Candidate Alzheimer's Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression.

MicroRNAs have been demonstrated as key regulators of gene expression in the etiology of a range of diseases including Alzheimer's disease (AD). Recently, we identified miR-483-5p as the most upregulated miRNA amongst a panel of miRNAs in blood plasma specific to prodromal, early-stage Alzheimer's disease patients. Here, we investigated the functional role of miR-483-5p in AD pathology. Using TargetScan and miRTarBase, we identified the microtubule-associated protein MAPT, often referred to as TAU, and the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), known to phosphorylate TAU, as predicted direct targets of miR-483-5p. Employing several functional assays, we found that miR-483-5p regulates ERK1 and ERK2 at both mRNA and protein levels, resulting in lower levels of phosphorylated forms of both kinases. Moreover, miR-483-5p-mediated repression of ERK1/2 resulted in reduced phosphorylation of TAU protein at epitopes associated with TAU neurofibrillary pathology in AD. These results indicate that upregulation of miR-483-5p can decrease phosphorylation of TAU via ERK pathway, representing a compensatory neuroprotective mechanism in AD pathology. This miR-483-5p/ERK1/TAU axis thus represents a novel target for intervention in AD.

A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis.

Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants.

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

A New Inhibitor of Tubulin Polymerization Kills Multiple Cancer Cell Types and Reveals p21-Mediated Mechanism Determining Cell Death after Mitotic Catastrophe.

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative-OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

microRNA diagnostic panel for Alzheimer's disease and epigenetic trade-off between neurodegeneration and cancer.

microRNAs (miRNAs) have been extensively studied as potential biomarkers for Alzheimer's disease (AD). Their profiles have been analyzed in blood, cerebrospinal fluid (CSF) and brain tissue. However, due to the high variability between the reported data, stemming from the lack of methodological standardization and the heterogeneity of AD, the most promising miRNA biomarker candidates have not been selected. Our literature review shows that out of 137 miRNAs found to be altered in AD blood, 36 have been replicated in at least one independent study, and out of 166 miRNAs reported as differential in AD CSF, 13 have been repeatedly found. Only 3 miRNAs have been consistently reported as altered in three analyzed specimens: blood, CSF and the brain (hsa-miR-146a, hsa-miR-125b, hsa-miR-135a). Nonetheless, all 36 repeatedly differential miRNAs in AD blood are promising as components of the diagnostic panel. Given their predicted functions, such miRNA panel may report multiple pathways contributing to AD pathology, enabling the design of personalized therapies. In addition, the analysis revealed that the miRNAs dysregulated in AD overlap highly with miRNAs implicated in cancer. However, the directions of the miRNA changes are usually opposite in cancer and AD, indicative of an epigenetic trade-off between the two diseases.

Oxidant/Antioxidant Imbalance in Alzheimer's Disease: Therapeutic and Diagnostic Prospects.

Alzheimer's disease (AD) is the most common cause of dementia and a great socioeconomic burden in the aging society. Compelling evidence demonstrates that molecular change characteristics for AD, such as oxidative stress and amyloid ß (Aß) oligomerization, precede by decades the onset of clinical dementia and that the disease represents a biological and clinical continuum of stages, from asymptomatic to severely impaired. Nevertheless, the sequence of the early molecular alterations and the interplay between them are incompletely understood. This review presents current knowledge about the oxidative stress-induced impairments and compromised oxidative stress defense mechanisms in AD brain and the cross-talk between various pathophysiological insults, with the focus on excessive reactive oxygen species (ROS) generation and Aß overproduction at the early stages of the disease. Prospects for AD therapies targeting oxidant/antioxidant imbalance are being discussed, as well as for the development of novel oxidative stress-related, blood-based biomarkers for early, noninvasive AD diagnostics.

Search for Alzheimer's disease biomarkers in blood cells: hypotheses-driven approach.

Current Alzheimer's disease (AD) diagnostics is based on cognitive testing, and detecting amyloid Aß and τ pathology by brain imaging and assays of cerebrospinal fluid. However, biomarkers identifying complex pathways contributing to pathology are lacking, especially for early AD. Preferably, such biomarkers should be more cost-effective and present in easily available diagnostic tissues, such as blood. Here, we summarize the recent findings of potential early AD molecular diagnostic biomarkers in blood platelets, lymphocytes and erythrocytes. We review molecular alterations which refer to such main hypotheses of AD pathogenesis as amyloid cascade, oxidative and mitochondrial stress, inflammation and alterations in cell cycle regulatory molecules. The major advantage of such biomarkers is the potential ability to indicate individualized therapies in AD patients.

Familial Alzheimer's Disease Lymphocytes Respond Differently Than Sporadic Cells to Oxidative Stress: Upregulated p53-p21 Signaling Linked with Presenilin 1 Mutants.

Familial (FAD) and sporadic (SAD) Alzheimer's disease do not share all pathomechanisms, but knowledge on their molecular differences is limited. We previously reported that cell cycle control distinguishes lymphocytes from SAD and FAD patients. Significant differences were found in p21 levels of SAD compared to FAD lymphocytes. Since p21 can also regulate apoptosis, the aim of this study was to compare the response of FAD and SAD lymphocytes to oxidative stress like 2-deoxy-D-ribose (2dRib) treatment and to investigate the role of p21 levels in this response. We report that FAD cells bearing seven different PS1 mutations are more resistant to 2dRib-induced cell death than control or SAD cells: FAD cells showed a lower apoptosis rate and a lower depolarization of the mitochondrial membrane. Despite that basal p21 cellular content was lower in FAD than in SAD cells, in response to 2dRib, p21 mRNA and protein levels significantly increased in FAD cells. Moreover, we found a higher cytosolic accumulation of p21 in FAD cells. The transcriptional activation of p21 was shown to be dependent on p53, as it can be blocked by PFT-α, and correlated with the increased phosphorylation of p53 at Serine 15. Our results suggest that in FAD lymphocytes, the p53-mediated increase in p21 transcription, together with a shift in the nucleocytoplasmic localization of p21, confers a survival advantage against 2dRib-induced apoptosis. This compensatory mechanism is absent in SAD cells. Thus, therapeutic and diagnostic designs should take into account possible differential apoptotic responses in SAD versus FAD cells.

Profile of 6 microRNA in blood plasma distinguish early stage Alzheimer's disease patients from non-demented subjects.

Alzheimer's disease (AD) is the most common age-related dementia. Among its major challenges is identifying molecular signatures characteristic for the early AD stage in patients with Mild Cognitive Impairment (MCI-AD), which could serve for deciphering the AD pathomechanism and also as non-invasive, easy-to-access biomarkers. Using qRT-PCR we compared the microRNA (miRNA) profiles in blood plasma of 15 MCI-AD patients, whose diagnoses were confirmed by cerebrospinal fluid (CSF) biomarkers, with 20 AD patients and 15 non-demented, age-matched individuals (CTR).To minimize methodological variability, we adhered to standardization of blood and CSF assays recommended by the international Joint Programming for Neurodegenerative Diseases (JPND) BIOMARKAPD consortium, and we employed commercially available Exiqon qRT-PCR-assays. In the first screening, we assessed 179 miRNAs of plasma. We confirmed 23 miRNAs reported earlier as AD biomarker candidates in blood and found 26 novel differential miRNAs between AD and control subjects. For representative 15 differential miRNAs, the TargetScan, MirTarBase and KEGG database analysis indicated putative protein targets among such AD hallmarks as MAPT (Tau), proteins involved in amyloidogenic proteolysis, and in apoptosis. These 15 miRNAs were verified in separate, subsequent subject groups. Finally, 6 miRNAs (3 not yet reported in AD context and 3 reported in AD blood) were selected as the most promising biomarker candidates differentiating early AD from controls with the highest fold changes (from 1.32 to 14.72), consistent significance, specificities from 0.78 to 1 and sensitivities from 0.75 to 1. (patent pending, PCT/IB2016/052440).

Oxidative Stress and Aberrant Cell Cycle in Alzheimer's Disease Lymphocytes: Diagnostic Prospects.

In Alzheimer's disease (AD), molecular changes are observed not only in patients' neurons but also in peripheral cells, such as blood lymphocytes. These include changes in the level of oxidative stress markers, mitochondria impairment, and aberrant cell cycle regulation in AD blood lymphocytes. While the concepts of early causes of AD are currently highly controversial, these findings provide support for the cell cycle hypothesis of AD pathomechanism and emphasize the systemic nature of the disease. Moreover, because of difficulties in studying dynamic processes in the human brain, lymphocytes seem to be useful for readout of AD molecular mechanisms. In addition, lymphocytes as easily accessible human cells have potential diagnostic value. We summarize current perspectives for the development of new therapeutic strategies based on oxidative stress and cell cycle dysregulation in AD, and for diagnostic methodologies involving new markers in AD lymphocytes.

Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species.

In vertebrates, several genes which are differentially expressed in various species, have been implicated in sex determination and gonadal differentiation. We used immunolocalization to study the expression pattern of three proteins AMH, DMRT1, RSPO1 involved in the sexual differentiation of gonads. The pattern of AMH, DMRT1 and RSPO1 expression was analyzed in X. laevis and in five other divergent anuran species: Bombina bombina, Bufo viridis, Hyla arborea, Rana arvalis and Rana temporaria during gonadal development. The pattern of expression of AMH in the developing testes of six studied anuran species was similar to that described for other vertebrates. AMH was strongly expressed in differentiating Sertoli cells. Interestingly, in B. viridis, R. arvalis and R. temporaria, AMH was also expressed in ovaries. In all studied species, DMRT1 was highly expressed in the developing testes, in both the somatic and germ cells. It was also expressed at low level in ovaries in all studied species, with the exception of H. arborea. RSPO1 was expressed in the developing ovaries, especially in the somatic cells, and was almost undetectable in developing testes in all examined anurans. These developmental expression patterns strongly suggest an involvement of AMH and DMRT1 in the development of male gonads and of RSPO1 in the female gonads. The differences in the expression patterns of these proteins in the gonads of different species might reflect the diversity of gonadal development patterns in anurans resulting from long lasting and diverged paths of their evolution.

Retinoic acid homeostasis regulates meiotic entry in developing anuran gonads and in Bidder's organ through Raldh2 and Cyp26b1 proteins.

The vitamin A (retinol) and its metabolites such as retinoic acid (RA) affect vertebrate gametogenesis. The level of RA in cells relies on the balance between its synthesis and degradation. The sex-dependent equilibrium is reached in different ways in various species. It is known that RA induces meiosis in developing gonads in mouse, chicken and urodel amphibians, but its role in anuran amphibians has not been studied. Here we show in six anuran species (Xenopus laevis, Bombina bombina, Hyla arborea, Bufo viridis, Rana arvalis and Rana temporaria) that cultured undifferentiated gonads were insensitive to RA treatment, but the RA induced ectopic meiosis in cultured larval testes. In larval testes of all studied species, the exogenous RA induced leptotene phase of I meiotic prophase in gonia, but only in H. arborea and B. viridis gonia progressed to zygotene phase. In the cultured developing ovaries, exogenous RA led to increase in the number of oocytes as compared to the control. Inhibition of either RA synthesis or RA-receptors prevented meiotic entry in larval gonads of all species. Exogenous RA rescued this inhibitory effect demonstrating that the balance in RA homeostasis plays a key role in meiotic entry in anuran gonads. The localization of two enzymes, Raldh2 and Cyp26b1, which antagonistically control RA levels and whose abundance suggests the sites of RA synthesis and degradation respectively, showed two distinct expression patterns specific for (i) X. laevis, H. arborea, R. arvalis, R. temporaria and (ii) B. bombina, B. viridis. Thus, RA, in correlation with specific expression patterns of Raldh2 and Cyp26b, induces meiosis during gonad development in anurans. In addition, in B. viridis, RA signalling seems important for development of the Bidder's organ containing oocytes both in males and females.