The Levels of cdk5 and p35 Proteins and Tau Phosphorylation Are Reduced during Neuronal Apoptosis

doi:10.1006/bbrc.2001.4240

Biochemical and Biophysical Research Communications

Volume 280, Issue 4, 2 February 2001, Pages 998-1002

https://doi.org/10.1006/bbrc.2001.4240 Get rights and content

Abstract

Cyclin-dependent kinase 5 (cdk5) is believed to be involved in the phosphorylation of tau protein. We studied the expression of the protein levels of cdk5 and the neuron-specific cdk5 activator p35 as well as cdk5 activity and tau phosphorylation during apoptosis in rat hippocampal neuronal cultures. We observed that in cells treated with etoposide, cyclosporin A, 4-hydroxynonenal (HNE), or okadaic acid, there was an early reduction in the protein levels of p35, and later also in cdk5 with all treatments except etoposide. The level of p25, a calpain cleavage product of p35 suggested to have increased ability to activate cdk5, was reduced paralleling the amount of p35. The changes in the p35 and p25 protein levels coincided with decreases in cdk5 activity and tau phosphorylation after treatment with HNE and etoposide. However, the relationship between the p35 and p25 levels and cdk5 activity was complex. We conclude that neuronal apoptosis is accompanied with a decrease in the levels of p35, p25, and cdk5, and tau phosphorylation. These changes may reinforce the neuronal damage.

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Free D-aspartate triggers NMDA receptor-dependent cell death in primary cortical neurons and perturbs JNK activation, Tau phosphorylation, and protein SUMOylation in the cerebral cortex of mice lacking D-aspartate oxidase activity
2019, Experimental Neurology
Citation Excerpt :
Further studies will be required to investigate the involvement of calcineurin and of other phosphatases such as PP2A and PP1 in regulating Tau phosphorylation in Ddo−/− mouse brain. Tau dephosphorylation has been reported to be associated with neuronal apoptosis (Amadoro et al., 2004; Canu et al., 1998; Kerokoski et al., 2001; Liu et al., 2010; Mills et al., 1998; Pristera et al., 2013; Rametti et al., 2004), oxidative stress (Ko et al., 1997) and glutamate-induced cell death (Lorio et al., 2001). In this latter case, Tau is also cleaved to a fragment (Lorio et al., 2001) like that seen in 100 μM D-Asp-treated primary cortical neurons.
In mammals, free d-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme d-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo^−/−) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo^−/− mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons. Moreover, observations obtained in Ddo^−/− animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls.
Cdk5 protein inhibition and Aβ42 increase BACE1 protein level in primary neurons by a post-transcriptional mechanism: Implications of CDK5 as a therapeutic target for Alzheimer disease
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The decrease in Cdk5 protein was unexpected. It has been shown that various apoptotic agents decrease Cdk5 and p35 levels (57), and we observed caspase 3 cleavage, suggesting that Cdk5 inhibition reduces Cdk5 levels through a caspase 3/apoptosis-dependent mechanism. Because our results with CP681301 and roscovitine treatment allowed us to exclude our first hypothesis that inhibition of Cdk5 activity would attenuate the Aβ42-induced BACE1 increase, we tested the possibility that inhibition of caspase 3 could block the BACE1 elevation.
The β-secretase enzyme BACE1 initiates production of the amyloid-β (Aβ) peptide that comprises plaques in Alzheimer disease (AD) brain. BACE1 levels are increased in AD, potentially accelerating Aβ generation, but the mechanisms of BACE1 elevation are not fully understood. Cdk5/p25 has been implicated in neurodegeneration and BACE1 regulation, suggesting therapeutic Cdk5 inhibition for AD. In addition, caspase 3 has been implicated in BACE1 elevation. Here, we show that the Cdk5 level and p25:p35 ratio were elevated and correlated with BACE1 level in brains of AD patients and 5XFAD transgenic mice. Mouse primary cortical neurons treated with Aβ42 oligomers had increased BACE1 level and p25:p35 ratio. Surprisingly, the Aβ42-induced BACE1 elevation was not blocked by Cdk5 inhibitors CP68130 and roscovitine, and instead the BACE1 level was increased greater than with Aβ42 treatment alone. Moreover, Cdk5 inhibitors alone elevated BACE1 in a time- and dose-dependent manner that coincided with increased caspase 3 cleavage and decreased Cdk5 level. Caspase 3 inhibitor benzyloxycarbonyl-VAD failed to prevent the Aβ42-induced BACE1 increase. Further experiments suggested that the Aβ42-induced BACE1 elevation was the result of a post-transcriptional mechanism. We conclude that Aβ42 may increase the BACE1 level independently of either Cdk5 or caspase 3 and that Cdk5 inhibition for AD may cause BACE1 elevation, a potentially negative therapeutic outcome.
Post-translational modifications of tau protein: Implications for Alzheimer's disease
2011, Neurochemistry International
Citation Excerpt :
A role of CDK5 in tau phosphorylation and NFT formation has been clearly established in transgenic mice (Cruz et al., 2003; Noble et al., 2003). Although CDK5 regulates APP (amyloid precursor protein) processing and tau hyperphosphorylation, its involvement in AD remains controversial (Iqbal et al., 2009; Kerokoski et al., 2001; Lopes et al., 2010; Van den Haute et al., 2001). MAPKs also phosphorylate tau (Feijoo et al., 2005; Sergeant et al., 2008) and are found in NFTs of AD brains (Zhu et al., 2000).
Alzheimer's disease (AD) belongs to a group of neurodegenerative diseases collectively designated as “tauopathies”, because they are characterized by the aggregation of abnormally phosphorylated tau protein. The mechanisms responsible for tau aggregation and its contribution to neurodegeneration are still unknown. Thereby, understanding the modes of regulation of tau is of high interest in the determination of the possible causes at the origin of the formation of tau aggregates and to elaborate protection strategies to cope with these pathological lesions. The regulation of tau takes place predominantly through post-translational modifications. Extensive reports have been published about tau phosphorylation; however, the other tau post-translational modifications have received much less attention. Here, we review the different types of post-translational modifications of tau including phosphorylation, glycosylation, glycation, prolyl-isomerization, cleavage or truncation, nitration, polyamination, ubiquitination, sumoylation, oxidation and aggregation, with a particular interest towards their relevance in AD.
Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation
2007, Journal of Biological Chemistry
Citation Excerpt :
On the other hand, calpain has also been thought to be a signaling protease, which generates limited proteolysed fragments that possess altered properties in comparison to their parent molecules (18). Cdk5-p25 is different from Cdk5-p35 with regard to solubility, cellular localization, stability, and kinase activity (6–9, 38). Thus, it is important to understand how proteolytic cleavage of p35 to p25 is regulated.
Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser⁸ and Thr¹³⁸ as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr¹³⁸ was dephosphorylated in adult rat, although both Ser⁸ and Thr¹³⁸ were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser⁸ p35 to dual phospho-Ser⁸/Thr¹³⁸ p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr¹³⁸ predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.
Very low levels of methylmercury induce cell death of cultured rat cerebellar neurons via calpain activation
2005, Toxicology
Methylmercury, an environmental neurotoxicant, induces the apoptotic death of cerebellar granule cells in vitro at a low concentration. To further understand the mechanism of cell death, we used a rat cerebellar granule cell culture system to investigate whether the calpain/cyclin-dependent kinase 5 (cdk5)/p35 cascade, an important cascade for neuronal apoptosis, is involved in the methylmercury-induced death. A noteworthy finding was that the cerebellar granular cell death was increased at a very low concentration of methylmercury, 30 nM, which is lower than that previously reported. The high sensitivity to methylmercury indicates that this culture system is useful for studying methylmercury toxicity at very low concentrations. Using this system, we here found that the methylmercury-induced death was inhibited by the calpain inhibitor II. Furthermore, it was shown that, in methylmercury-exposed cells, α-fodrin and tau, calpain substrates, were cleaved to the fragments that disappeared by treatment with the calpain inhibitor II. We next assayed and showed that the intracellular Ca²⁺ concentration in cerebellar granule cells increased after methylmercury exposure in a time- and dose-dependent manner, significantly even at 30 nM. These results indicated that a very low concentration of methylmercury causes the intracellular Ca²⁺ concentration to increase, activates calpain in the cells, and then induces cell death. We further found that the p35 protein was also processed to p25 that forms the cdk5–p25 complex, a hyperactive kinase for tau. However, an immunoblot using the anti-phosphorylated tau antibody showed that there was no increase of phosphorylated tau in methylmercury-exposed cells. These results suggested that methylmercury-induced cell death via calpain activation should not involve the stimulation of tau phosphorylation activity.
Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediate glutamate-induced cleavage of the cyclin-dependent kinase 5 (cdk5) activator p35 in cultured rat hippocampal neurons
2004, Neuroscience Letters
Cyclin-dependent kinase 5 (cdk5) regulates crucial neurobiological events, and deregulation of cdk5 has been implicated in several neurodegenerative disorders. The deregulation is suggested to occur due to cleavage of the cdk5 activator protein p35 to a smaller p25 fragment by the calcium-activated protease calpain. Here we have elucidated the role of different calcium-permeable ionotropic glutamate receptors in the induction of p35 cleavage in cultured rat hippocampal neurons. The glutamate receptor agonists glutamic acid, N-methyl-d-aspartate (NMDA), kainic acid, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were all able to induce p35 cleavage, in a manner depending on extracellular calcium. The effect of glutamate was mediated by NMDA receptors, as it was prevented by the NMDA antagonist MK-801. Cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization, enhanced glutamate-induced p35 cleavage. In immature 6-day-old cultures the non-NMDA agonist kainic acid provoked p35 cleavage, whereas glutamate and NMDA were ineffective. The data suggest that both NMDA and non-NMDA receptors are able to induce p35 cleavage. Different factors, such as maturation state of neurons or desensitization properties of non-NMDA receptors, may determine which receptor predominantly mediates the effect of glutamate on p35 cleavage.

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¹: To whom correspondence should be addressed at University of Kuopio, Dept. of Neuroscience and Neurology, P.O. Box 1627, 70211 Kuopio, Finland. Fax: +358-17-162048. E-mail: [email protected].

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Regular ArticleThe Levels of cdk5 and p35 Proteins and Tau Phosphorylation Are Reduced during Neuronal Apoptosis

Abstract

J. Neurol. Sci.

Brain Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Am. J. Pathol.

Neurosci. Lett.

Dev. Biol.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Brain Res.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3β and cyclin-dependent kinase 5, a component of TPK II

Acta Neuropathol.

A brain-specific activator of cyclin-dependent kinase 5

Nature

Regular Article
The Levels of cdk5 and p35 Proteins and Tau Phosphorylation Are Reduced during Neuronal Apoptosis