The mTOR kinase inhibitor Everolimus decreases S6 kinase phosphorylation but fails to reduce mutant huntingtin levels in brain and is not neuroprotective in the R6/2 mouse model of Huntington's disease

Background

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion within the huntingtin gene. Mutant huntingtin protein misfolds and accumulates within neurons where it mediates its toxic effects. Promoting mutant huntingtin clearance by activating macroautophagy is one approach for treating Huntington's disease (HD). In this study, we evaluated the mTOR kinase inhibitor and macroautophagy promoting drug everolimus in the R6/2 mouse model of HD.

Results

Everolimus decreased phosphorylation of the mTOR target protein S6 kinase indicating brain penetration. However, everolimus did not activate brain macroautophagy as measured by LC3B Western blot analysis. Everolimus protected against early declines in motor performance; however, we found no evidence for neuroprotection as determined by brain pathology. In muscle but not brain, everolimus significantly decreased soluble mutant huntingtin levels.

Conclusions

Our data suggests that beneficial behavioral effects of everolimus in R6/2 mice result primarily from effects on muscle. Even though everolimus significantly modulated its target brain S6 kinase, this did not decrease mutant huntingtin levels or provide neuroprotection.

     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H₂O₂, nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2''-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H₂O₂ and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair.     

Hospital admissions for skin and soft tissue infections in a population with endemic scabies: A prospective study in Fiji, 2018–2019

Scabies is an important predisposing factor for impetigo but its role in more serious skin and soft tissue infections (SSTIs) is not well understood. Information is limited on incidence of SSTIs in the presence of endemic scabies. We conducted a prospective study of hospital admissions for SSTIs in the Northern Division of Fiji (population: 131,914). Prospective surveillance for admissions with impetigo, abscess, cellulitis, wound infection, pyomyositis, necrotizing fasciitis, infected scabies, and crusted scabies was conducted at the Division’s referral hospital between 2018 to 2019. Information was collected on demographic characteristics, clinical features, microbiology, treatment and outcomes. Over the study period, 788 SSTI admissions were recorded corresponding to a population incidence 647 per 100,000 person-years (95%CI 571–660). Incidence was highest at the extremes of age with peak incidence in children aged <5 years (908 per 100,000) and those aged ≥65 years (1127 per 100,000). Incidence was 1.7 times higher among the Indigenous Fijian population (753 per 100,000) compared to other ethnicities (442 per 100,000). Overall case fatality rate was 3.3%, and 10.8% for those aged ≥65 years. Scabies was diagnosed concurrently in 7.6% of all patients and in 24.6% of admitted children <5 years. There is a very high burden of hospital admissions for SSTIs in Fiji compared to high-income settings especially among the youngest, oldest and indigenous population which is concordant with scabies and impetigo distribution in this population. Our findings highlight the need for strategies to reduce the burden of SSTIs in Fiji and similar settings.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.