Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5

Ahn, Jae Suk; Musacchio, Andrea; Mapelli, Marina; Ni, Jake; Scinto, Leonard F. M.; Stein, Ross L.; Kosik, Kenneth S.; Yeh, Li-An

doi:10.1177/1087057103260594

Cited by 13 publications

(14 citation statements)

References 39 publications

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“…We calculated a CDK5 K M of 4 μM with histone as substrate. This value is comparable to the previously described K M of 4 μM in vivo tau (Jae Suk Ahn et al, 2004) and K M of 10 μM in vitro for histone (Min Liu et al, 2008). A 45–50% reduction in the histone phosphorylation rate with CDK5 obtained from cerebral tissue of CDK5miR treated mice was associated with a comparable reduction of CDK5 in the same tissues.…”

Section: Discussionsupporting

confidence: 89%

Silencing of CDK5 Reduces Neurofibrillary Tangles in Transgenic Alzheimer's Mice

Piedrahita¹,

Hernández²,

et al. 2010

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Alzheimer's disease is a major cause of dementia for which treatments remain unsatisfactory. Cyclin-dependent kinase 5 (CDK5) is a relevant kinase that has been hypothesized to contribute to the tau pathology. Several classes of chemical inhibitors for CDK5 have been developed, but they generally lack the specificity to distinguish among various ATP-dependent kinases. Therefore, the efficacy of these compounds when tested in animal models cannot definitively be attributed to an effect on CDK5. However, RNA interference (RNAi) targeting of CDK5 is specific and can be used to validate CDK5 as a possible treatment target. We delivered a CDK5 RNAi by lentiviral or adenoassociated viral vectors and analyzed the results in vitro and in vivo. Silencing of CDK5 reduces the phosphorylation of tau in primary neuronal cultures and in the brain of wild-type C57BL/6 mice. Furthermore, the knockdown of CDK5 strongly decreased the number of neurofibrillary tangles in the hippocampi of triple-transgenic mice (3ϫTg-AD mice). Our data suggest that this downregulation may be attributable to the reduction of the CDK5 availability in the tissue, without affecting the CDK5 kinase activity. In summary, our findings validate CDK5 as a reasonable therapeutic target for ameliorating tau pathology.

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Section: Discussionsupporting

confidence: 89%

Silencing of CDK5 Reduces Neurofibrillary Tangles in Transgenic Alzheimer's Mice

Piedrahita¹,

Hernández²,

et al. 2010

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“…Most methods used to detect phosphorylation of signaling molecules use phosphorylation-specific antibodies that selectively bind to individual phosphorylated sites. Such antibodies are used in, for example, Western blotting (3,4), ELISAs (5,6), dot blots (7), protein arrays (8), FACS (9) and fluorescence microscopy (10). There are two principal limitations of such methods.…”

mentioning

confidence: 99%

Isoelectric focusing technology quantifies protein signaling in 25 cells

O'Neill¹,

Bhamidipati²,

Bi³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

187

234

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A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. Highresolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.cell signaling ͉ immunoassay ͉ phosphorylation ͉ Western blot ͉ microfluidic

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“…Several potent leads for medicinal chemistry have previously been identified for different projects from screens of this library. [12][13][14][15] Compounds (MW = 350-550) were prespotted in 0.3 µl DMSO (Sigma-Aldrich, St. Louis, MO; ∼1.7 mM) on 384-well plates, which also contained wells with only DMSO as vehicle controls. DMEM (90 µl/well) was added to defrosted compound plates using a dispenser unit.…”

Section: Compound Library and Htsmentioning

confidence: 99%

Identifying Druglike Inhibitors of Myelin-Reactive T Cells by Phenotypic High-Throughput Screening of a Small-Molecule Library

Rossi¹,

Padmanaban²,

Ni³

et al. 2007

SLAS Discovery

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Inflammatory T cells that are reactive to myelin protein components of the CNS play a critical role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The authors have previously generated mice that predominantly harbor T cells transgenic for a T-cell receptor (TCR) that is specific to the myelin proteolipid protein (PLP) 139-151 and that spontaneously develop MS-like paralysis. T cells from healthy transgenic mice respond to stimulation with PLP139-151 in a highly specific manner by proliferation and secretion of proinflammatory cytokines such as interleukin (IL)-2 and interferon (INF)-gamma in vitro. To identify druglike compounds that may inhibit inflammatory T-cell responses, the authors have developed a high-throughput screening assay with primary T cells from PLP TCR transgenic mice. They have screened 41,184 small-molecule compounds that follow Lipinski's rules for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells. To this end, the screen identified 6 nontoxic compounds with a molecular weight <500 that inhibited inflammatory responses in PLP-reactive T cells in a concentration-dependent fashion. The identified compounds represent valid leads that may be developed into novel therapeutics for MS that could be administered orally.

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Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5

Cited by 13 publications

References 39 publications

Silencing of CDK5 Reduces Neurofibrillary Tangles in Transgenic Alzheimer's Mice

Silencing of CDK5 Reduces Neurofibrillary Tangles in Transgenic Alzheimer's Mice

Isoelectric focusing technology quantifies protein signaling in 25 cells

Identifying Druglike Inhibitors of Myelin-Reactive T Cells by Phenotypic High-Throughput Screening of a Small-Molecule Library

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