Coexpression of human cdk5 and its activator p35 with human protein tau in neurons in brain of triple transgenic mice

The potential contribution of cyclin-dependent protein kinase 5 (cdk5) to hyperphosphorylate protein tau, as claimed in Alzheimer's disease, was investigated in vivo. We generated single, double, and triple transgenic mice that coexpress human cdk5 and its activator p35 as well as human protein tau in cerebral neurons. Whereas expression and increased cdk5-kinase activity was obtained, as measured in vitro and demonstrated in vivo, neither murine nor human protein tau was appreciably phosphorylated in the brain of double and triple transgenic mice. These mice behaved and reproduced normally. Silver impregnation and immunohistochemistry of brain sections demonstrated that neurofilament proteins became redistributed in apical dendrites of cortical neurons. This suggested a cytoskeletal effect, but no other relevant brain pathology became apparent. These observations indicate that cdk5/p35 is not a major protein tau kinase and that cdk5/p35 did not cause neurodegeneration in mouse brain, as opposed to cdk5/p25.     

p35/Cyclin-dependent kinase 5 is required for protection against β-amyloid-induced cell death but not tau phosphorylation by ceramide

Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.     

Kinases and phosphatases and tau sites involved in Alzheimer neurofibrillary degeneration

Microtubule associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer's disease (AD) and related tauopathies; in this form it is the major protein subunit of paired helical filaments (PHF)/neurofibrillary tangles. However, the nature of protein kinases and phosphatases and tau sites involved in this lesion has been elusive. We investigated self-assembly and microtubule assembly promoting activities of hyperphosphorylated tau isolated from Alzheimer disease brain cytosol, the AD abnormally hyperphosphorylated tau (AD P-tau) before and after dephosphorylation by phosphoseryl/phosphothreonyl protein phosphatase-2A (PP-2A), and then rephosphorylation by cyclic AMP-dependent protein kinase (PKA), calcium, calmodulin-dependent protein kinase II (CaMKII), glycogen synthase kinase-3beta (GSK-3beta) and cyclin-dependent protein kinase 5 (cdk5) in different kinase combinations. We found that (i) dephosphorylation of AD P-tau by PP-2A inhibits its polymerization into PHF/straight filaments (SF) and restores its binding and ability to promote assembly of tubulin into microtubules; (ii) rephosphorylation of PP-2A-dephosphorylated AD P-tau by sequential phosphorylation by PKA, CaMKII and GSK-3beta or cdk5, and as well as by cdk5 and GSK-3beta, promotes its self-assembly into tangles of PHF similar to those seen in Alzheimer brain, and (iii) phosphorylation of tau sites required for this pathology are Thr231 and Ser262, along with several sites flanking the microtubule binding repeat region. Phosphorylation of recombinant human brain tau(441) yielded similar results as the PP-2A dephosphorylated AD P-tau, except that mostly SF were formed. The conditions for the abnormal hyperphosphorylation of tau that promoted its self-assembly also induced the microtubule assembly inhibitory activity. These findings suggest that activation of PP-2A or inhibition of either both GSK-3beta and cdk5 or one of these two kinases plus PKA or CaMKII might be required to inhibit Alzheimer neurofibrillary degeneration.     

p35nck5a and cyclin-dependent kinase 5 colocalize in Lewy bodies of brains with Parkinson’s disease

We examined the immunohistochemical localization of p35^nck5a, the regulatory subunit of cyclin-dependent kinase 5 (cdk5), in brains obtained postmortem from patients with Parkinson’s disease (PD) and controls. We found p35^nck5a immunoreactivity in Lewy bodies (LB) in the substantia nigra, locus ceruleus, and neocortex of brains from patients with PD. In addition, p35^nck5a was colocalized with cdk5 immunoreactivity in LB. Cdk5 is the kinase most likely to be responsible for the phosphorylation of neurofilament proteins of LB, which is a crucial step for the formation of the insoluble LB fibrils. Since p35^nck5a regulates the catalytic activity of cdk5 by forming a heterodimer with cdk5, the present results strongly support the hypothesis that a cdk5-p35^nck5a complex is involved in the formation of LB fibrils. Received: 6 December 1996 / Revised, accepted: 31 January 1997     

Axonopathy, tau abnormalities, and dyskinesia, but no neurofibrillary tangles in p25-transgenic mice

Neurofibrillary tangles, one of the pathologic hallmarks of Alzheimer's disease (AD), are composed of abnormally polymerized tau protein. The hyperphosphorylation of tau alters its normal cellular function and is thought to promote the formation of neurofibrillary tangles. Growing evidence suggests that cyclin-dependent kinase 5 (cdk5) plays a role in tau phosphorylation, but the function of the enzyme in tangle formation remains uncertain. In AD, cdk5 is constitutively activated by p25, a highly stable, 25kD protein thought to be increased in the AD brain. To test the hypothesis that p25/cdk5 interactions promote neurofibrillary pathology, we created transgenic mouse lines that overexpress the human p25 protein specifically in neurons. Mice with high transgenic p25 expression have augmented cdk5 activity and develop severe hindlimb semiparalysis and mild forelimb dyskinesia beginning at approximately 3 months of age. Immunohistochemical and ultrastructural analyses showed widespread axonal degeneration with focal accumulation of tau in various regions of the brain and, to a lesser extent, the spinal cord. However, there was no evidence of neurofibrillary tangles in neuronal somata or axons, nor were paired helical filaments evident ultrastructurally. These studies confirm that p25 overexpression can lead to tau abnormalities and axonal degeneration in vivo but do not support the hypothesis that p25-related induction of cdk5 is a primary event in the genesis of neurofibrillary tangles.     

Colocalization and fluorescence resonance energy transfer between cdk5 and AT8 suggests a close association in pre-neurofibrillary tangles and neurofibrillary tangles

Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase that, when activated, induces neurite outgrowth. Recent in vitro studies have shown that cdk5 phosphorylates tau at serine 199, serine 202, and threonine 205 and that p25, an activator of cdk5, is increased in Alzheimer disease (AD). Since tau is hyperphosphorylated at these sites in neurofibrillary tangles, we examined brain tissue from patients with AD and normal elderly control cases to determine whether cdk5 and these phosphoepitopes colocalize in neurofibrillary tangles. Adjacent temporal lobe sections were double immunostained with a polyclonal anti-cdk5 and monoclonal AT8 (which recognizes phosphorylated serine 199, serine 202, and threonine 205 in tau) antibodies. A subset of AT8 phosphotau-positive neurons was immunoreactive for cdk5 in entorhinal (area 28) and perirhinal (area 35) cortices and CA1 of the hippocampus. We assessed the ratio of cdk5-positive cells to AT8-positive cells and found that there is a higher degree of colocalization in pre-neurofibrillary tangles as opposed to intraneuronal and extraneuronal neurofibrillary tangles. We further examined colocalization using fluorescence resonance energy transfer. This suggests a close, stable intermolecular association between cdk5 and phosphorylated tau, consistent with phosphorylation of tau by cdk5 in AD brain.     

Lipoperoxidation is selectively involved in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by extensive neurofibrillary tangle (NFT) formation and neuronal loss in selective neuronal populations. Currently, no clues to the biological events underlying the pathological process have emerged. In Alzheimer disease (AD), which shares with PSP the occurrence of NFTs, advanced glycation end products (AGEs) as well as oxidation adducts have been found to be increased in association with neurofibrillary pathology. The presence and the amount of lipid and protein oxidation markers, as well as of pyrraline and pentosidine. 2 major AGEs, was assessed by biochemical, immunochemical, and immunocytochemical analysis in midbrain tissue from 5 PSP cases, 6 sporadic AD cases, and 6 age-matched control cases. The levels of 4-hydroxynonenal (HNE) and thiobarbituric acid reactive substances (TBARS), 2 major products of lipid peroxidation, were significantly increased by 1.6-fold (p < 0.04) and 3.9-fold (p < 0.01), respectively, in PSP compared with control tissues, whereas in AD only TBARS were significantly increased. In PSP tissue the intensity of neuronal HNE immunoreactivity was proportional to the extent of abnormal aggregated tau protein. The amount of protein oxidation products and AGEs was instead similar in PSP and control tissues. In AD, a higher but not significant level of pyrraline and pentosidine was measured, whereas the level of carbonyl groups was doubled. These findings indicate that in PSP, unlike in AD, lipid peroxidation is selectively associated with NFT formation. The intraneuronal accumulation of toxic aldehydes may contribute to hamper tau degradation, leading to its aggregation in the PSP specific abnormal filaments.     

p35/cdk5 binds and phosphorylates beta-catenin and regulates beta-catenin/presenilin-1 interaction

The neuronal cyclin-dependent kinase p35/cdk5 comprises a catalytic subunit (cdk5) and an activator subunit (p35). To identify novel p35/cdk5 substrates, we utilized the yeast two-hybrid system to screen for human p35 binding partners. From one such screen, we identified beta-catenin as an interacting protein. Confirmation that p35 binds to beta-catenin was obtained by using glutathione S-transferase (GST)-beta-catenin fusion proteins that interacted with both endogenous and transfected p35, and by showing that beta-catenin was present in p35 immunoprecipitates. p35 and beta-catenin also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that p35/cdk5 phosphorylates beta-catenin. beta-catenin also binds to presenilin-1 and altered beta-catenin/presenilin-1 interactions may be mechanistic in Alzheimer's disease (AD). Abnormal p35/cdk5 activity has also been suggested to contribute to AD. We therefore investigated how modulation of p35/cdk5 activity influenced beta-catenin/presenilin-1 interactions. Inhibition of p35/cdk5 with roscovitine did not alter the steady state levels of either beta-catenin or presenilin-1 but reduced the amount of presenilin-1 bound to beta-catenin. Thus, p35/cdk5 binds and phosphorylates beta-catenin and regulates its binding to presenilin-1. The findings reported here therefore provide a novel molecular framework to connect p35/cdk5 with beta-catenin and presenilin-1 in AD.     

Tau neurofibrillary pathology and microtubule stability

We previously reported that nonomolar concentrations of Taxol and several structurally diverse microtubule (MT)-stabilizing agents significantly enhanced the survival of neurons in the presence of fibrils of amyloid beta peptide (Abeta). Pretreatment of neurons with MT-stabilizing drugs also blocked Abeta-induced activation of tau hyperphosphorylation. Although tau is a substrate for several kinases, we initially focused on cdk5, as this tau kinase has been shown to be activated in Abeta-treated neurons and Alzheimer's disease (AD) brain. In an in vitro kinase assay, Taxol inhibited activation of cdk5 by Abeta. In addition, the proposed cellular cascade in which calpain activation leads to cleavage of the cdk5 regulator, p35, to the strong kinase activator p25 was also prevented. Taxol did not directly inhibit the activity of either cdk5 or calpain, indicating that other cellular components are required for the effect of the drug on Abeta activation of tau phosphorylation. Our results suggest that drugs that interact with MTs can alter signaling events in neurons, possibly because some MTs play a role in organizing protein complexes involved in responses to Abeta. Thus the cytoskeletal network may serve as a biosensor of cellular well-being.     

Cdk5 as a drug target for the treatment of Alzheimer's disease

Cyclin-dependent kinase-5 (cdk5) is suggested to play a role in tau phosphorylation and contribute to the pathogenesis of Alzheimer’s disease (AD). One of its activators, p25, is dramatically increased in AD brains where p25 and cdk5 are colocalized with neurofibrillary tangles. Several animal models have shown a correlation of p25/cdk5 activities with tau phosphorylation. Overexpression of p25/cdk5 in nueronal cultures not only leads to tau phosphorylation but also cytoskeletal abnormalities and neurodegeneration. Therefore, cdk5 kinase inhibitors are potential therapeutic agents for the treatment of AD. Availability of potent, selective, brain permeable cdk5 inhibitors and relevant animal models in which their efficacy can be treated will be critical in the development of these inhibitors.     

Increase in the relative expression of tau with four microtubule binding repeat regions in frontotemporal lobar degeneration and progressive supranuclear palsy brains

Some cases of familial frontotemporal dementia (FTD) leading to frontotemporal lobar degeneration (FTLD) are caused by mutations in tau on chromosome 17 (FTDP-17). Certain mutations alter the ratio between four (4R tau) and three (3R tau) repeat tau isoforms whereas cases with progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) mainly have 4R tau brain pathology. We assessed tau mRNA and protein levels in frontal cortex from 15 sporadic FTLD, 21 PSP, 5 CBD, 15 Alzheimer’s disease (AD) and 16 control brains. Moreover, we investigated the disease association and possible tau splicing effects of the tau H1 haplotype. Cases with FTLD and PSP had lower tau mRNA levels than control brains. When analyzing 4R tau and 3R tau mRNA separately, control subjects displayed a 4R tau/3R tau ratio of 0.48. Surprisingly, FTLD brains displayed a more elevated ratio (1.32) than PSP brains (1.12). Also, several FTLD and PSP cases had higher 4R tau/3R tau mRNA than FTDP-17 cases, included as reference tissues, and the ratio increase was seen regardless of underlying histopathology, i.e. both for tau-positive and tau-negative FTLD cases. Furthermore, total tau protein levels were slightly decreased in both FTLD and AD as compared to control subjects. Finally, we confirmed the association of tau H1 with PSP, but could not find any haplotype-related effect on tau exon 10 splicing. In conclusion, we demonstrated increased but largely variable 4R tau/3R tau mRNA ratios in FTLD and PSP cases, suggesting heterogeneous pathophysiological processes within these disorders.     

Cdk5 and munc-18/p67 co-localization in early stage neurofibrillary tangles-bearing neurons in Alzheimer type dementia brains

Hyperphosphorylation of tau protein occurs during the formation of paired helical filament (PHF) in the brain with Alzheimer's disease. As previously reported, cyclin-dependent kinase (cdk) 5 can phosphorylate tau at the site of abnormally phosphorylated in PHF. To characterize the relationship between cdk5 and PHF-tau, we investigated the localization of cdk5 and its regulator, p67 (munc 18), in the hippocampus and temporal lobes from 12 Alzheimer type dementia (ATD) patients and 5 controls using immunohistochemical procedures. The specificity of antibodies was confirmed with Western blot analysis. Anti-cdk5 antibody diffusely stained the perikarya of some tau2-positive or neurofibrillary tangle (NFT)-bearing neurons in ATD brains, while cdk5-positive staining was scarcely found in control brains. Anti-p67 antibody also showed stronger immunoreactivity of pyramidal neurons in ATD brains than in control brains. Double immunostaining with anti-cdk5 and anti-p67 antibodies revealed co-localization of both molecules in some pyramidal neurons. These findings suggest that cdk5 is activated by p67 at the early stage of NFT formation and accelerates NFT formation. In cdk5-positive and p67-negative neurons, cdk5 may be activated by other regulator molecules such as p35. In addition, cdk5-positive reactive astrocytes were found close to cdk5-positive NFT-bearing neurons m ATD brains but not in control brains, suggesting a correlation between NFT and reactive astrocytes.     

Localization and expression of cdc2 and cdk4 in Alzheimer brain tissue.

Two regulators of the eukaryotic cell cycle, cell division cycle 2 (cdc2) and cyclin-dependent kinase 4 (cdk4), have been reported to be related to Alzheimer's disease (AD) pathology, and especially to hyperphosphorylated tau protein. Using well-characterized polyclonal antibodies which recognize the C termini of cdc2 kinase and cdk4, we examined by immunohistochemistry brain tissues from patients with non-neurological conditions, AD and cerebral infarction. Semiquantitative mRNA analysis by RT-PCR was also done using non-neurological and AD brains. In AD, as previously reported, the antibody to cdc2 showed positive staining of a few intracytoplasmic neurofibrillary tangles (NFTs). In addition, this antibody gave positive immunolabelling in astrocytes and capillaries in all brains studied. In both AD and cerebral infarct cases, the staining of astrocytes was more intense than in non-neurological brain tissue. In all cases, the antibodies to cdk4 showed positive immunolabelling in the nuclei of all cell types except neurons. In AD tissue, the antibody showed additional staining of neuronal nuclei and cytoplasm. In contrast to a previous report, we did not find positive labelling of NFTs with the anti-cdk4 antibody. In infarct areas, particularly strong nuclear staining in glial cells was seen. The relative levels of cdk4 mRNA in AD brains were higher than those in controls. These data suggest that cdc2 kinase appears in glial cells capable of cell division and may play a role in the regulation of amyloid precursor protein processing and NFT formation in neurons. As suggested in a report on rat brain, neuronal expression of cdk4 may reflect some pathological process in damaged cells in AD.     

Epistasis between tau phosphorylation regulating genes (CDK5R1 and GSK‐3β) and Alzheimer’s disease risk

Objective – Glycogen synthase kinase‐3β (GSK‐3β) and cyclin‐dependent kinase 5 (CDK5) have been implicated as two major protein kinases involved in the abnormal hyperphosphorylation of tau in Alzheimer’s disease (AD) brain, and the development of neurofibrillary tangles. CDK5 regulatory subunit 1 (CDK5R1) encodes for p35, a protein required for activation of CDK5. As both CDK5R1 and GSK‐3β genes are related to phosphorylation of tau, we examined the combined contribution of these genes to the susceptibility for AD. Methods – In a case–control study in 283 AD patients and 263 healthy controls, we examined the combined effects between CDK5R1 (3′‐UTR, rs735555) and GSK‐3β (?50, rs334558) polymorphisms on susceptibility to AD. Results – Subjects carrying both the CDK5R1 (3′‐UTR, rs735555) AA genotype and the GSK‐3β (?50, rs334558) CC genotype had a 12.5‐fold decrease in AD risk (adjusted by age, sex and APOE status OR = 0.08, 95% CI = 0.01–0.76, P = 0.03), suggesting synergistic effects (epistasis) between both genes. Conclusion – These data support a role for tau phosphorylation regulating genes in risk for AD.     

Deregulation of cdk5 in Hippocampal sclerosis

Hippocampal sclerosis (HS) is the most common cause of chronic medically refractory epilepsy in adults. Histologically, HS is characterized by segmental neuronal loss and gliosis. Although neuronal loss is important to the pathophysiology of HS, the molecular mechanisms underlying the neuronal loss remain uncertain. Recently, it has been appreciated that proteins important in neurodevelopment may also have a role in neurodegeneration. Cyclin-dependent kinase 5 (cdk5), known to be crucial in development of the normal cerebral cortex, has now been shown as pivotal in several cell death paradigms, including apoptosis and necrosis. Deregulation of cdk5 by p25 causes hyperphosphorylation of tau and may contribute to pathology in several neurodegenerative conditions. Furthermore, it has been shown that after transient forebrain ischemia, cdk5 causes specific death of CA1 neurons in the rat hippocampus by direct phosphorylation of the NR2A subunit of the NMDA receptor and subsequent excitotoxicity. Because apoptosis, necrosis, and excitotoxicity are all thought to contribute to neuronal loss in HS, we hypothesized that abnormalities of the cdk5 pathway would accompany this disorder. Surgically resected cases of HS with adjacent histologically normal lateral temporal cortex were examined for cdk5 and its activator p35/p25. We consistently found increased immunoreactivity for p35/p25 in surviving neurons within areas of neuronal loss compared with areas where neurons were preserved. Western blots showed the ratio of p25 to p35 to be greater in diseased hippocampi than in the adjacent histologically normal temporal lobe. Histone-based kinase assays demonstrated increased activity of the p25-cdk5 complex in HS compared with the temporal lobe despite neuronal loss in the hippocampal samples. Our results suggest that p25 is pathologically increased in HS and that deregulation of cdk5 by p25 might contribute to neuronal death in this condition.     

Cdk5 inhibits anterograde axonal transport of neurofilaments but not that of tau by inhibition of mitogen-activated protein kinase activity

Cyclin-dependent kinase 5 (cdk5) inhibits neurofilament (NF) anterograde axonal transport while p42/44 mitogen-activated protein kinase (MAPk) promotes it. Since cdk5 is known to inhibit MAP kinase activity, we examined whether or not cdk5 inhibits anterograde NF transport via inhibition of MAPk activity. To accomplish this, we manipulated the activity of these kinases in differentiated NB2a/d1 cells, and monitored anterograde axonal transport of green fluorescent protein-conjugated-NF-M (GFP-M) and cyan fluorescent protein-conjugated (CFP)-tau. The cdk5 inhibitor roscovitine increased anterograde axonal transport of GFP-M and CFP-tau; transfection with cdk5/p25 inhibited transport of both. Inhibition of MAPk activity by PD98059 or expression of dominant-negative MAPk inhibited anterograde GFP-M transport, while expression of constitutively active MAPk enhanced it; these treatments did not affect CFP-tau transport. PD98059 prevented roscovitine-mediated enhancement of GFP-M transport, but did not prevent enhancement of CFP-tau transport. Co-transfection with constitutively activated MAPk prevented the inhibition of GFP-M transport that normally accompanied transfection with cdk5/p25, but did not prevent inhibition of tau transport by cdk5/p25. Finally, the extent of inhibition of GFP-M axonal transport by PD98059 was not additive to that derived from transfection with cdk5/p35, and the increase in NF transport that accompanies roscovitine treatment was not additive to that derived from transfection with constitutively activated MAPk, suggesting that the influence of these kinases on NF transport was within the same, rather than distinct, pathways. These findings suggest that axonal transport of tau and NFs is under the control of distinct kinase cascades, and that cdk5 inhibits NF transport at least in part by inhibiting MAPk.     

S/P and T/P phosphorylation is critical for tau neurotoxicity in Drosophila

The microtubule-associated protein tau is hyperphosphorylated abnormally in AD and related neurodegenerative disorders. Many phospho epitopes created by proline directed kinases (SP/TP sites) show relative specificity for disease states. To test whether phosphorylation at the disease-associated SP/TP sites affects tau toxicity in vivo, we expressed a form of tau in Drosophila in which all SP/TP sites are mutated to alanine. We find that blocking phosphorylation at SP/TP motifs markedly reduces tau toxicity in vivo. Using phosphorylation-specific antibodies, we identify a positive correlation between increased phosphorylation at disease-associated sites and neurotoxicity. We use the phosphorylation-incompetent version of tau to show that kinase and phosphatase modifiers of tau neurotoxicity, including cdk5/p35, the JNK kinase hemipterous and PP2A act via SP/TP phosphorylation sites. We provide direct evidence in an animal model system to support the role of phosphorylation at SP/TP sites in playing a critical role in tau neurotoxicity.     

PHF-like tau phosphorylation in mammalian hibernation is not associated with p25-formation

In Alzheimer’s disease and related disorders, hyperphosphorylation of tau is associated with an increased activity of cyclin dependent kinase 5 (cdk5). Elevated cdk5 activity is thought to be due to the formation of p25 and thereby represents a critical element in the dysregulation of tau phosphorylation under pathological conditions. However, there is still a controversy regarding the correlation of p25 generation and tau pathology. Recently, we demonstrated physiological, paired helical filament-like tau phosphorylation that reversibly occurs in hibernating mammals. Here we used this model to test whether the tau phosphorylation in hibernation is associated with the formation of p25. Analysing brain material of arctic ground squirrels and Syrian hamsters we found no evidence for a hibernation dependent generation of p25. Hence, we suppose that phosphorylation of tau does not require the formation of p25. Instead we suggest that the truncation of p35 to p25 represents a characteristic of pathological alterations and may contribute to aggregation and deposition of hyperphosphorylated tau.     

New approaches to the discovery of cdk5 inhibitors

Cyclin-dependent kinase 5 (cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. This family is known for its role in the cell cycle, but cdk5 differs due to its interaction with activators p35 or p39, both abundant in post-mitotic neurons. Cdk5 is not known to have a role in cell cycle regulation at all, but is known to be an important modulator of neuronal activity. Cdk5 has been an attractive target for CNS diseases for a number of years. Among its attractions is the possibility that inhibitors will prevent the pathological phosphorylation of tau and neurofibrillary pathology in both Alzheimer's disease and tauopathies. More recently, there has been evidence that cdk5 is involved in the processing of pain and therefore inhibitors would also have potential therapeutic value for acute pain. Several classes of potent chemical inhibitors for cdk5 have been identified but most are competitive with the ATP binding site, resulting in a lack of specificity among the other cyclin-dependent kinases as well as other ATP-dependent kinases. We are working to discover specific inhibitors that might disrupt the interaction of tau and cdk5 at sites other than the ATP binding site. We are screening our compound library of 110,000 compounds using the full length tau as a substrate and will separate ATP competitive from non-competitive binders. In addition, we are taking a computational approach with virtual screening to identify non-ATP-competitive binders. These two approaches may lead to the discovery of site-specific inhibitors for tau and cdk5 interactions rather than competitive inhibitors for ATP binding. The hope is that non-ATP competitive compounds will more likely be selective and will be better therapeutics.