Precision proteoform design for 4R tau isoform selective templated aggregation.

Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.

PSEN1 E280A Cholinergic-like Neurons and Cerebral Spheroids Derived from Mesenchymal Stromal Cells and from Induced Pluripotent Stem Cells Are Neuropathologically Equivalent.

Alzheimer's disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jelly's mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPß fragments, produce eAß42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening. Design, Setting, and Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated. Exposures: Testing for SARS-CoV-2. Main Outcomes and Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected. Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures. Conclusions and Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.

The proline-rich domain promotes Tau liquid-liquid phase separation in cells.

Tau protein in vitro can undergo liquid-liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.

A farnesyltransferase inhibitor activates lysosomes and reduces tau pathology in mice with tauopathy.

Tau inclusions are a shared feature of many neurodegenerative diseases, among them frontotemporal dementia caused by tau mutations. Treatment approaches for these conditions include targeting posttranslational modifications of tau proteins, maintaining a steady-state amount of tau, and preventing its tendency to aggregate. We discovered a new regulatory pathway for tau degradation that operates through the farnesylated protein, Rhes, a GTPase in the Ras family. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib reduced Rhes and decreased brain atrophy, tau inclusions, tau sumoylation, and tau ubiquitination in the rTg4510 mouse model of tauopathy. In addition, lonafarnib treatment attenuated behavioral abnormalities in rTg4510 mice and reduced microgliosis in mouse brain. Direct reduction of Rhes in the rTg4510 mouse by siRNA reproduced the results observed with lonafarnib treatment. The mechanism of lonafarnib action mediated by Rhes to reduce tau pathology was shown to operate through activation of lysosomes. We finally showed in mouse brain and in human induced pluripotent stem cell-derived neurons a normal developmental increase in Rhes that was initially suppressed by tau mutations. The known safety of lonafarnib revealed in human clinical trials for cancer suggests that this drug could be repurposed for treating tauopathies.

Non-contact monitoring of extra-cellular field potentials with a multi-electrode array.

Developing tools to enable non-invasive, high-throughput electrophysiology measurements of large functional-networks of electrogenic cells used as in vitro disease models for the heart and brain remains an outstanding challenge for preclinical drug discovery, where failures are costly and can prove to be fatal during clinical trials. Here we demonstrate, for the first time, that it is possible to perform non-contact monitoring of extra-cellular field potentials with a multi-electrode array (MEA). To do this preliminary demonstration we built a prototype with a custom mechanical stage to micro-position cells grown on conventional glass coverslips over the recording surface of a MEA sensor. The prototype can monitor extra-cellular fields generated by multi-cellular networks in a non-contact configuration, enabling a single MEA sensor to probe different cultures in succession, without fouling or degrading its sensitive electronic surface. This first demonstration with easy to culture cardiomyocyte cells and a prototype device points to the exciting possibility for instrument development leading to more efficient and cost-effective drug screening paradigms for cardiovascular and neurological diseases.

Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs).

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimer's disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tau-heparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse brain slice culture. Together, these results suggest novel strategies to halt tau transmission.

A Primate lncRNA Mediates Notch Signaling during Neuronal Development by Sequestering miRNA.

Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex.

MUC1* ligand, NM23-H1, is a novel growth factor that maintains human stem cells in a more naïve state.

We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.

MicroRNA profiling reveals two distinct p53-related human pluripotent stem cell states.

Reprogramming methodologies have provided multiple routes for achieving pluripotency. However, pluripotency is generally considered to be an almost singular state, with subtle differences described between induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs). We profiled miRNA expression levels across 49 human cell lines, including ESCs, iPSCs, differentiated cells, and cancer cell lines. We found that the resulting miRNA profiles divided the iPSCs and hESCs examined into two distinct categories irrespective of the cell line origin. The miRNAs that defined these two pluripotency categories also distinguished cancer cells from differentiated cells. Transcriptome analysis suggested that several gene sets related to p53 distinguished these categories, and overexpression of the p53-targeting miRNAs miR-92 and miR-141 in iPSCs was sufficient to change their classification status. Thus, our results suggest a subdivision of pluripotent stem cell states that is independent of their origin but related to p53 network status.

Silencing of CDK5 reduces neurofibrillary tangles in transgenic alzheimer's mice.

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 (3×Tg-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.

The Amphimedon queenslandica genome and the evolution of animal complexity.

Sponges are an ancient group of animals that diverged from other metazoans over 600 million years ago. Here we present the draft genome sequence of Amphimedon queenslandica, a demosponge from the Great Barrier Reef, and show that it is remarkably similar to other animal genomes in content, structure and organization. Comparative analysis enabled by the sequencing of the sponge genome reveals genomic events linked to the origin and early evolution of animals, including the appearance, expansion and diversification of pan-metazoan transcription factor, signalling pathway and structural genes. This diverse 'toolkit' of genes correlates with critical aspects of all metazoan body plans, and comprises cell cycle control and growth, development, somatic- and germ-cell specification, cell adhesion, innate immunity and allorecognition. Notably, many of the genes associated with the emergence of animals are also implicated in cancer, which arises from defects in basic processes associated with metazoan multicellularity.

MUC1* mediates the growth of human pluripotent stem cells.

The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast "feeder cells". Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells.

MicroRNA dysregulation is observed in different types of cancer. MiR-21 up-regulation has been reported for the majority of cancers profiled to date; however, knowledge is limited on the mechanism of action of miR-21, including identification of functionally important targets that contribute to its proproliferative and antiapoptotic actions. In this study, we show for the first time that miR-21 targets multiple important components of the p53, transforming growth factor-beta (TGF-beta), and mitochondrial apoptosis tumor-suppressive pathways. Down-regulation of miR-21 in glioblastoma cells leads to derepression of these pathways, causing repression of growth, increased apoptosis, and cell cycle arrest. These phenotypes are dependent on two of the miR-21 targets validated in this study, HNRPK and TAp63. These findings establish miR-21 as an important oncogene that targets a network of p53, TGF-beta, and mitochondrial apoptosis tumor suppressor genes in glioblastoma cells.

MicroRNAs: regulators of oncogenesis and stemness.

MicroRNAs (miRNAs) are essential post-transcriptional regulators that determine cell identity and fate. Aberrant expression of miRNAs can lead to diseases, including cancer. Expression of many miRNAs in the de-differentiated brain tumor cancer stem cells resembles that of neural stem cells. In this issue of BMC Medicine, Silber et al provide evidence of the expression of such miRNAs and their potential to mediate differentiation in both stem cell populations. In this commentary, we discuss the known functions of miRNAs in cancer and stem cells, their therapeutic potential and how the findings of Silber et al provide insight into the role of miR-124/miR-137 dysregulation in glioblastomas.

Heterogeneous dysregulation of microRNAs across the autism spectrum.

microRNAs (miRNAs) are approximately 21 nt transcripts capable of regulating the expression of many mRNAs and are abundant in the brain. miRNAs have a role in several complex diseases including cancer as well as some neurological diseases such as Tourette's syndrome and Fragile x syndrome. As a genetically complex disease, dysregulation of miRNA expression might be a feature of autism spectrum disorders (ASDs). Using multiplex quantitative polymerase chain reaction (PCR), we compared the expression of 466 human miRNAs from postmortem cerebellar cortex tissue of individuals with ASD (n = 13) and a control set of non-autistic cerebellar samples (n = 13). While most miRNAs levels showed little variation across all samples suggesting that autism does not induce global dysfunction of miRNA expression, some miRNAs among the autistic samples were expressed at significantly different levels compared to the mean control value. Twenty-eight miRNAs were expressed at significantly different levels compared to the non-autism control set in at least one of the autism samples. To validate the finding, we reversed the analysis and compared each non-autism control to a single mean value for each miRNA across all autism cases. In this analysis, the number of dysregulated miRNAs fell from 28 to 9 miRNAs. Among the predicted targets of dysregulated miRNAs are genes that are known genetic causes of autism such Neurexin and SHANK3. This study finds that altered miRNA expression levels are observed in postmortem cerebellar cortex from autism patients, a finding which suggests that dysregulation of miRNAs may contribute to autism spectrum phenotype.

Detection of a microRNA signal in an in vivo expression set of mRNAs.

BACKGROUND: microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of a miRNA to the target mRNA. As a result, translation of the target mRNA is inhibited and the mRNA may be destabilized. The inhibitory effects of miRNAs have been linked to diverse cellular processes including malignant proliferation, apoptosis, development, differentiation, and metabolic processes. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. METHODOLOGY/PRINCIPAL FINDINGS: RNA was extracted from 12 human primary brain tumor biopsies. These samples were used to determine genome-wide mRNA expression levels by microarray analysis and a miRNA profile by real-time reverse transcription PCR. Correlation coefficients were determined for all possible mRNA-miRNA pairs and the distribution of these correlations compared to the random distribution. An excess of high positive and negative correlation pairs were observed at the tails of these distributions. Most of these highest correlation pairs do not contain sufficiently complementary sequences to predict a target relationship; nor do they lie in physical proximity to each other. However, by examining pairs in which the significance of the correlation coefficients is modestly relaxed, negative correlations do tend to predict targets and positive correlations tend to predict physically proximate pairs. A subset of high correlation pairs were experimentally validated by over-expressing or suppressing a miRNA and measuring the correlated mRNAs. CONCLUSIONS/SIGNIFICANCE: Sufficient information exists within a set of tumor samples to detect endogenous correlations between miRNA and mRNA levels. Based on the validations the causal arrow for these correlations is likely to be directed from the miRNAs to the mRNAs. From these data sets, we inferred and validated a tumor suppression pathway linked to miR-181c.

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.