The Vac14p–Fig4p complex acts independently of Vac7p and couples PI3,5P2 synthesis and turnover

Phosphoinositide-signaling lipids function in diverse cellular pathways. Dynamic changes in the levels of these signaling lipids regulate multiple processes. In particular, when Saccharomyces cerevisiae cells are exposed to hyperosmotic shock, PI3,5P2 (phosphatidylinositol [PI] 3,5-bisphosphate) levels transiently increase 20-fold. This causes the vacuole to undergo multiple acute changes. Control of PI3,5P2 levels occurs through regulation of both its synthesis and turnover. Synthesis is catalyzed by the PI3P 5-kinase Fab1p, and turnover is catalyzed by the PI3,5P2 5-phosphatase Fig4p. In this study, we show that two putative Fab1p activators, Vac7p and Vac14p, independently regulate Fab1p activity. Although Vac7p only regulates Fab1p, surprisingly, we find that Vac14 regulates both Fab1p and Fig4p. Moreover, Fig4p itself functions in both PI3,5P2 synthesis and turnover. In both the absence and presence of Vac7p, the Vac14p–Fig4p complex controls the hyperosmotic shock–induced increase in PI3,5P2 levels. These findings suggest that the dynamic changes in PI3,5P2 are controlled through a tight coupling of synthesis and turnover

Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy.

BackgroundThe trans-neuronal propagation of tau has been implicated in the progression of tau-mediated neurodegeneration. There is critical knowledge gap in understanding how tau is released and transmitted, and how that is dysregulated in diseases. Previously, we reported that lysine acetyltransferase p300/CBP acetylates tau and regulates its degradation and toxicity. However, whether p300/CBP is involved in regulation of tau secretion and propagation is unknown.MethodWe investigated the relationship between p300/CBP activity, the autophagy-lysosomal pathway (ALP) and tau secretion in mouse models of tauopathy and in cultured rodent and human neurons. Through a high-through-put compound screen, we identified a new p300 inhibitor that promotes autophagic flux and reduces tau secretion. Using fibril-induced tau spreading models in vitro and in vivo, we examined how p300/CBP regulates tau propagation.ResultsIncreased p300/CBP activity was associated with aberrant accumulation of ALP markers in a tau transgenic mouse model. p300/CBP hyperactivation blocked autophagic flux and increased tau secretion in neurons. Conversely, inhibiting p300/CBP promoted autophagic flux, reduced tau secretion, and reduced tau propagation in fibril-induced tau spreading models in vitro and in vivo.ConclusionsWe report that p300/CBP, a lysine acetyltransferase aberrantly activated in tauopathies, causes impairment in ALP, leading to excess tau secretion. This effect, together with increased intracellular tau accumulation, contributes to enhanced spreading of tau. Our findings suggest that inhibition of p300/CBP as a novel approach to correct ALP dysfunction and block disease progression in tauopathy

Multimodality labeling strategies for the investigation of nanocrystalline cellulose biodistribution in a mouse model of breast cancer

Methods We have developed a nuclear and fluorescence labeling strategy for nanocrystalline cellulose (CNC), an emerging biomaterial with versatile chemistry and facile preparation from renewable sources. We modified CNC through 1,1′-carbonyldiimidazole (CDI) activation with radiometal chelators desferrioxamine B and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), allowing for the labeling with zirconium-89 (t½ = 78.41 h) and copper-64 (t½ = 12.70 h), respectively, for non-invasive positron emission tomography (PET) imaging. The far-red fluorescent dye Cy5 was added for ex vivo optical imaging, microscopy and flow cytometry. The multimodal CNC were evaluated in the syngeneic orthotopic 4T1 tumor model of human stage IV breast cancer. Results Modified CNC exhibited low cytotoxicity in RAW 264.7 macrophages over 96 h, and high radiolabel stability in vitro. After systemic administration, radiolabeled CNC were rapidly sequestered to the organs of the reticulo-endothelial system (RES), indicating immune recognition and no passive tumor targeting by the enhanced permeability and retention (EPR) effect. Modification with NOTA was a more favorable strategy in terms of radiolabeling yield, specific radioactivity, and both the radiolabel and dispersion stability in physiological conditions. Flow cytometry analysis of Cy5-positive immune cells from the spleen and tumor corroborated the uptake of CNC to phagocytic cells. Conclusions Future studies on the in vivo behavior of CNC should be concentrated on improving the nanomaterial stability and circulation half-life under physiological conditions and optimizing further the labeling yields for the multimodality imaging strategy presented. Advances in knowledge Our studies constitute one of the first accounts of a multimodality nuclear and fluorescent probe for the evaluation of CNC biodistribution in vivo and outline the pitfalls in radiometal labeling strategies for future evaluation of targeted CNC-based drug delivery systems. Implications for patient care Quantitative and sensitive molecular imaging methods provide information on the structure–activity relationships of the nanomaterial and guide the translation from in vitro models to clinically relevant animal models.Peer reviewe

Hedgehog pathway dysregulation contributes to the pathogenesis of human gastrointestinal stromal tumors via GLI-mediated activation of KIT expression.

Gastrointestinal stromal tumors (GIST) arise within the interstitial cell of Cajal (ICC) lineage due to activating KIT/PDGFRA mutations. Both ICC and GIST possess primary cilia (PC), which coordinate PDGFRA and Hedgehog signaling, regulators of gastrointestinal mesenchymal development. Therefore, we hypothesized that Hedgehog signaling may be altered in human GIST and controls KIT expression. Quantitative RT-PCR, microarrays, and next generation sequencing were used to describe Hedgehog/PC-related genes in purified human ICC and GIST. Genetic and pharmacologic approaches were employed to investigate the effects of GLI manipulation on KIT expression and GIST cell viability. We report that Hedgehog pathway and PC components are expressed in ICC and GIST and subject to dysregulation during GIST oncogenesis, irrespective of KIT/PDGFRA mutation status. Using genomic profiling, 10.2% of 186 GIST studied had potentially deleterious genomic alterations in 5 Hedgehog-related genes analyzed, including in the PTCH1 tumor suppressor (1.6%). Expression of the predominantly repressive GLI isoform, GLI3, was inversely correlated with KIT mRNA levels in GIST cells and non-KIT/non-PDGFRA mutant GIST. Overexpression of the 83-kDa repressive form of GLI3 or small interfering RNA-mediated knockdown of the activating isoforms GLI1/2 reduced KIT mRNA. Treatment with GLI1/2 inhibitors, including arsenic trioxide, significantly increased GLI3 binding to the KIT promoter, decreased KIT expression, and reduced viability in imatinib-sensitive and imatinib-resistant GIST cells. These data offer new evidence that genes necessary for Hedgehog signaling and PC function in ICC are dysregulated in GIST. Hedgehog signaling activates KIT expression irrespective of mutation status, offering a novel approach to treat imatinib-resistant GIST

Visualization of the Genesis and Fate of Isotype-switched B Cells during a Primary Immune Response

The life history of isotype-switched B cells is unclear, in part, because of an inability to detect rare antigen-specific B cells at early times during the immune response. To address this issue, a small population of B cells carrying targeted antibody transgenes capable of class switching was monitored in immunized mice. After contacting helper T cells, the first switched B cells appeared in follicles rather than in the red pulp, as was expected. Later, some of the switched B cells transiently occupied the red pulp and marginal zone, whereas others persisted in germinal centers (GCs). Antigen-experienced IgM B cells were rarely found in GCs, indicating that these cells switched rapidly after entering GCs or did not persist in this environment

An Investigation of Clinical Correlates of Lewy Bodies in Autopsy-Proven Alzheimer Disease

Background: Studies of patients meeting clinical and pathologic criteria for Alzheimer disease (AD) have not consistently found associations between the presence of Lewy bodies (LBs) at postmortem examination and a higher frequency during life of the clinical features of dementia with LBs. Objective: To evaluate the clinical correlates of LBs in patients with AD. Design and Methods: Fifty-one patients were diagnosed as having probable AD during life and met pathologic criteria for AD. Semiquantitative ratings for LBs were obtained in 4 brain regions: substantia nigra, cingulate, insular cortex, and hippocampus. The patients had been followed up semiannually for up to 9.9 years before death, and clinical features associated with dementia with LBs, including extrapyramidal signs and visual hallucinations, were assessed at each study visit. Logistic regression analyses determined whether patients who had LBs were more likely than those without LBs to express specific clinical signs during follow-up. Cox analyses determined whether patients with LBs developed clinical signs or died earlier. Generalized estimating equations were used to compare rates of cognitive or functional change. Results: Nineteen of the 51 patients had at least 1 LB in one of the studied regions. In no case was a significant relation noted between LBs and the presence of a measured clinical sign. No LB measure was associated with an increased risk of developing any of the evaluated clinical signs earlier in the disease. There was no association between the presence of LBs and more rapid mortality or more rapid disease progression. Conclusions: In patients diagnosed as having AD during life, we did not observe a relation of LBs noted during postmortem examination with the presence of any clinical feature that we assessed or with the rapidity of disease progression. The relation between LBs and specific clinical manifestations may be tenuous in these patients

Rapid online assessment of reading ability

Published18 March 2021An accurate model of the factors that contribute to individual differences in reading ability depends on data collection in large, diverse and representative samples of research participants. However, that is rarely feasible due to the constraints imposed by standardized measures of reading ability which require test administration by trained clinicians or researchers. Here we explore whether a simple, two-alternative forced choice, time limited lexical decision task (LDT), self-delivered through the webbrowser, can serve as an accurate and reliable measure of reading ability. We found that performance on the LDT is highly correlated with scores on standardized measures of reading ability such as the Woodcock-Johnson Letter Word Identification test (r = 0.91, disattenuated r = 0.94). Importantly, the LDT reading ability measure is highly reliable (r = 0.97). After optimizing the list of words and pseudowords based on item response theory, we found that a short experiment with 76 trials (2–3 min) provides a reliable (r = 0.95) measure of reading ability. Thus, the self-administered, Rapid Online Assessment of Reading ability (ROAR) developed here overcomes the constraints of resourceintensive, in-person reading assessment, and provides an efficient and automated tool for effective online research into the mechanisms of reading (dis)ability.We would like to thank the Pavlovia and PsychoPy team for their support on the browser-based experiments. This work was funded by NIH NICHD R01HD09586101, research grants from Microsoft and Jacobs Foundation Research Fellowship to J.D.Y

Metabolic remodeling of white adipose tissue in obesity

Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower V̇o2, V̇co2, and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity