Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

An inhibitor with GSK3ß and DYRK1A dual inhibitory properties reduces Tau hyperphosphorylation and ameliorates disease in models of Alzheimer's disease.

Since Alzheimer's disease (AD) is a complex and multifactorial neuropathology, the discovery of multi-targeted inhibitors has gradually demonstrated greater therapeutic potential. Neurofibrillary tangles (NFTs), the main neuropathologic hallmarks of AD, are mainly associated with hyperphosphorylation of the microtubule-associated protein Tau. The overexpression of GSK3ß and DYRK1A has been recognized as an important contributor to hyperphosphorylation of Tau, leading to the strategy of using dual-targets inhibitors for the treatment of this disorder. ZDWX-12 and ZDWX-25, as harmine derivatives, were found good inhibition on dual targets in our previous study. Here, we firstly evaluated the inhibition effect of Tau hyperphosphorylation using two compounds by HEK293-Tau P301L cell-based model and okadaic acid (OKA)-induced mouse model. We found that ZDWX-25 was more effective than ZDWX-12. Then, based on comprehensively investigations on ZDWX-25 in vitro and in vivo, 1) the capability of ZDWX-25 to show a reduction in phosphorylation of multiple Tau epitopes in OKA-induced neurodegeneration cell models, and 2) the effect of reduction on NFTs by 3xTg-AD mouse model under administration of ZDWX-25, an orally bioavailable, brain-penetrant dual-targets inhibitor with low toxicity. Our data highlight that ZDWX-25 is a promising drug for treating AD.

The effects of protein phosphatase inhibitors on the duration of central sensitization of rat dorsal horn neurons following injection of capsaicin.

Protein kinases and phosphatases catalyze opposing reactions of phosphorylation and dephosphorylation, which may modulate the function of crucial signaling proteins in central nervous system. This is an important mechanism in the regulation of intracellular signal transduction pathways in nociceptive neurons. To explore the role of protein phosphatase in central sensitization of spinal nociceptive neurons following peripheral noxious stimulation, using electrophysiological recording techniques, we investigated the role of two inhibitors of protein phosphatase type 2A (PP2A), fostriecin and okadaic acid (OA), on the responses of dorsal horn neurons to mechanical stimuli in anesthetized rats following intradermal injection of capsaicin. Central sensitization was initiated by injection of capsaicin into the plantar surface of the left paw. A microdialysis fiber was implanted in the spinal cord dorsal horn for perfusion of ACSF and inhibitors of PP2A, fostriecin and okadaic acid. We found that in ACSF pretreated animals, the responses to innocuous and noxious stimuli following capsaicin injection increased over a period of 15 min after injection and had mostly recovered by 60 min later. However, pre- or post-treatment with the phosphatase inhibitors, fostriecin or OA, significantly enhanced the effects of capsaicin injection by prolonging the responses to more than 3 hours. These results confirm that blockade of protein phosphatase activity may potentiate central sensitization of nociceptive transmission in the spinal cord following capsaicin injection and indicate that protein phosphatase type 2A may be involved in determining the duration of capsaicin-induced central sensitization.

Induction of apoptosis and inhibition of papilloma formation may signal a new role for okadaic acid.

Okadaic acid (OA), a tumor promoter in the mouse skin carcinogenesis model, has been shown to induce apoptosis in tumor cell lines that harbor H-ras mutations. We examined the effects of OA on mouse keratinocytes with (308) and without (C50) H-ras mutation in vitro and in an in vivo system. Following exposure to varying concentrations of OA over time, the effects of OA in vitro were assessed using microscopic, biochemical and flow cytometric techniques. OA effects on the cells included incorporation of propidium iodide, externalization of phosphatidylserine, and development of hypodiploidy. 308 cells demonstrated typical DNA ladder formation, rapid chromatin and nuclear condensation, while C50 cells demonstrated delayed chromatin condensation and nuclear fragmentation, but no DNA ladder formation. In vivo, OA elicited delayed papilloma formation and reduced tumor multiplicity. Though its mechanism of action is not fully known, we found that OA-induced inhibition of the clonal expansion of initiated cells may be related to the presence or absence of H-ras mutation.

Induction of apoptosis in human oral squamous carcinoma cell lines by protein phosphatase inhibitors.

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in oral epithelial cells we examined the effects of protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CA), on cultured human oral squamous carcinoma (SCC) cell line, SCC-25 cells. After reaching subconfluence these cells were exposed to varying concentrations of the protein phosphatase inhibitors, OA and CA. Both OA and CA induced cell death in SCC-25 cells in a dose-dependent fashion as determined by phase-contrast microscopy and WST-1 cell viability assay. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin was observed. DNA ladder formation also was detected in SCC-25 cells by treatment with OA and CA. The induced nuclear fragmentation and DNA ladder formation were dose-dependent with maximal effect at concentrations of 20 nM OA and 2 nM CA, respectively. OA also induced DNA ladder formation in other human oral SCC cell lines, SCCKN and SCCTF. To further determine if new gene transcription and protein synthesis are required for OA-induced apoptosis in SCC-25 cells, the cells were treated for 48 h with varying concentrations of cycloheximide in the presence of 20 nM OA. Cycloheximide did not protect the cells against OA-induced cytotoxicity and DNA ladder formation. Based on the known selectivity of OA and CA, the present results indicate that the pathway of the apoptosis in the cultured oral SCC cells is in part regulated by protein phosphatase type 1 and type 2A. Our results also indicate that new protein synthesis is not involved in OA-induced apoptosis in SCC-25 cells.

Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid.

We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level.

Phosphorylation status of matrix metalloproteinase 2 in myocardial ischaemia-reperfusion injury.

OBJECTIVE: To investigate whether alterations in the phosphorylation status of matrix metalloproteinase 2 (MMP-2) in the heart may be protective in the setting of ischaemia-reperfusion (IR) injury. DESIGN: In-vitro heart function and biochemical research study. SETTING: University basic science laboratory. INTERVENTIONS: Male Sprague-Dawley rats, weighing 250-350 g. Isolated rat hearts were perfused at constant pressure either aerobically for 75 min or subjected to 20 min of global, no-flow ischaemia followed by 30 min of reperfusion. MAIN OUTCOME MEASURES: Heart mechanical function, MMP-2 activity and troponin I levels. RESULTS: The serine/threonine phosphatase inhibitor okadaic acid (OA) improved the recovery of mechanical function compared with control IR hearts and prevented the loss of troponin I. OA significantly reduced protein phosphatase 2A, but not protein phosphatase 1, activity in perfused hearts. IR stimulated the activation and release of MMP-2 into the coronary effluent in the first 2 min of reperfusion. This was accompanied by a decrease in the remaining activity and protein level of MMP-2 in heart tissue determined at the end of the reperfusion. OA did not alter the IR-stimulated release of MMP-2 into the coronary effluent, but reduced the decrease in MMP-2 in reperfused hearts. The immunoprecipitation of heart homogenates using anti-phosphoserine antibody showed that MMP-2 is phosphorylated. The dephosphorylation of MMP-2 by alkaline phosphatase treatment of homogenates prepared from IR hearts treated with OA significantly increased MMP-2 activity. CONCLUSIONS: These results suggest that the phosphorylation status of MMP-2 is important in its contribution to myocardial IR injury.

Okadaic acid-mediated induction of the c-fos gene in estrogen receptor-negative human breast carcinoma cells utilized, in part, posttranscriptional mechanisms involving adenosine-uridine-rich elements.

Signal transduction via modulation of phosphorylation after selective inhibition of protein phosphatase (PP) 1 and/or PP2A appears to play a role in okadaic acid (OA)-mediated effects. Treatment of several estrogen receptor-negative human breast carcinoma (HBC) cells with 100 nM OA resulted in induction of c-fos, c-myc, and cyclin-dependent kinase inhibitor p21WAF1/CIP1 genes. Transfections of various luciferase reporter constructs in HBC cells revealed involvement of activator protein-1-dependent as well as -independent pathways in induction of the c-fos gene by OA. MDA-MB-468 HBC cells were stably transfected with plasmids expressing luciferase, chimeric luciferase- c-fos 3' untranslated region (3'UTR), or chimeric luciferase-p21WAF1/CIP 3'UTR mRNAs. Expression of chimeric luciferase-c-fos and luciferase-p21WAF1/CIP1 mRNAs was elevated by OA in several independent sublines. Actinomycin D chase experiments revealed an enhanced rate of decay of luciferase-c-fos mRNA, whereas treatment with OA caused approximately 3.5-fold enhanced stability of the chimeric luciferase-c-fos mRNA only. By transfecting different plasmids containing deletions of c-fos 3'UTR, OA-responsive sequences were mapped to an 86-nucleotide, AU-rich region. UV cross-linking experiments using HBC cell cytosolic proteins showed multiple complexes with the AU-rich region subfragments of c-fos, as well as c-myc and p21WAF1/CIP1 mRNAs. OA enhanced binding of a novel Mr approximately 75,000 protein present in the cytosolic extracts of HBC cells to the AU-rich RNA probes of all of the above three genes. Taken together, OA regulation of HBC cell gene expression involves the activator protein-1 pathway, as well as enhanced binding of a novel Mr approximately 75,000 protein to an AU-rich region of the 3'UTRs of the target genes.