Full-length, glycosylated NSP4 is localized to plasma membrane caveolae by a novel raft isolation technique

Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.     

Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus

Mammalian hibernation is characterized by prolonged torpor bouts interspersed by brief arousal periods. Adequate antioxidant defenses are needed both to sustain cell viability over weeks of deep torpor and to defend against high rates of oxyradical formation associated with massive oxygen-based thermogenesis during arousal. The present study shows that up-regulation of peroxiredoxins contributes to antioxidant defense during torpor in thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Expression levels of three isozymes of the 2-Cys peroxiredoxin (Prdx) family were quantified by Western blotting, the results showing 4.0- and 12.9-fold increases in Prdx1 protein in brown adipose tissue (BAT) and heart, respectively, during hibernation compared with euthermia. Comparable increases in Prdx2 were 2.4- and 3.7-fold whereas Prdx3 rose by 3.1-fold in heart of torpid animals. Total 2-Cys peroxiredoxin enzymatic activity also rose during hibernation by 1.5-fold in heart and 3.5-fold in BAT. Furthermore, RT-PCR showed that prdx2 mRNA levels increased by 1.7- and 3.7-fold in BAT and heart, respectively, during hibernation. A partial nucleotide sequence of prdx2 from ground squirrels was obtained by PCR amplification, the deduced amino acid sequence showing 96-97% identity with Prdx2 from other mammals. Some unique amino acid substitutions were identified that might contribute to stabilizing Prdx2 conformation at the near 0 degrees C body temperatures during torpor.     

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μm. The K_i value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μm. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μm) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μm) required to reverse behavioral deficits and pathology in Tau transgenic mice.     

Intertidal community responses to field‐based experimental warming

As the climate warms, there is little doubt that ecosystems of the future will look different from those we see today. However, community responses to warming in the field are poorly understood. We examined the effects of field‐based warming on intertidal communities in the Salish Sea, which is a regional thermal ‘hot spot’ and therefore a model system for studying thermally stressed communities. We manipulated temperature at three tidal heights by deploying black‐ and white‐bordered settlement plates. Black plates increased in situ substratum temperature by an average of 2.6°C (maximum temperature, 40.9°C). Barnacles fared poorly on black plates in all zones. When overall thermal stress was highest (summer in the high intertidal zone) herbivores were absent. In lower tidal zones, herbivores were abundant on white plates but were scarce on black plates. The total percent cover of algae was unaffected by the temperature treatment, despite the fact that macroalgae were expected to be the least thermally tolerant functional group. However, we did find that ephemeral green algae exhibited a delay in phenology on black plates. We also found that species richness declined and invertebrate assemblage structure was altered due to warming. Results from this year long experiment suggest that communities in thermally stressful habitats respond to warming via the interplay between species‐specific thermal responses and secondary adaptive strategies such as behavioral microhabitat selection. Declines in diversity and changes in the invertebrate assemblage were due to the decline of local thermally‐stressed species and the lack of replacement by warm‐adapted species. Given the low variation in the species pool along the northeast Pacific coastline, the arrival of warm‐adapted species to the Salish Sea may not occur over relevant time scales, leaving local communities depauperate.