Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format.

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer's disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting ß-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

Vitamin C supplementation for pregnant smoking women and pulmonary function in their newborn infants: a randomized clinical trial.

IMPORTANCE: Maternal smoking during pregnancy adversely affects offspring lung development, with lifelong decreases in pulmonary function and increased asthma risk. In a primate model, vitamin C blocked some of the in-utero effects of nicotine on lung development and offspring pulmonary function. OBJECTIVE: To determine if newborns of pregnant smokers randomized to receive daily vitamin C would have improved results of pulmonary function tests (PFTs) and decreased wheezing compared with those randomized to placebo. DESIGN, SETTING, AND PARTICIPANTS: Randomized, double-blind trial conducted in 3 sites in the Pacific Northwest between March 2007 and January 2011. One hundred fifty-nine newborns of randomized pregnant smokers (76 vitamin C treated and 83 placebo treated) and 76 newborns of pregnant nonsmokers were studied with newborn PFTs. Follow-up assessment including wheezing was assessed through age 1 year, and PFTs were performed at age 1 year. INTERVENTIONS: Pregnant women were randomized to receive vitamin C (500 mg/d) (n = 89) or placebo (n = 90). MAIN OUTCOMES AND MEASURES: The primary outcome was measurement of newborn pulmonary function (ratio of the time to peak tidal expiratory flow to expiratory time [TPTEF:TE] and passive respiratory compliance per kilogram [Crs/kg]) within 72 hours of age. Secondary outcomes included incidence of wheezing through age 1 year and PFT results at age 1 year. A subgroup of pregnant smokers and nonsmokers had genotyping performed. RESULTS: Newborns of women randomized to vitamin C (n = 76), compared with those randomized to placebo (n = 83), had improved pulmonary function as measured by TPTEF:TE (0.383 vs 0.345 [adjusted 95% CI for difference, 0.011-0.062]; P = .006) and Crs/kg (1.32 vs 1.20 mL/cm H2O/kg [95% CI, 0.02-0.20]; P = .01). Offspring of women randomized to vitamin C had significantly decreased wheezing through age 1 year (15/70 [21%] vs 31/77 [40%]; relative risk, 0.56 [95% CI, 0.33-0.95]; P = .03). There were no significant differences in the 1-year PFT results between the vitamin C and placebo groups. The effect of maternal smoking on newborn lung function was associated with maternal genotype for the α5 nicotinic receptor (rs16969968) (P < .001 for interaction). CONCLUSIONS AND RELEVANCE: Supplemental vitamin C taken by pregnant smokers improved newborn PFT results and decreased wheezing through 1 year in the offspring. Vitamin C in pregnant smokers may be an inexpensive and simple approach to decrease the effects of smoking in pregnancy on newborn pulmonary function and respiratory morbidities. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00632476.

Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain.

Uptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

Health professional's knowledge and awareness of perinatal depression: results of a national survey.

INTRODUCTION: Postnatal depression affects 14% of women, occurring also antenatally, with potential long-term consequences, making it an important disorder to detect and manage early. In this study we sought to examine knowledge and awareness of perinatal depression in health professionals involved in perinatal care throughout Australia prior to the implementation of a comprehensive screening program, aimed at improving detection and access to appropriate management. METHODS: A random sample of General Practitioners (GPs) and Maternal Child Health Nurses (MCHNs) and Midwives, in regions throughout Australia to be subsequently targeted by a screening and education program, were invited to participate. Responses to a hypothetical vignette and a knowledge questionnaire, as well as details of experience were completed. FINDINGS: Questionnaires were completed by 246 GPs, 338 MCHNs and 569 midwives, with overall response rates; GP's 23%; MCHN's 55% and midwives 57%. Although knowledge level was similar among professional groups, MCHNs had higher levels of awareness of perinatal depression. Both GPs and MCHNs were more likely than midwives to recognize the need for providing help to women with emotional distress. Depression was more likely to be considered postnatally than antenatally in all groups, with GPs most likely to provide this diagnosis. GPs had a significant propensity to recommend antidepressants, and midwives to select non-specific medications. CONCLUSIONS: Health professionals responding to this survey had a high awareness and similar knowledge base. Further education on antenatal depression and the safety risks and alternatives to medication is important for all groups, but particularly important for midwives and GPs. The latter is especially relevant given the preference for women with perinatal depression not to use pharmacological interventions to treat their emotional distress.

Dietary vitamin C intake is inversely related to cough and wheeze in young smokers.

We aimed to investigate whether dietary vitamin C intake, an important antioxidant, is inversely related to self-reported respiratory symptoms in young adults of a community. A random sample of 4300 subjects, aged 20-44 years, living in Bergen, Norway, received a postal questionnaire on respiratory symptoms; 80% responded. Vitamin C intake (mg per week) was estimated from a food-frequency questionnaire asking how often the subject, during the last year, had consumed units of orange juice, oranges, potatoes, carrots and tomatoes. Significant differences in the intake of vitamin C were observed across smoking categories with current smokers having the lowest intake, while there was no variation by gender, age or occupational dust exposure. Dietary vitamin C intake was in univariate analyses inversely related to "morning cough", "chronic cough", "wheeze" and "wheeze ever". After adjusting for gender, age, body mass index, "occupational exposure" pack-years as well as having and stratified on smoking habits in multiple logistic regression analyses, the relationship between dietary vitamin C intake and "cough" and "wheeze" tended to be associated to smoking. The odds ratio (OR) for "morning cough" was 0.68 (95% CI: 0.35-0.95), "chronic cough" OR 0.69 (95% CI: 0.47-1.04) and "wheeze ever" OR 0.75 (95% CI: 0.56-1.01) in current-smokers with dietary vitamin C intake in the upper (> or =395 mg/ week) vs. the lower (<209 mg/week) tertile. The OR for "wheeze" was 0.56 (95% CI: 0.35-0.88) in ex-smokers. The magnitude ofthese effects remained after excluding subjects with supplementary vitamin C intake (n=199) from the statistical analyses. Among young Norwegian adults, having a low prevalence of asthma and high prevalences of smoking-related respiratory symptoms, dietary vitamin C intake may act as an antioxidant and thereby reduce cough and wheeze in smokers having high oxidant stress.