscovitine (Absource Diagnostics) and the wingless‐type (WNT)‐pathway inhibitor IWP‐2 (Sigma‐Aldrich) were used as indicated.

Techniques: Phospho-proteomics, Western Blot, Flow Cytometry, Inhibition, Fluorescence, Control

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: The regulatory subunits of CDK5.

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Clinical Proteomics, Membrane, Binding Assay, Activity Assay

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: The mechanism of Cdk5 activation. The mechanism of Cdk5 activation. CDK5 alone is an inert catalyst subunit. CDK5 is activated by the p35 CDK5 activator and moves to the membrane as p35 binds to the membrane through myristoylation of the N-terminal region. p35 is a short-lived protein that is broken down by proteasomes. When cells are stressed or met with death signal, calpain is activated and cuts p35 into p25 C-terminal fragments. The deletion of p10 prolongs the half-life of p25. CDK5/p25 can be separate from the membrane and phosphorylate additional proteins. (modified from Kimura et al. ).

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Activation Assay, Membrane, Modification

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: The regulation of CDK5 by posttranslational modification.

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Modification, Phospho-proteomics, Activity Assay, Activation Assay, Migration

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: The modulation of transcription factors by CDK5.

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Phospho-proteomics

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: Schematic of kinase pathway phosphorylating EPRS Ser886 and Ser999. IFN-γ–activated CDK5 phosphorylate EPRS, leading to the formation of the GAIT complex. This inhibits inflammatory mRNA translation. Abbreviations- interferon-gamma (IFNγ), glutamyl-prolyl tRNA synthetase (EPRS), IFN-γ–activated inhibitor of translation (GAIT), tRNA multisynthetase complex (MSC). (modified from Arif et al. ).

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Modification

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: A summary of the various cyclin-dependent kinase 5 (CDK5)-mediated biological processes. CDK5 plays important roles not only in the central nervous system but also in different biological processes. Functions in the central nervous system include synaptic function, axon guidance, cell adhesion, and neurodegenerative diseases. Functions outside of the central nervous system include androgen production, cell cycle, cancer cell proliferation/apoptosis, and tumour metastasis. Abbreviations- microtubule-associated proteins 1B (MAP1B), focal adhesion kinase (FAK), doublecortin (DCX), p21 activated kinase 1 (Pak-1), Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE-1), Eph receptor A4 (EphA4), transforming growth factor-β1′ (TGF-β1), retinoblastoma (Rb), E2F transcription factor 1 (E2F1). (modified from Shupp et al. ).

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Modification

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: Impacts of CDK5 on the hallmarks of cancer: Uncontrolled proliferative signalling, Overcome growth suppressors, escaping antitumor immune system evasion, replicative immortality, tumour promoting inflammation, invasion and metastasis, inducing angiogenesis, genomic instability and mutation, resisting programmed cell death, metabolic reprogramming. Through the effect on the target protein, CDK5 is conducive to tumour development and metastasis. Abbreviations: phorbol-12-myristate-13-acetate-induced protein 1 (Noxa), Signal transducer and activator of transcription 3 (STAT3), androgen receptor (AR), retinoblastoma (Rb), bridging integrator 1 (BIN1), programmed cell death ligand 1 (PD-L1), focal adhesion kinase (FAK), targeting protein for Xklp2 (TPX2), hypoxia-inducible factor-1α (HIF-1α), ataxia-telangiectasia mutated (ATM). (Line 516–525). This figure is adapted and modified from Lenjisa et al., and Stecca et al. [ , ].

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: Mutagenesis, Modification

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: Structures of CDK5 inhibitors: roscovitine, flavopiridol, dinaciclib, olomoucine, purvalanol A, indurubin-3′, 20-223, AT7519.

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques:

Journal: Cancers

Article Title: The Role of CDK5 in Tumours and Tumour Microenvironments

doi: 10.3390/cancers13010101

Figure Lengend Snippet: IC 50 values of CDK5 inhibitors.

Article Snippet: We classify the CDK5 inhibitors as early pan CDK inhibitors, multitarget CDK5 inhibitors, and selective CDK5 inhibitors according to report of Whittaker et al.’s ( ) [ ].

Techniques: