Differential control of clustering of the sodium channels Na(v)1.2 and Na(v)1.6 at developing CNS nodes of Ranvier

Na(v)1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na(v)1.2 and its beta2 subunit, but not Na(v)1.6 or beta1, are clustered in developing central nervous system nodes and that clustering of Na(v)1.2 and Na(v)1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na(v)1.2 alpha and beta2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na(v)1.6 or beta1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na(v)1.2 and Na(v)1.6 channels is differentially controlled and suggest that myelination induces Na(v)1.6 clustering.     

Sleep-Wake Cycle Dysfunction in the TgCRND8 Mouse Model of Alzheimer’s Disease: From Early to Advanced Pathological Stages

In addition to cognitive decline, individuals affected by Alzheimer’s disease (AD) can experience important neuropsychiatric symptoms including sleep disturbances. We characterized the sleep-wake cycle in the TgCRND8 mouse model of AD, which overexpresses a mutant human form of amyloid precursor protein resulting in high levels of β-amyloid and plaque formation by 3 months of age. Polysomnographic recordings in freely-moving mice were conducted to study sleep-wake cycle architecture at 3, 7 and 11 months of age and corresponding levels of β-amyloid in brain regions regulating sleep-wake states were measured. At all ages, TgCRND8 mice showed increased wakefulness and reduced non-rapid eye movement (NREM) sleep during the resting and active phases. Increased wakefulness in TgCRND8 mice was accompanied by a shift in the waking power spectrum towards fast frequency oscillations in the beta (14-20 Hz) and low gamma range (20-50 Hz). Given the phenotype of hyperarousal observed in TgCRND8 mice, the role of noradrenergic transmission in the promotion of arousal, and previous work reporting an early disruption of the noradrenergic system in TgCRND8, we tested the effects of the alpha-1-adrenoreceptor antagonist, prazosin, on sleep-wake patterns in TgCRND8 and non-transgenic (NTg) mice. We found that a lower dose (2 mg/kg) of prazosin increased NREM sleep in NTg but not in TgCRND8 mice, whereas a higher dose (5 mg/kg) increased NREM sleep in both genotypes, suggesting altered sensitivity to noradrenergic blockade in TgCRND8 mice. Collectively our results demonstrate that amyloidosis in TgCRND8 mice is associated with sleep-wake cycle dysfunction, characterized by hyperarousal, validating this model as a tool towards understanding the relationship between β-amyloid overproduction and disrupted sleep-wake patterns in AD.     

Early Spring,Severe Frost Events,and Drought Induce Rapid Carbon Loss in High Elevation Meadows

By the end of the 20^th century, the onset of spring in the Sierra Nevada mountain range of California has been occurring on average three weeks earlier than historic records. Superimposed on this trend is an increase in the presence of highly anomalous “extreme” years, where spring arrives either significantly late or early. The timing of the onset of continuous snowpack coupled to the date at which the snowmelt season is initiated play an important role in the development and sustainability of mountain ecosystems. In this study, we assess the impact of extreme winter precipitation variation on aboveground net primary productivity and soil respiration over three years (2011 to 2013). We found that the duration of snow cover, particularly the timing of the onset of a continuous snowpack and presence of early spring frost events contributed to a dramatic change in ecosystem processes. We found an average 100% increase in soil respiration in 2012 and 2103, compared to 2011, and an average 39% decline in aboveground net primary productivity observed over the same time period. The overall growing season length increased by 57 days in 2012 and 61 days in 2013. These results demonstrate the dependency of these keystone ecosystems on a stable climate and indicate that even small changes in climate can potentially alter their resiliency.     

Peptides That Bind Specifically to an Antibody from a Chronic Lymphocytic Leukemia Clone Expressing Unmutated Immunoglobulin Variable Region Genes

Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the disease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) engage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined the binding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable domains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected peptide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in families based on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb revealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (micromolar K_D), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations (nanomolar K_D) and with immunoglobulin heavy chain variable (IGHV)-mutated antibodies selected by environmental antigens. This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthy subjects with monoclonal B lymphocytosis.     

Loss of vasomotor responsiveness to the mu-opioid receptor ligand endomorphin-1 in adjuvant monoarthritic rat knee joints

Endomorphin-1 is a short-chain neuropeptide with a high affinity fo the mu-opioid receptor and has recently been localized in acutely inflamed knee joints where it was found to reduce inflammation. The present study examined the propensity of endomorphin-1 to modulate synovial blood flow in normal and adjuvant-inflamed at knee joints. Under deep urethane anesthesia, endomorphin-1 was topically applied to exposed normal and 1 wk adjuvant monoarthritic knee joints (0.1 ml bolus; 10(-12)-10(-9) mol). Relative changes in articular blood flow were measured by laser Doppler perfusion imaging and vascular resistances in response to the opioid were calculated. In normal knees, endomorphin-1 caused a dose-dependent increase in synovial vascular resistance and this effect was significantly inhibited by the specific mu-opioid receptor antagonist d-Phe-Cys-Tyr-d-Trp-O n-Thr-Pen-Th amide (CTOP) (P < 0.0001, 2-factor ANOVA, n = 5-7). One week after adjuvant inflammation, the hypoaemic effect of endomorphin-1 was completely abolished (P < 0.0001, 2-factor ANOVA, n = 5-7). Immunohistochemical analysis of normal and adjuvant-inflamed joints showed a ninefold increase in endomorphin-1 levels in the monoarthritic knee compared with normal control. Western blotting and immunohistochemistry revealed a moderate number of mu-opioid receptors in normal knees; however, mu-opioid receptors were almost undetectable in arthritic joints. These findings demonstrate that peripheral administration of endomorphin-1 reduces knee joint blood flow and this effect is not sustainable during advanced inflammation. The loss of this hypoaemic response appears to be due to down regulation of mu-opioid receptors as a consequence of endomorphin-1 accumulation within the arthritic joint.     

Stimulation of the acute-phase response in simian virus 40-hepatocyte cell lines.

Seven simian virus 40 (SV40)-hepatocyte cell lines were characterized with respect to the ability to express eight liver acute-phase genes. cDNA clones corresponding to albumin, serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, alpha-, beta-, and gamma-fibrinogen, and alpha 1-major-acute-phase protein mRNAs were used in Northern (RNA) or slot blot analyses. In the noninduced state, six of the seven cell lines showed significant (i.e., liverlike) levels of constitutive expression of all genes examined except that expression of haptoglobin mRNA was considerable lower than in the normal liver. To examine whether these immortalized liver cells can respond appropriately to inflammatory mediators, cells were treated with conditioned medium from activated human monocytes or mixed lymphocyte cultures. Results showed that these SV40-hepatocyte cell lines responded to the conditioned media in culture by down-regulating albumin gene expression and up-regulating other acute-phase genes in a time- and dose-dependent manner. These results indicate that the SV40-hepatocytes retained not only the ability to express a number of acute-phase genes but also the ability to respond to external stimuli. The usefulness of these cell lines for analysis of the molecular mechanisms involved in the regulation of these acute-phase genes is discussed.     

Loss of Extended Synaptotagmins ESyt2 and ESyt3 does not affect mouse development or viability,but in vitro cell migration and survival under stress are affected

The Extended Synaptotagmins (Esyts) are a family of multi-C2 domain membrane proteins with orthologs in organisms from yeast to human. Three Esyt genes exist in mouse and human and these have most recently been implicated in the formation of junctions between endoplasmic reticulum and plasma membrane, as well as the Ca²⁺ dependent replenishment of membrane phospholipids. The data are consistent with a function in extracellular signal transduction and cell adhesion, and indeed Esyt2 was previously implicated in both these functions in Xenopus. Despite this, little is known of the function of the Esyts in vivo. We have generated mouse lines carrying homozygous deletions in one or both of the genes encoding the highly homologous Esyt2 and Esyt3 proteins. Surprisingly, esyt2^?/?/esyt3^?/? mice develop normally and are both viable and fertile. In contrast, esyt2^?/?/esyt3^?/? mouse embryonic fibroblasts display a reduced ability to migrate in standard in vitro assays, and are less resistant to stringent culture conditions and to oxidative stress than equivalent wild type fibroblasts.