P25 Accumulation Research Articles

Comparison of the Proteome of Huh7 Cells Transfected with Hepatitis B Virus Subgenotype A1, with or without G1862T.

HBeAg is a non-structural, secreted protein of hepatitis B virus (HBV). Its p25 precursor is post-translationally modified in the endoplasmic reticulum. The G1862T precore mutation leads to the accumulation of P25 in the endoplasmic reticulum and activation of unfolded protein response. Using mass spectrometry, comparative proteome profiling of Huh-7 cells transfected with wildtype (WT) or G1862T revealed significantly differentially expressed proteins resulting in 12 dysregulated pathways unique to WT-transfected cells and 7 shared between cells transfected with either WT or G1862T. Except for the p38 MAPK signalling pathway, WT showed a higher number of DEPs than G1862T-transfected cells in all remaining six shared pathways. Two signalling pathways: oxidative stress and cell cycle signalling were differentially expressed only in cells transfected with G1862T. Fifteen pathways were dysregulated in G1862T-transfected cells compared to WT. The 15 dysregulated pathways were involved in the following processes: MAPK signalling, DNA synthesis and methylation, and extracellular matrix organization. Moreover, proteins involved in DNA synthesis signalling (replication protein A (RPA) and DNA primase (PRIM2)) were significantly upregulated in G1862T compared to WT. This upregulation was confirmed by mRNA quantification of both genes and immunofluorescent confocal microscopy for RPA only. The dysregulation of the pathways involved in these processes may lead to immune evasion, persistence, and uncontrolled proliferation, which are hallmarks of cancer.

NbALY916 is involved in potato virus X P25‐triggered cell death in Nicotiana benthamiana

Systemic necrosis often occurs during viral infection of plants and is thought mainly to be the result of long‐term stress induced by viral infection. Potato virus X (PVX) encodes the P25 pathogenicity factor that triggers a necrotic reaction during PVX‐potato virus Ysynergistic coinfection. In this study, we discovered that NbALY916, a multifunctional nuclear protein, could interact with P25. When NbALY916 expression was reduced by tobacco rattle virus (TRV)‐based virus‐induced gene silencing, the accumulation of P25 was increased, which would be expected to cause more severe necrosis. However, silencing of NbALY916 reduced the extent of cell death caused by P25. Furthermore, we found that overexpression of NbALY916 increased the accumulation of H2O2 and triggered more extensive cell death when coexpressed with P25, even though accumulation of P25 was itself reduced by the increased expression of NbALY916. Furthermore, transient expression of P25 specifically induced the expression of NbALY916 mRNA, but not the mRNAs of three other ALYs in Nicotiana benthamiana. In addition, we showed that silencing of NbALY916 or transient overexpression of NbALY916 affected the infection of PVX in N. benthamiana. Our results reveal that NbALY916 has an antiviral role that, in the case of PVX, operates by inducing the accumulation of H2O2 and mediating the degradation of P25.

Cdk5 Inhibitory Peptide Prevents Loss of Neurons and Alleviates Behavioral Changes in p25 Transgenic Mice.

Accumulation of p25 is thought to be a causative risk factor for Alzheimer's disease (AD). As a cleaved product of p35, p25 binds to cyclin-dependent kinase 5 (Cdk5) and leads to the hyperactivity of Cdk5. Then, Cdk5/p25 phosphorylates many pathological substrates related to neurodegenerative diseases. p25 transgenic (Tg) mouse model recaptures some pathological changes of AD, including tau hyperphosphorylation, neurofibrillary tangles, neuroinflammation, and neuronal death, which can be prevented by transgenic expression of Cdk5 inhibitory peptide (CIP) before the insult of p25. In the present study, we would like to know whether adeno-associated virus serotype-9 (AAV9)-mediated CIP can protect neurons after insult of p25 in p25Tg mice. Administration of AAV9-CIP or control virus were delivered in the brain of p25Tg mice via intracerebroventricular infusions following the induction of p25. Western blotting, immunohistochemistry and immunofluorescence assessment, and animal behavioral evaluation were performed. Brain atrophy, neuronal death, tau phosphorylation and inflammation in the hippocampus, and cognitive decline were observed in p25Tg mice. Administration of CIP but not the control virus in p25Tg mice reduced levels of tau phosphorylation and inflammation in the hippocampus, which is correlated with inhibition of brain atrophy and neuronal apoptosis in the hippocampus, and improvement of cognitive decline. Our results provide further evidence that the neurotoxicity of p25 can be alleviated by CIP.

Brain targeting of 9c,11t-Conjugated Linoleic Acid, a natural calpain inhibitor, preserves memory and reduces A\u03b2 and P25 accumulation in 5XFAD mice

Deregulation of Cyclin-dependent kinase 5 (CDK5) by binding to the activated calpain product p25, is associated with the onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Conjugated Linoleic Acid (CLA), a calpain inhibitor, is a metabolite of Punicic Acid (PA), the main component of Pomegranate seed oil (PSO). We have shown recently that long-term administration of Nano-PSO, a nanodroplet formulation of PSO, delays mitochondrial damage and disease advance in a mouse model of genetic Creutzfeldt Jacob disease (CJD). In this project, we first demonstrated that treatment of mice with Nano-PSO, but not with natural PSO, results in the accumulation of CLA in their brains. Next, we tested the cognitive, biochemical and pathological effects of long-term administration of Nano-PSO to 5XFAD mice, modeling for Alzheimer’s disease. We show that Nano-PSO treatment prevented age-related cognitive deterioration and mitochondrial oxidative damage in 5XFAD mice. Also, brains of the Nano-PSO treated mice presented reduced accumulation of Aβ and of p25, a calpain product, and increased expression of COX IV-1, a key mitochondrial enzyme. We conclude that administration of Nano-PSO results in the brain targeting of CLA, and suggest that this treatment may prevent/delay the onset of neurodegenerative diseases, such as AD and CJD.

Multidimensional co-segmentation of longitudinal brain MRI ensembles in the presence of a neurodegenerative process

MRI Segmentation of a pathological brain poses a significant challenge, as the available anatomical priors that provide top-down information to aid segmentation are inadequate in the presence of abnormalities. This problem is further complicated for longitudinal data capturing impaired brain development or neurodegenerative conditions, since the dynamic of brain atrophies has to be considered as well. For these cases, the absence of compatible annotated training examples renders the commonly used multi-atlas or machine-learning approaches impractical.We present a novel segmentation approach that accounts for the lack of labeled data via multi-region multi-subject co-segmentation (MMCoSeg) of longitudinal MRI sequences. The underlying, unknown anatomy is learned throughout an iterative process, in which the segmentation of a region is supported both by the segmentation of the neighboring regions, which share common boundaries, and by the segmentation of corresponding regions, in the other jointly segmented images. A 4D multi-region atlas that models the spatio-temporal deformations and can be adapted to different subjects undergoing similar degeneration processes is reconstructed concurrently.An inducible mouse model of p25 accumulation (the CK-p25 mouse) that displays key pathological hallmarks of Alzheimer disease (AD) is used as a gold-standard to test the proposed algorithm by providing a conditional control of rapid neurodegeneration. Applying the MMCoSeg to a cohort of CK-p25 mice and littermate controls yields promising segmentation results that demonstrate high compatibility with expertise manual annotations. An extensive comparative analysis with respect to current well-established, atlas-based segmentation methods highlights the advantage of the proposed approach, which provides accurate segmentation of longitudinal brain MRIs in pathological conditions, where only very few annotated examples are available.

Inhibition of p25/Cdk5 Attenuates Tauopathy in Mouse and iPSC Models of Frontotemporal Dementia.

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation.SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

From Cdk5 To Alzheimer‘s Disease: Future Challenges

Dr Li-Huei Tsai was born in Taipei, Taiwan. In 1986, she began her PhD at the University of Texas Southwestern (TX, USA). Under the direction of Bradford Ozanne, she graduated in 1990 and joined Ed Harlow’s laboratory at Cold Spring Harbor Laboratory and Massachusetts General Hospital (MA, USA) for postdoctoral training. She was appointed Assistant Professor of Pathology at Harvard Medical School (MA, USA) in 1994, and was promoted to Professor of Pathology in 2002. In 2006, she relocated her laboratory to Massachusetts Institute of Technology (MA, USA) and became the Picower Professor of Neuroscience. Dr Tsai began directing the Neurobiology Program at the Stanley Center for Psychiatric Research in 2007 and she was named Director of the Picower Institute for Learning and Memory in 2009. Dr Tsai aims to elucidate the cellular and molecular mechanisms contributing to the development and manifestation of the pathology and symptoms of Alzheimer’s disease. She demonstrated that dysregulation of cyclin-dependent kinase 5, as the result of p25 accumulation, contributes to increased β-amyloid peptide production, tau pathology and impaired synaptic plasticity. Dr Tsai created the inducible CK-p25 mouse model that exhibits robust neurodegeneration and memory impairment, and used this model to explore novel approaches for therapeutic intervention. She made the unexpected finding that nonselective HDAC inhibitors facilitate the recovery of learning and memory, after massive neuronal loss has occurred in the CK-p25 mice, and they markedly induce dendritic growth and synapse formation. Subsequently HDAC2 was identified to play an essential role in regulating synaptic plasticity and memory formation by binding to the promoters of genes implicated in synaptogenesis, synaptic plasticity and memory. She is currently investigating how dysregulation of chromatin remodeling contributes to the pathogenesis of neurodegeneration and cognitive impairment.

Crosstalk between cdk5 and MEK–ERK signalling upon opioid receptor stimulation leads to upregulation of activator p25 and MEK1 inhibition in rat brain

Cyclin-dependent kinase 5 (cdk5) participates in opioid receptor signalling through complex molecular mechanisms. The acute effects of selective μ-(fentanyl) and δ-(SNC-80) opioid receptor agonists, as well as the chronic effects of morphine (the prototypic opiate agonist mainly acting at μ-receptors), modulating cdk5 and activators p35/p25 and their interactions with neurotoxic/apoptotic factors, dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP-32) and extracellular signal-regulated kinase (ERK) were quantified (Western Blot analyses) in the rat corpus striatum and/or cerebral cortex. To assess the involved mechanisms, MDL28170 was used to inhibit calpain activity and SL327 to disrupt MEK (ERK kinase)-ERK activation. Acute fentanyl (0.1mg/kg) and SNC-80 (10mg/kg) induced rapid (7–60min) 2- to 4-fold increases of p25 content, without induction of cdk5/p25 pro-apoptotic c-Jun NH2-terminal protein kinase or aberrant cleavage of poly(ADP-ribose)-polymerase-1, a hallmark of apoptosis. In contrast, fentanyl and SNC-80 stimulated cdk5-mediated p-Thr75 DARPP-32 (+116–166%; PKA inhibition) and p-Thr286 MEK1 (+21–82%; MEK inactivation), and this latter effect resulted in uncoupling of MEK to ERK signals. Calpain inhibition with MDL28170 (cleavage of p35 to p25) attenuated fentanyl-induced p25 accumulation (−57%), but not the stimulation of p-Thr286 MEK1 or p-Thr75 DARPP-32. MEK–ERK inhibition with SL327 fully prevented fentanyl-induced p25 upregulation. Notably, chronic morphine treatment (10–100mg/kg for 6days) also increased p25 content and p25/p35 ratio (and activated/inactivated MEK1) in rat brain cortex, which indicated that p25 upregulation persisted under the sustained stimulation of μ-opioid receptors. The results demonstrate that the acute stimulation of opioid receptors leads to upregulation of p25 activator through a MEK–ERK and calpain-dependent pathway, and to disruption of MEK–ERK signalling by a cdk5/p35-induced MEK1 inhibition. Moreover, the effects induced by the sustained stimulation of μ-receptors with morphine suggest the participation of cdk5/p25 complex in opiate-induced long-term neuroplasticity.

Dopaminergic and Glutamatergic Signaling Crosstalk in Huntington's Disease Neurodegeneration: The Role of p25/Cyclin-Dependent Kinase 5

Altered glutamatergic and dopaminergic signaling has been proposed as contributing to the specific striatal cell death observed in Huntington's disease (HD). However, the precise mechanisms by which mutant huntingtin sensitize striatal cells to dopamine and glutamate inputs remain unclear. Here, we demonstrate in knock-in HD striatal cells that mutant huntingtin enhances dopamine-mediated striatal cell death via dopamine D(1) receptors. Moreover, we show that NMDA receptors specifically potentiate the vulnerability of mutant huntingtin striatal cells to dopamine toxicity as pretreatment with NMDA increased D(1)R-induced cell death in mutant but not wild-type cells. As potential underlying mechanism of increased striatal vulnerability, we identified aberrant cyclin-dependent kinase 5 (Cdk5) activation. We demonstrate that enhanced Cdk5 phosphorylation and increased calpain-mediated conversion of the Cdk5 activator p35 into p25 may account for the deregulation of Cdk5 associated to dopamine and glutamate receptor activation in knock-in HD striatal cells. Moreover, supporting a detrimental role of Cdk5 in striatal cell death, neuronal loss can be widely prevented by roscovitine, a potent Cdk5 inhibitor. Significantly, reduced Cdk5 expression together with enhanced Cdk5 phosphorylation and p25 accumulation also occurs in the striatum of mutant Hdh(Q111) mice and HD human brain suggesting the relevance of deregulated Cdk5 pathway in HD pathology. These findings provide new insights into the molecular mechanisms underlying the selective vulnerability of striatal cells in HD and identify p25/Cdk5 as an important mediator of dopamine and glutamate neurotoxicity associated to HD.

Rapsyn Interaction with Calpain Stabilizes AChR Clusters at the Neuromuscular Junction

Agrin induces, whereas acetylcholine (ACh) disperses, ACh receptor (AChR) clusters during neuromuscular synaptogenesis. Such counteractive interaction leads to eventual dispersal of nonsynaptic AChR-rich sites and formation of receptor clusters at the postjunctional membrane. However, the underlying mechanisms are not well understood. Here we show that calpain, a calcium-dependent protease, is activated by the cholinergic stimulation and is required for induced dispersion of AChR clusters. Interestingly, the AChR-associated protein rapsyn interacted with calpain in an agrin-dependent manner, and this interaction inhibited the protease activity of calpain. Disrupting the endogenous rapsyn/calpain interaction enhanced CCh-induced dispersion of AChR clusters. Moreover, the loss of AChR clusters in agrin mutant mice was partially rescued by the inhibition of calpain via overexpressing calpastatin, an endogenous calpain inhibitor, or injecting calpeptin, a cell-permeable calpain inhibitor. These results demonstrate that calpain participates in ACh-induced dispersion of AChR clusters, and rapsyn stabilizes AChR clusters by suppressing calpain activity.

Calorie restriction ameliorates neurodegenerative phenotypes in forebrain-specific presenilin-1 and presenilin-2 double knockout mice

Conditional double knockout of presenilin-1 and presenilin-2 (cDKO) in forebrain of mice led to brain atrophy, tau hyperphosphorylation, synaptic dysfunction and cognitive deficit. These brain changes recapitulated most of the neurodegenerative phenotypes of Alzheimer's disease (AD). In this report, we have investigated the effects of 4-month calorie restriction (CR) regimen on different phenotypes in cDKO mice. We found that CR improved novel object recognition and contextual fear conditioning memory in the cDKO mice. Histological and biochemical analysis showed that CR attenuated ventricle enlargement, caspase-3 activation and astrogliosis. In addition, the induction of tau hyperphosphorylation in the cDKO mice was reduced by CR, possibly through reduction of p25 accumulation and aberrant CDK5 activation. Finally, DNA microarray analysis demonstrated that CR could increase the expression of neurogenesis related genes and decrease the expression of inflammation related genes in the hippocampus of cDKO mice. The possible molecular mechanisms of the CR effects on alleviating AD pathogenesis have been discussed.

Involvement of Cdk5/p25 in Digoxin-triggered Prostate Cancer Cell Apoptosis

Cardiac digitalis has been considered to be a treatment for breast cancer. Our previous study indicates that digoxin, one member in digitalis, decreases the proliferation of prostate cancer cells, but the mechanisms remain unclear. In the present study, Ca(2+) proved to be an important factor in digoxin-triggered prostate cancer cell death. Because cyclin-dependent kinase (Cdk)5 and p35 cleavage (p25 formation) have been reported to be targets of intracellular Ca(2+), and subsequently correlated to apoptosis, we not only demonstrated first that Cdk5, p35, and p25 proteins were all expressed in prostate cancer cells (including lymph node carcinoma of the prostate (LNCaP) and DU-145 cells), but also showed where p25 formation and Cdk5 kinase activity were affected by treatment with digoxin. The inhibitor of p35 cleavage (calpeptin) was used to reduce p25 formation, and the result suggested that p25 accumulation might be the major cause of digoxin-triggered LNCaP cell death. Butyrolactone-I and roscovitine, two Cdk5 kinase inhibitors, were also found to prevent digoxin-triggered LNCaP cell death. In addition, treatment of siRNA-Cdk5 diminished digoxin-triggered cell death, as compared with the treatments of siRNA-Cdk1 or siRNA-Cdk2, which implies the specific involvement of Cdk5 in digoxin-triggered cell death. Caspase inhibitor set and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay were used to demonstrate that digoxin-triggered LNCaP cell apoptosis through Cdk5 activation. These results suggest that Cdk5/p35 and p25 are novel players in digoxin-triggered prostate cancer cell apoptosis and, therefore, become potential therapeutic targets.

Aberrant Cdk5 Activation by p25 Triggers Pathological Events Leading to Neurodegeneration and Neurofibrillary Tangles

Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. Here, we generated inducible transgenic mouse lines overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directed Cdk5 to pathological substrates. These animals exhibited neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau was hyperphosphorylated at many epitopes, aggregated tau accumulated, and neurofibrillary pathology developed progressively in these animals. Our cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology.

A survey of Cdk5 activator p35 and p25 levels in Alzheimer’s disease brains

P25, a calpain cleavage product of the cyclin-dependent kinase 5 (Cdk5) activator p35, causes prolonged activation of Cdk5. Although p25 has been shown to accumulate in brains of patients with Alzheimer’s disease (AD), it is not known whether p25 accumulation in AD is brain region-specific. We analyzed the amounts of p25 and p35 in human autopsy samples from multiple brain regions including frontal cortex, inferior parietal cortex and hippocampus using immunoblotting assays. Our results show that the p25–p35 indices are higher in AD than in the control groups in all three brain regions. The most significant difference in p25–p35 indices between AD and control groups is in the frontal cortex. No significant difference in calpain activity between AD and control groups is observed, indicating that postmortem calpain activation cannot account for the elevation of p25/p35 ratios in AD brains. Our results support the notion that p25 accumulation deregulates Cdk5 activity in AD brains, and the deregulated Cdk5 activity may contribute to the pathogenesis of AD.

Involvement of cyclin dependent kinase5 activator p25 on tau phosphorylation in mouse brain

P35 or its truncated fragment p25 is required for cyclin dependent kinase (Cdk)5 activation. It has been reported that p25 is accumulated in the brain of Alzheimer's disease (AD) patients and that p25/Cdk5 induces high phosphorylation of tau and apoptosis in cultured neurons (Nature 402 (1999) 615). Our investigation of AD brain did not show specific accumulation of p25. Exposure to Ca ionophore (A23187) at 10 −6 M induced p25 accumulation in rat primary hippocampal neurons, causing neuronal death without showing hyperphosphorylation of tau. Transgenic mice expressing p25 showed the accumulation of p25 but neither hyperphosphorylation of tau nor neuronal death was shown in these mice. The feature of these mice was the progression of cell growth in pituitary gland. These results suggest that overexpression of p25 lead to the activation of cell cycle but not to the direct phosphorylation of tau.

Oxygen free radical injury is sufficient to cause some Alzheimer-type molecular abnormalities in human CNS neuronal cells.

Cell loss and neuritic/cytoskeletal lesions represent two of the major categories of dementia-associated structural abnormalities in Alzheimer's disease (AD). Cell loss is ultimately mediated by apoptosis and mitochondrial DNA damage due to enhanced sensitivity to oxidative stress, but the mechanism responsible for the neuritic/cytoskeletal lesions including the abnormal proliferation of cortical neurites is not known. This study examines the potential role of oxygen free radical injury as a factor contributing to both cell death and neuritic sprouting cascades in AD. PNET2 human neuronal cells were treated with H2O2 (8 micro M to 88 micro M) for 24 hours and then analyzed for viability, DNA damage, and pro-apoptosis, survival, and sprouting gene expression and signaling. H2O2-treatment resulted in dose-dependent increases in cell death due to genomic and mitochondrial DNA damage associated with increased levels of 8-OHdG and the p53 and CD95 pro-apoptosis genes, reduced levels of the Bcl-2 survival gene, activation of JNK and p38 stress kinases, and inhibition of PI3 kinase survival signaling. However, the H2O2-treated cells also manifested increased expression of growth and sprouting molecules, including GAP-43, nitric oxide synthase 3, neuronal thread protein (NTP; approximately 17 kD and approximately 21 kD forms), proliferating cell nuclear antigen, and phospho-Erk MAPK, and normal levels of the AD-associated approximately 41 kD NTP species, cyclin dependent kinase 5 (cdk-5), and phospho-tau. In addition, the H2O2-treated cells had increased levels of p25, the catalytically active and stable cleavage product of p35, which regulates cdk-5 activity. Previous studies demonstrated p25 accumulation in AD brains and p25-induced hyperphosphorylation of tau and neuronal apoptosis. The findings herein suggest that oxygen free radical injury in human CNS neuronal cells is sufficient to cause some but not all of the pro-death and pro-sprouting molecular abnormalities that occur in AD.

Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.

Cyclin-dependent kinase 5 (Cdk5) is required for proper development of the mammalian central nervous system. To be activated, Cdk5 has to associate with its regulatory subunit, p35. We have found that p25, a truncated form of p35, accumulates in neurons in the brains of patients with Alzheimer's disease. This accumulation correlates with an increase in Cdk5 kinase activity. Unlike p35, p25 is not readily degraded, and binding of p25 to Cdk5 constitutively activates Cdk5, changes its cellular location and alters its substrate specificity. In vivo the p25/Cdk5 complex hyperphosphorylates tau, which reduces tau's ability to associate with microtubules. Moreover, expression of the p25/Cdk5 complex in cultured primary neurons induces cytoskeletal disruption, morphological degeneration and apoptosis. These findings indicate that cleavage of p35, followed by accumulation of p25, may be involved in the pathogenesis of cytoskeletal abnormalities and neuronal death in neurodegenerative diseases.