The Amyloid Precursor Protein Intracellular Domain-Fe65 Multiprotein Complexes: A Challenge to the Amyloid Hypothesis for Alzheimer's Disease?

Since its proposal in 1994, the amyloid cascade hypothesis has prevailed as the mainstream research subject on the molecular mechanisms leading to the Alzheimer's disease (AD). Most of the field had been historically based on the role of the different forms of aggregation of β-amyloid peptide (Aβ). However, a soluble intracellular fragment termed amyloid precursor protein (APP) intracellular domain (AICD) is produced in conjunction with Aβ fragments. This peptide had been shown to be highly toxic in both culture neurons and transgenic mice models. With the advent of this new toxic fragment, the centerpiece for the ethiology of the disease may be changed. This paper discusses the potential role of multiprotein complexes between the AICD and its adapter protein Fe65 and how this could be a potentially important new agent in the neurodegeneration observed in the AD

Bioinformatics Approaches for Predicting Kinase–Substrate Relationships

Protein phosphorylation, catalyzed by protein kinases, is the main posttranslational modification in eukaryotes, regulating essential aspects of cellular function. Using mass spectrometry techniques, a profound knowledge has been achieved in the localization of phosphorylated residues at proteomic scale. Although it is still largely unknown, the protein kinases are responsible for such modifications. To fill this gap, many computational algorithms have been developed, which are capable to predict kinase–substrate relationships. The greatest difficulty for these approaches is to model the complex nature that determines kinase–substrate specificity. The vast majority of predictors is based on the linear primary sequence pattern that surrounds phosphorylation sites. However, in the intracellular environment the protein kinase specificity is influenced by contextual factors, such as protein–protein interactions, substrates co-expression patterns, and subcellular localization. Only recently, the development of phosphorylation predictors has begun to incorporate these variables, significantly improving specificity of these methods. An accurate modeling of kinase–substrate relationships could be the greatest contribution of bioinformatics to understand physiological cell signaling and its pathological impairment

New Players in Neuronal Iron Homeostasis: Insights from CRISPRi Studies

Selective regional iron accumulation is a hallmark of several neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. The underlying mechanisms of neuronal iron dyshomeostasis have been studied, mainly in a gene-by-gene approach. However, recent high-content phenotypic screens using CRISPR/Cas9-based gene perturbations allow for the identification of new pathways that contribute to iron accumulation in neuronal cells. Herein, we perform a bioinformatic analysis of a CRISPR-based screening of lysosomal iron accumulation and the functional genomics of human neurons derived from induced pluripotent stem cells (iPSCs). Consistent with previous studies, we identified mitochondrial electron transport chain dysfunction as one of the main mechanisms triggering iron accumulation, although we substantially expanded the gene set causing this phenomenon, encompassing mitochondrial complexes I to IV, several associated assembly factors, and coenzyme Q biosynthetic enzymes. Similarly, the loss of numerous genes participating through the complete macroautophagic process elicit iron accumulation. As a novelty, we found that the impaired synthesis of glycophosphatidylinositol (GPI) and GPI-anchored protein trafficking also trigger iron accumulation in a cell-autonomous manner. Finally, the loss of critical components of the iron transporters trafficking machinery, including MON2 and PD-associated gene VPS35, also contribute to increased neuronal levels. Our analysis suggests that neuronal iron accumulation can arise from the dysfunction of an expanded, previously uncharacterized array of molecular pathways

Dissecting the role of redox signaling in neuronal development

The generation of abnormally high levels of reactive oxygen species (ROS) is linked to cellular dysfunction, including neuronal toxicity and neurodegeneration. However, physiological ROS production modulates redox-sensitive roles of several molecules such as transcription factors, signaling proteins, and cytoskeletal components. Changes in the functions of redox-sensitive proteins may be important for defining key aspects of stem cell proliferation and differentiation, neuronal maturation, and neuronal plasticity. In neurons, most of the studies have been focused on the pathological implications of such modifications and only very recently their essential roles in neuronal development and plasticity has been recognized. In this review, we discuss the participation of NADPH oxidases (NOXs) and a family of protein-methionine sulfoxide oxidases, named molecule interacting with CasLs, as regulated enzymatic sources of ROS production in neurons, and describes the contribution of ROS signaling to neurogenesis and differentiation, neurite outgrowth, and neuronal plasticity.Fil: Bórquez, Daniel A.. Universidad de Chile; Chile. Universidad Diego Portales; ChileFil: Urrutia, Pamela J.. Universidad de Chile; ChileFil: Wilson Rodriguez, Carlos. Universidad de Chile; ChileFil: Van Zundert, Brigitte. Universidad Andrés Bello; ChileFil: Nuñez, Marco Tulio. Universidad de Chile; ChileFil: González Billault, Christian. Universidad de Chile; Chile. Center For Geroscience, Brain Health And Metabolism; Chil

RENDIMIENTO DE CORDEROS EN CRECIMIENTO ALIMENTADOS CON ENSILADOS DE POLLINAZA, CERDAZA Y UREA CON MELAZA DE CAÑA O UN SUBPRODUCTO DE PANADERÍA

Las excretas pecuarias están constituidas por fracciones de alimentos no digeridas y por otros nutrientes que se incorporan en el tubo digestivo de los animales. Su producción y acumulación es fuente de contaminación ambiental, pero también son una fuente valiosa de nitrógeno y minerales en la alimentación de rumiantes. El objetivo de esta investigación fue evaluar dietas con ensilados de rastrojo de maíz, melaza de caña (MEL) y subproducto de panadería (SPP) como fuentes de carbohidratos hidrosolubles (C), mezclados con cerdaza fresca (CF), pollinaza deshidratada (PO) y urea agrícola (UR) como fuentes de nitrógeno (N), y su efecto en el crecimiento y las características de la canal de corderos. Los corderos recibieron durante 60 d dietas con 145 g PC kg-1 MS y 10 MJ EM kg-1 MS, más 400 g kg-1 (MS) de ensilado. Después, los corderos se sacrificaron para medir variables productivas y calidad de la canal. El consumo de MS (CMS) se analizó con medidas repetidas; para rendimiento, engrasamiento y morfometría se empleó el diseño de bloques al azar con arreglo factorial 2 x 3. Las variables cualitativas se analizaron con la prueba Kruskal-Wallis. Las medias de cuadrados mínimos de CMS a los 60 d no presentaron diferencias (p>0.05) entre tratamientos. Sin embargo, hubo efecto (p£0.05) de la fuente de N sobre ganancia diaria de peso (GDP) e interacción de NxC para ancho de pierna y grosor de grasa torácica. La fuente de C afectó (p£0.05) el rendimiento del perímetro de pierna, área de chuleta y profundidad de grasa subcutánea. La conformación de la canal fue inferior (p£0.05) para la combinación SPP con UR; con los otros tratamientos se obtuvieron grados de conformación de R a U. El tratamiento SPP con CF causó las mayores (p£0.05) coberturas de grasa externa, color de carne rosa claro y color de grasa crema. La conclusión es que los corderos alimentados con ensilado de CF y SPP tuvieron canales con mejor conformación y engrasamiento

Tuba activates cdc42 during neuronal polarization downstream of the small gtpase rab8a

The acquisition of neuronal polarity is a complex molecular process that depends on changes in cytoskeletal dynamics and directed membrane traffic, regulated by the Rho and Rab families of small GTPases, respectively. However, during axon specification, a molecular link that couples these protein families has yet to be identified. In this paper, we describe a new positive feedback loop between Rab8a and Cdc42, coupled by Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), that ensures a single axon generation in rodent hippocampal neurons from embryos of either sex. Accordingly, Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Although Rab8 and Tuba do not interact physically, the activity of Rab8 is essential to generate a proximal to distal axonal gradient of Tuba in cultured neurons. Tuba-associated and Rab8a-associated polarity defects are also evidenced in vivo, since dominant negative (DN) Rab8a or Tuba knock-down impairs cortical neuronal migration in mice. Our results suggest that Tuba coordinates directed vesicular traffic and cytoskeleton dynamics during neuronal polarization.Fil: Urrutia, Pamela J. Universidad de Chile; Chile. Geroscience Center for Brain Health and Metabolism; ChileFil: Bodaleo, Felipe. Geroscience Center for Brain Health and Metabolism; Chile. Universidad de Chile; ChileFil: Bórquez, Daniel A.. Universidad Diego Portales; ChileFil: Homma, Yuta. Tohoku University; JapónFil: Rozés Salvador, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Villablanca, Cristopher. Universidad de Chile; Chile. Geroscience Center for Brain Health and Metabolism; ChileFil: Conde, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Fukuda, Mitsunori. Tohoku University; JapónFil: González Billault, Christian. Universidad de Chile; Chile. Geroscience Center for Brain Health and Metabolism; Chil

Identifying counties at risk of high overdose mortality burden during the emerging fentanyl epidemic in the USA: a predictive statistical modelling study.

BackgroundThe emergence of fentanyl around 2013 represented a new, deadly stage of the opioid epidemic in the USA. We aimed to develop a statistical regression approach to identify counties at the highest risk of high overdose mortality in the subsequent years by predicting annual county-level overdose death rates across the contiguous USA and to validate our approach against observed overdose mortality data collected between 2013 and 2018.MethodsWe fit mixed-effects negative binomial regression models to predict overdose death rates in the subsequent year for 2013-18 for all contiguous state counties in the USA (ie, excluding Alaska and Hawaii). We used publicly available county-level data related to health-care access, drug markets, socio-demographics, and the geographical spread of opioid overdose as model predictors. The crude number of county-level overdose deaths was extracted from restricted US Centers for Disease Control and Prevention mortality records. To predict county-level overdose rates for the year 201X: (1) a model was trained on county-level predictor data for the years 2010-201(X-2) paired with county-level overdose deaths for the year 2011-201(X-1); (2) county-level predictor data for the year 201(X-1) was fed into the model to predict the 201X county-level crude number of overdose deaths; and (3) the latter were converted to a population-adjusted rate. For comparison, we generated a benchmark set of predictions by applying the observed slope of change in overdose death rates in the previous year to 201(X-1) rates. To assess the predictive performance of the model, we compared predicted values (of both the model and benchmark) to observed values by (1) calculating the mean average error, root mean squared error, and Spearman's correlation coefficient and (2) assessing the proportion of counties in the top decile (10%) of overdose death rates that were correctly predicted as such. Finally, in a post-hoc analysis, we sought to identify variables with greatest predictive utility.FindingsBetween 2013 and 2018, among the 3106 US counties included, our modelling approach outperformed the benchmark strategy across all metrics. The observed average county-level overdose death rate rose from 11·8 per 100 000 people in 2013 to 15·4 in 2017 before falling to 14·6 in 2018. Our negative binomal modelling approach similarly identified an increasing trend, predicting an average 11·8 deaths per 100 000 in 2013, up to 15·1 in 2017, and increasing further to 16·4 in 2018. The benchmark model over-predicted average death rates each year, ranging from 13·0 per 100 000 in 2013 to 18·3 in 2018. Our modelling approach successfully ranked counties by overdose death rate identifying between 42% and 57% of counties in the top decile of overdose mortality (compared with 29% and 43% using the benchmark) each year and identified 194 of the 808 counties with emergent overdose outbreaks (ie, newly entered the top decile) across the study period, versus 31 using the benchmark. In the post-hoc analysis, we identified geospatial proximity of overdose in nearby counties, opioid prescription rate, presence of an urgent care facility, and several economic indicators as the variables with the greatest predictive utility.InterpretationOur model shows that a regression approach can effectively predict county-level overdose death rates and serve as a risk assessment tool to identify future high mortality counties throughout an emerging drug use epidemic.FundingNational Institute on Drug Abuse

Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate