Ataxin-1 fusion partners alter polyQ lethality and aggregation

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.     

The db mutation improves memory in younger mice in a model of Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Lepr^db/db) induces mixed or vascular dementia in mature to middle-aged APP^ΔNL/ΔNL x PS1^P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aβ content, which we have not observed at older ages. This was unlikely to be related to altered Aβ synthesis, as both β- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1β mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.     

School performance in Danish children exposed to maternal type 1 diabetes in utero: A nationwide retrospective cohort study

BackgroundConflicting results have been reported concerning possible adverse effects on the cognitive function of offspring of mothers with type 1 diabetes (O-mT1D). Previous studies have included offspring of parents from the background population (O-BP), but not offspring of fathers with type 1 diabetes (O-fT1D) as the unexposed reference group.Methods and findingsThis is a population-based retrospective cohort study from 2010 to 2016. Nationally standardized school test scores (range, 1 to 100) were obtained for public school grades 2, 3, 4, 6, and 8 in O-mT1D and compared with those in O-fT1D and O-BP. Of the 622,073 included children, 2,144 were O-mT1D, and 3,474 were O-fT1D. Multiple linear regression models were used to compare outcomes, including the covariates offspring with type 1 diabetes, parity, number of siblings, offspring sex, smoking during pregnancy, parental age, and socioeconomic factors. Mean test scores were 54.2 (standard deviation, SD 24.8) in O-mT1D, 54.4 (SD 24.8) in O-fT1D, and 56.4 (SD 24.7) in O-BP. In adjusted analyses, the mean differences in test scores were −1.59 (95% CI −2.48 to −0.71, p < 0.001) between O-mT1D and O-BP and −0.78 (95% CI −1.48 to −0.08, p = 0.03) between O-fT1D and O-BP. No significant difference in the adjusted mean test scores was found between O-mT1D and O-fT1D (p = 0.16). The study’s limitation was no access to measures of glycemic control during pregnancy.ConclusionsO-mT1D achieved lower test scores than O-BP but similar test scores compared with O-fT1D. Glycemic control during pregnancy is essential to prevent various adverse pregnancy outcomes in women with type 1 diabetes. However, the present study reduces previous concerns regarding adverse effects of in utero hyperglycemia on offspring cognitive function.

Anne Lærke Spangmose and colleagues examine the association between school performance and exposure to maternal or paternal type 1 diabetes in utero in Denmark.     

RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.     

Tandem immunoaffinity purification of protein complexes from Caenorhabditis elegans

The accurate determination of DNA concentration is essential for many processes in molecular biology and physiology and includes both gel- and cuvette-based methods. The recently introduced fluorescent dye, PicoGreen, has several advantages over other methods because it is sensitive and specific for double-stranded DNA (dsDNA). The dye is excited at 480 nm and emits at 520 nm when bound to dsDNA. This report describes the construction and use of PicoGmeter, a simple, inexpensive, fixed-wavelength fluorometer suitable for measuring PicoGreen fluorescence. PicoGmeter employs a blue light emitting diode (LED) for excitation and a photodiode to measure fluorescence. When compared to a commercially available instrument, PTI DeltaScan, the PicoGmeter performed admirably. Calibration curves for both instruments were superimposeable. Moreover, there was no significant difference between concentrations of DNA estimated by both instruments. A Bland and Altman analysis revealed that the PicoGmeter was equivalent to the PTI DeltaScan for estimating dsDNA concentration by the PicoGreen method. This simple, inexpensive, battery-operated fluorometer will allow investigators to employ the PicoGreen method without incurring the cost of purchasing a spectrofluorometer.