Sleep-wake body temperature regulates tau secretion in mice and correlates with CSF and plasma tau in humans.

The sleep-wake cycle regulates interstitial fluid and cerebrospinal fluid (CSF) tau levels in both mouse and human by mechanisms that remain unestablished. Here, we reveal a novel pathway by which wakefulness increases extracellular tau levels in mouse and humans. In mice, higher body temperature (BT) associated with wakefulness and sleep deprivation increased CSF tau. In vitro, wakefulness temperatures upregulated tau secretion via a temperature-dependent increase in activity and expression of unconventional protein secretion pathway-1 components, namely caspase-3-mediated C-terminal cleavage of tau (TauC3), and membrane expression of PIP2 and syndecan-3. In humans, the increase in both CSF and plasma tau levels observed post-wakefulness correlated with BT increase during wakefulness. Our findings suggest sleep-wake variation in BT may contribute to regulating extracellular tau levels, highlighting the importance of thermoregulation in pathways linking sleep disturbance to neurodegeneration, and the potential for thermal intervention to prevent or delay tau-mediated neurodegeneration.

Temperature-induced Artifacts in Tau Phosphorylation: Implications for Reliable Alzheimer's Disease Research.

In preclinical research on Alzheimer's disease and related tauopathies, tau phosphorylation analysis is routinely employed in both cellular and animal models. However, recognizing the sensitivity of tau phosphorylation to various extrinsic factors, notably temperature, is vital for experimental accuracy. Hypothermia can trigger tau hyperphosphorylation, while hyperthermia leads to its dephosphorylation. Nevertheless, the rapidity of tau phosphorylation in response to unintentional temperature variations remains unknown. In cell cultures, the most significant temperature change occurs when the cells are removed from the incubator before harvesting, and in animal models, during anesthesia prior to euthanasia. In this study, we investigate the kinetics of tau phosphorylation in N2a and SH-SY5Y neuronal cell lines, as well as in mice exposed to anesthesia. We observed changes in tau phosphorylation within the few seconds upon transferring cell cultures from their 37°C incubator to room temperature conditions. However, cells placed directly on ice post-incubation exhibited negligible phosphorylation changes. In vivo, isoflurane anesthesia rapidly resulted in tau hyperphosphorylation within the few seconds needed to lose the pedal withdrawal reflex in mice. These findings emphasize the critical importance of preventing temperature variation in researches focused on tau. To ensure accurate results, we recommend avoiding anesthesia before euthanasia and promptly placing cells on ice after removal from the incubator. By controlling temperature fluctuations, the reliability and validity of tau phosphorylation studies can be significantly enhanced.

Sauna-like conditions or menthol treatment reduce tau phosphorylation through mild hyperthermia.

In Alzheimer's disease (AD), hyper-phosphorylation and aggregation of tau correlate with clinical progression and represent a valid therapeutic target. A recent 20-year prospective study revealed an association between moderate to high frequency of Finnish sauna bathing and a lower incidence of dementia and AD, but the molecular mechanisms underlying these benefits remain uncertain. Here, we tested the hypothesis that sauna-like conditions could lower tau phosphorylation by increasing body temperature. We observed a decrease in tau phosphorylation in wild-type and hTau mice as well as in neuron-like cells when exposed to higher temperatures. These effects were correlated with specific changes in phosphatase and kinase activities, but not with inflammatory or heat shock responses. We also used a drug strategy to promote thermogenesis: topical application of menthol, which led to a sustained increase in body temperature in hTau mice, concomitant with a significant decrease in tau phosphorylation. Our results suggest that sauna-like conditions or menthol treatment could lower tau pathology through mild hyperthermia, and may provide promising therapeutic strategies for AD and other tauopathies.

Circadian and sleep/wake-dependent variations in tau phosphorylation are driven by temperature.

STUDY OBJECTIVES: Aggregates of hyperphosphorylated tau protein are a hallmark of Alzheimer's disease (AD) and other tauopathies. Sleep disturbances are common in AD patients, and insufficient sleep may be a risk factor for AD. Recent evidence suggests that tau phosphorylation is dysregulated by sleep disturbances in mice. However, the physiological regulation of tau phosphorylation during the sleep-wake cycle is currently unknown. We thus aimed to determine whether tau phosphorylation is regulated by circadian rhythms, inherently linked to the sleep-wake cycle. METHODS: To answer these questions, we analyzed by Western blotting tau protein and associated kinases and phosphatases in the brains of awake, sleeping, and sleep-deprived B6 mice. We also recorded their temperature. RESULTS: We found that tau phosphorylation undergoes sleep-driven circadian variations as it is hyperphosphorylated during sleep but not during acute sleep deprivation. Moreover, we demonstrate that the mechanism behind these changes involves temperature, as tau phosphorylation was inversely correlated with circadian- and sleep deprivation-induced variations in body temperature, and prevented by housing the animals at a warmer temperature. Notably, similar changes in tau phosphorylation were reproduced in neuronal cells exposed to temperatures recorded during the sleep-wake cycle. Our results also suggest that inhibition of protein phosphatase 2A (PP2A) may explain the hyperphosphorylation of tau during sleep-induced hypothermia. CONCLUSION: Taken together, our results demonstrate that tau phosphorylation follows a circadian rhythm driven mostly by body temperature and sleep, and provide the physiological basis for further understanding how sleep deregulation can affect tau and ultimately AD pathology.

Molecular risk stratification in advanced heart failure patients.

Risk stratification in advanced heart failure (HF) is crucial for the individualization of therapeutic strategy, in particular for heart transplantation and ventricular assist device implantation. We tested the hypothesis that cardiac gene expression profiling can distinguish between HF patients with different disease severity. We obtained tissue samples from both left (LV) and right (RV) ventricle of explanted hearts of 44 patients undergoing cardiac transplantation or ventricular assist device placement. Gene expression profiles were obtained using an in-house microarray containing 4217 muscular organ-relevant genes. Based on their clinical status, patients were classified into three HF-severity groups: deteriorating (n= 12), intermediate (n= 19) and stable (n= 13). Two-class statistical analysis of gene expression profiles of deteriorating and stable patients identified a 170-gene and a 129-gene predictor for LV and RV samples, respectively. The LV molecular predictor identified patients with stable and deteriorating status with a sensitivity of 88% and 92%, and a specificity of 100% and 96%, respectively. The RV molecular predictor identified patients with stable and deteriorating status with a sensitivity of 100% and 96%, and a specificity of 100% and 100%, respectively. The molecular prediction was reproducible across biological replicates in LV and RV samples. Gene expression profiling has the potential to reproducibly detect HF patients with highest HF severity with high sensitivity and specificity. In addition, not only LV but also RV samples could be used for molecular risk stratification with similar predictive power.

Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions.

BACKGROUND: Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. RESULTS: EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. CONCLUSION: This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions.

Differentially-expressed genes in pig Longissimus muscles with contrasting levels of fat, as identified by combined transcriptomic, reverse transcription PCR, and proteomic analyses.

Intramuscular fat content is important for many meat quality parameters. This work is aimed at identifying functional categories of genes associated with natural variation among individuals in intramuscular fat content to help the design of genetic schemes for high marbling potential. Taking advantage of the global nature of transcriptomic and proteomic technologies, 40 genes were identified as differently expressed between high fat and low fat pig Longissimus muscles at slaughter weight. They are involved in metabolic processes, cell communication, binding, and response to stimulus. Using real-time PCR in muscle biopsies taken earlier in the fattening period, the group with a high intramuscular fat content was also characterized by the down-expression of genes playing a negative role in adipogenesis, such as architectural transcription factor high-motility hook A1, mitogen activated protein-kinase14, and cyclin D1. These results suggest that interindividual variability in intramuscular fat content might arise essentially from differences in early adipogenesis.

Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups.

Terminal phosphate groups on double-stranded DNA probes bind strongly to glass substrates coated with a zirconium phosphonate monolayer, and probes immobilized in this way as microarrays can be used to detect protein targets. The sensitivity of the microarray was shown to be enhanced by the use of a polyguanine segment ((G)n , n > or = 5) as a spacer between the phosphate linker and the protein interaction domain. More importantly, the presence of phosphate linkers on both ends of the dsDNA probes leads to significant enhancement of target capture. The relevant characteristics of the different probes when bound to the surface were determined, by the original use of a combination of surface characterization techniques (XPS, AFM, and Sarfus). In this context, the location of the phosphate linkers in the duplex probes was found to result in different probe surface coverage and presentation on the surface, which affect subsequent interactions with the target protein.

Testing and improving experimental parameters for the use of low molecular weight targets in array-CGH experiments.

Array-comparative genomic hybridization (CGH) has evolved as a useful technique for the detection and characterization of deletions, and, to a lesser extent, of duplications. The resolution of the technique is dictated by the genomic distance between targets spotted on the microarray, and by the targets' sizes. The use of region-specific, high-resolution microarrays is a specific goal when studying regions that are prone to rearrangements, such as those involved in deletion syndromes. The aim of the present study was to evaluate the best experimental conditions to be used for array-CGH analysis using low molecular weight (LMW) targets. The parameters tested were: the target concentration, the way LMW targets are prepared (either as linearized plasmids or as purified PCR products), and the way the targets are attached to the array-CGH slide (in a random fashion on amino-silane coated slides, or by one amino-modified end on epoxysilane-coated slides). As a test case, we constructed a microarray harboring LMW targets located in the CREBBP gene, mutations of which cause the Rubinstein-Taybi syndrome (RTS). From 10 to 15% of RTS patients have a CREBBP deletion. We showed that aminosilane- and epoxysilane-coated slides were equally efficient with targets above 1,000 bp in size. On the other hand, with the smallest targets, especially those below 500 bp, epoxysilane-coated slides were superior to aminosilane-coated slides, which did not allow deletion detection. Use of the high resolution array allowed us to map intragenic breakpoints with precision and to identify a very small deletion and a duplication that were not detected by the currently available techniques for finding CREBBP deletions.

Transcriptome profiling uncovers metabolic and regulatory processes occurring during the transition from desiccation-sensitive to desiccation-tolerant stages in Medicago truncatula seeds.

To investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds, 16086-element microarrays were used to monitor changes in the transcriptome of desiccation-sensitive 3-mm-long radicles of Medicago truncatula seeds at different time points during incubation in a polyethylene glycol (PEG) solution at -1.7 MPa, resulting in a gradual re-establishment of DT. Gene profiling was also performed on embryos before and after the acquisition of DT during maturation. More than 1300 genes were differentially expressed during the PEG incubation. A large number of genes involved in C metabolism are expressed during the re-establishment of DT. Quantification of C reserves confirms that lipids, starch and oligosaccharides were mobilised, coinciding with the production of sucrose during the early osmotic adjustment. Several clusters of gene profiles were identified with different time-scales. Genes expressed early during the PEG incubation belonged to classes involved in early stress and adaptation responses. Interestingly, several regulatory genes typically expressed during abiotic/drought stresses were also upregulated during maturation, arguing for the partial overlap of ABA-dependent and -independent regulatory pathways involved in both drought and DT. At later time points, in parallel to the re-establishment of DT, upregulated genes are comparable with those involved in late seed maturation. Concomitantly, a massive repression of genes belonging to numerous classes occurred, including cell cycle, biogenesis, primary and energy metabolism. The re-establishment of DT in the germinated radicles appears to concur with a partial return to the quiescent state prior to germination.

Hepatocyte iron loading capacity is associated with differentiation and repression of motility in the HepaRG cell line.

High liver iron content is a risk factor for developing hepatocellular carcinoma (HCC). However, HCC cells are always iron-poor. Therefore, an association between hepatocyte iron storage capacity and differentiation is suggested. To characterize biological processes involved in iron loading capacity, we used a cDNA microarray to study the differentiation of the human HepaRG cell line, from undifferentiated proliferative cells to hepatocyte differentiated cells. We were able to identify genes modulated along HepaRG differentiation, leading us to propose new genes not previously associated with HCC. Moreover, using Gene Ontology annotations, we demonstrated that HepaRG hepatocyte iron loading capacity occurred both with the repression of genes involved in cell motility, signal transduction, and biosynthesis and with the appearance of genes linked to lipid metabolism and immune response. These results provide new insights in the understanding of the relationship between iron and hepatocyte differentiation during iron-related hepatic diseases.

Profiling dendritic cell maturation with dedicated microarrays.

Dendritic cell (DC) maturation is the process by which immature DC in the periphery differentiate into fully competent antigen-presenting cells that initiate the T cell response. However, DC respond to many distinct maturation stimuli, and different types of mature DC induce qualitatively different T cell responses. As DC maturation involves the coordinated regulation of hundreds of genes, comprehensive assessment of DC maturation status would ideally involve monitoring the expression of all of these transcripts. However, whole-genome microarrays are not well-suited for routine phenotyping of DC, as the vast majority of genes represented on such chips are not relevant to DC biology, and their cost limits their use for most laboratories. We therefore developed a DC-dedicated microarray, or "DC Chip", incorporating probes for 121 genes up-regulated during DC maturation, 93 genes down-regulated during maturation, 14 DC-specific genes, and 90 other genes with known or probable immune functions. These microarrays were used to study the kinetics of DC maturation and the differences in maturation profiles among five healthy donors after stimulation with tumor necrosis factor-alpha + polyI:C. Results obtained with the DC Chip were consistent with flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction, as well as previously published data. Furthermore, the coordinated regulation of a cluster of genes (indoleamine dioxygenase, kynureninase, kynurenine monoxygenase, tryptophanyl tRNA synthetase, and 3-hydroxyanthranilate 3,4-dioxygenase) involved in tryptophan metabolism was observed. These data demonstrate the use of the DC Chip for monitoring the molecular processes involved in the orientation of the immune response by DC.

Transcriptome variations in human CaCo-2 cells: a model for enterocyte differentiation and its link to iron absorption.

Complete clinical expression of the HFE1 hemochromatosis is very likely modulated by genes linked to duodenal iron absorption, whose level is conditioned by unknown processes taking place during enterocyte differentiation. We carried out a transcriptomic study on CaCo-2 cells used as a model of enterocyte differentiation in vitro. Of the 720 genes on the microarrays, 80, 50, and 56 were significantly down-regulated up-regulated, and invariant during differentiation. With regard to iron metabolism, we showed that HEPH, SLC11A2, SLC11A3, and TF are significantly up-regulated, while ATP7B and SLC39A1 (and SFT) are down-regulated and ACO1, dCYTb, FECH, and FTH1 show constant expression. Ontological annotations highlight the decrease in the expression of cell cycle and DNA metabolism associated genes as well as transcription, protein metabolism, signal transduction, and nucleocytoplasmic transport associated genes, whereas there are increases in the expression of genes linked to cell adhesion, lipid and xenobiotic metabolism, iron transport and homeostasis, and immune response.

New approach to oligonucleotide microarrays using zirconium phosphonate-modified surfaces.

A new approach to oligonucleotide arrays is demonstrated that utilizes zirconium phosphonate-derivatized glass slides. The active slides are prepared by binding Zr(4+) to surfaces terminated with organophosphonate groups previously deposited using either Langmuir-Blodgett or self-assembled monolayer methods. Oligonucleotide probes modified with a terminal phosphate bind strongly to the active zirconium phosphonate monolayer, and arrays for detecting fluorescent targets have been prepared using commercial spotting and scanning instruments. Preferred binding to the surface of the terminal phosphate of the modified probes instead of the internal phosphate diester groups is demonstrated and shown to yield increased fluorescence intensity after hybridization with labeled targets. A significant decrease in background signal is achieved by treating the slides with bovine serum albumin after spotting and before hybridization. A further increase in fluorescence after hybridization is observed when using a poly-guanine spacer between the probe oligomer and the terminal phosphate. Combining these modifications, an intensity ratio of nearly 1000 is achieved when comparing 5'-phosphate-modified 33-mer probes with unmodified probes upon hybridization with fluorescent targets.