Expression of complement system components during aging and amyloid deposition in APP transgenic mice

<p>Abstract</p> <p>Background</p> <p>A causal role of the complement system in Alzheimer's disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components.</p> <p>Methods</p> <p>APP23 mice expressing human APP751 with the Swedish double mutation as well as C57BL/6 mice were used at different ages. mRNA was quantified by Realtime PCR and the age- as well as amyloid induced changes determined. The protein levels of complement C1q and C3 were analysed by Western blotting. Histology was done to test for amyloid plaque association and activation of the complement cascade.</p> <p>Results</p> <p>High mRNA levels were detected for C1q and some inhibitory complement components. The expression of most activating components starting at C3 was low. Expression of C1q, C3, C4, C5 and factor B mRNA increased with age in control C57BL/6 mice. C1q and C3 mRNA showed a substantial additional elevation during amyloid formation in APP23 mice. This increase was confirmed on the protein level using Western blotting, whereas immunohistology indicated a recruitment of complement to amyloid plaques up to the C3 convertase.</p> <p>Conclusion</p> <p>Early but not late components of the mouse complement system show an age-dependent increase in expression. The response to amyloid deposition is comparatively smaller. The low expression of C3 and C5 and failure to upregulate C5 and downstream components differs from human AD brain and likely contributes to the lack of full complement activation in APP transgenic mice.</p

Diagnostico ecogeográfico da ocorrência de jabuticabeiras nativas no sudoeste do Paraná

The jabuticaba tree is a native fruit specie of restrict occurrence in the southwest region of the state of Paraná, Brazil. This observation carried the hypothesis that jabuticaba trees have edafoclimatic preference. This study aimed to investigate the ecogeografic and edafoclimatic conditions of the sites of naturally occurrence of jabuticaba trees on southwest region of Parana, Brazil. The study was conducted at 14 sites of occurrence of jabuticaba tree. Were recorded 4,036 adult plants of the Plinia cauliflora specie, in 201.9 hectares in the Araucaria Forest Biome. The sites were located mostly between 650 and 850 m of altitude and the plants were located always in the highest part of the toposequence site with minus probability of frost. Jabuticaba trees that occur in sites with lower altitude and higher average annual temperature are bigger than others from occurrence in sites with higher altitude and lower temperature. The soils where the jabuticaba trees grow up were clayey, strongly acidic (pH almost 4.0), with high content of organic matter and iron, aluminum high-saturation, low levels of phosphorus and very low saturation of bases.A jabuticabeira é uma fruteira nativa de ocorrência não generalizada na região sudoeste do Paraná, o que leva à hipótese de que esta espécie apresenta algumas preferências edafoclimáticas. Este trabalho teve como objetivo investigar as condições ecogeográficas e edafoclimáticas dos ambientes de ocorrência natural da jabuticabeira na região sudoeste do Paraná. O estudo foi efetuado em 14 sítios de ocorrência dessa espécie. Foram registradas 4.036 plantas adultas pertencentes à espécie Plinia cauliflora, em 201,9 a de mata do Ecossistema Floresta com Araucária. Os sítios estão localizados predominantemente entre 650 e 850 m de altitude, e as plantas localizam-se sempre na parte mais alta da topossequência, local menos propenso à ocorrência de geadas. Jabuticabeiras que ocorrem em sítios de menor altitude e com maior temperatura média anual são maiores que aquelas em sítios de maior altitude e menor temperatura. Os solos onde ocorrem as jabuticabeiras são argilosos, fortemente ácidos (pH próximo a 4,0), com alto teor de matéria orgânica e ferro, alta saturação de alumínio, baixo teor de fósforo e muito baixo índice de saturação de bases

Fruit weight and cycle length repeatability of plum and peach trees

O objetivo deste trabalho foi comparar o peso de fruto e a duração do ciclo entre cultivares de ameixeira e pessegueiro, estimar os coeficientes de repetibilidade e de determinação e o número de medições, para uma predição segura dessas características, e comparar diferentes métodos de estimação da repetibilidade. Os caracteres foram avaliados em 5 safras, em 9 cultivares de ameixeira (Prunus salicina) e 33 cultivares de pessegueiro (Prunus persica), em delineamento inteiramente casualizado. O coeficiente de repetibilidade foi estimado pelos métodos de análise de variância, componentes principais e análise estrutural. Foi observado o maior peso de fruto nas cultivares de ameixeira Harry Pickstone e Pluma 7 e na cultivar de pessegueiro Santa Áurea. A menor duração do ciclo ocorreu nas cultivares de ameixeira América, Irati e Methley e na cultivar de pessegueiro Granada. Os coeficientes de repetibilidade e de determinação foram altos para ambos os caracteres em ameixeira e pessegueiro. Não houve diferenças no coeficiente de repetibilidade entre os métodos utilizados. São necessárias de uma a três medições de peso de fruto, e de uma a duas medições da duração do ciclo, para obtenção dos valores genotípicos reais das cultivares de ameixeira e pessegueiro, respectivamente, com coeficiente de determinação de 90%.The objective of this work was to compare fruit weight and length of cycle, among cultivars of plum and peach, besides estimating the repeatability and determination coefficients and the number of measurements, for a safe prediction of these characteristics, and to compare different methods to estimate the repeatability. The traits were evaluated in 5 productive cycle for 9 plum cultivars (Prunus salicina) and 33 peach cultivars (Prunus persica), in a completely randomized design. The repeatability coefficient was estimated by methods of analysis of variance, principal components and structural analysis. The greates fruit weight was observed for Harry Pickstone and Pluma 7 plum cultivars and for Santa Aurea peach cultivar. The shortest lenght of cycle occurred in America, Irati and Methley plum cultivars and in Granada peach cultivar. The repeatability and determination coefficients were high for both traits in plum and in peach trees. There were no differences in the repeatability coefficient estimation among the methods used. One to three measurements of fruit weight and one and two measurements of length of cycle are necessary for obtaining the real genotypic values of plum and peach cultivars, respectively, with a determination coefficient of 90%

Indução de resistência à podridão-parda em pêssegos pelo uso de eliciadores em pós-colheita

The objective of this work was to evaluate the effects of acibenzolar-S-methyl and harpin protein elicitors, applied in post-harvest, in systemic resistance induction to brown-rot, on peaches. A completely randomized design was utilized, with four replicates, in a split plot scheme – injured or noninjured peaches –, and treatments were constituted of : acibenzolar-S-methyl (50 mg of a.i. L-1), two commercial products with harpin protein (80 mg of a.i. L-1); and control (distilled water). Peaches were sprayed individually with 1 mL of aqueous solution containing the treatments and, after 12 hours, the inoculation of Monilinia fructicola was carried out (0.2 mL of spore suspension with 105 spores mL-1, in each side of the fruit). Sixty hours past inoculation, the size of lesions, esporulation and percentage of control were evaluated. The contents of total protein, total phenol, total and reducing sugar, besides activity of phenylalanine ammonia-lyase (PAL), were determined. The elicitors induce resistance to M. fructicola, with reduction in fungal development. Elicitor's increased the evaluated biochemical parameters and the activity of PAL, which was related to the size reduction of lesions in peaches. The elicitors could be used in integrated management of brown-rot, in post-harvest applications.O objetivo deste trabalho foi avaliar o efeito dos eliciadores acibenzolar-S-metil e proteína harpina, aplicados em pós-colheita, na indução de resistência sistêmica à podridão-parda em pêssegos. O delineamento experimental foi inteiramente casualizado, com quatro repetições, em parcelas subdivididas – com e sem ferimentos provocados aos frutos –, e os tratamentos foram constituídos por: acibenzolar-S-metil (50 mg do i.a. L-1), dois produtos comerciais com proteína harpina (80 mg do i.a. L-1) e uma testemunha (água destilada). Os frutos foram pulverizados, individualmente, com 1 mL de solução aquosa com os tratamentos e, após 12 horas, efetuou-se a inoculação com Monilinia fructicola (0,2 mL da suspensão com concentração de 105 esporos mL-1, em cada lado do fruto). Após 60 horas da inoculação, avaliaram-se: a área lesionada, a esporulação e o percentual de controle. Determinaram-se os teores de proteínas totais, açúcares redutores e totais, fenóis, além da atividade da enzima fenilalanina amônia-liase (FAL). Os eliciadores induziram resistência dos frutos a M. fructicola, com redução do desenvolvimento do fungo. O uso dos eliciadores aumentou os teores dos parâmetros bioquímicos avaliados e a atividade da FAL, que esteve relacionada à redução da área lesionada em pêssegos. Os indutores podem contribuir para o manejo integrado da podridão-parda em pêssegos, em pós-colheita

Protocol suggested for dna extraction of jabuticaba trees

O presente trabalho teve por objetivo adaptar o protocolo descrito por Soto (2001), testar diferentes fontes (tecido vegetal) e estabelecer condi\ue7\uf5es de coleta e pr\ue9-tratamento de amostras para extra\ue7\ue3o de DNA de jabuticabeira ( Plinia cauliflora (DC.) Berg, Myrtaceae). Utilizaram-se folhas em desenvolvimento, folhas completamente expandidas, c\ue2mbio do caule e pec\uedolo foliar. O protocolo testado pode ser utilizado para extra\ue7\ue3o de DNA de jabuticabeira, pois proporcionou isolamento de DNA de boa qualidade e possibilitou amplifica\ue7\ue3o por meio de PCR (\u201cRea\ue7\ue3o de Polimerase em Cadeia\u201d). Para evitar a oxida\ue7\ue3o de folhas \ue9 necess\ue1rio realizar a macera\ue7\ue3o com nitrog\ueanio l\uedquido.This study aimed to adapt the protocol described by Soto (2001), to test different sources (plant tissue) and to establish conditions for collection and pre-treatment of samples for extracting DNA from jabuticaba trees ( Plinia cauliflora (DC.) Berg, Myrtaceae). For this purpose, developing and expanded leaves, cambium of the stem and leaf stalk were used. The protocol tested can be used for DNA extraction from jabuticaba trees, because it provided DNA isolation of good quality and allowed the amplification of DNA through of PCR (Polymerase Chain Reaction). The use of liquid nitrogen for the maceration of leaves is necessary to prevent oxidation

Neuropathology in Mice Expressing Mouse Alpha-Synuclein

α-Synuclein (αSN) in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD) and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN), which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occured in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are strikingly similar to those evoked by expression of human wildtype or mutant forms

In vivo assessment of Lewy body and beta-amyloid copathologies in idiopathic normal pressure hydrocephalus: prevalence and associations with clinical features and surgery outcome

Background: Idiopathic normal pressure hydrocephalus (iNPH) is a clinico-radiological syndrome of elderly individuals likely sustained by different neurodegenerative changes as copathologies. Since iNPH is a potentially reversible condition, assessing neurodegenerative pathologies in vitam through CSF biomarkers and their influence on clinical features and surgical outcome represents crucial steps.Methods: We measured a-synuclein seeding activity related to Lewy body (LB) pathology by the real-time quaking-induced conversion assay (RT-QuIC) and Alzheimer disease core biomarkers (proteins total-tau, phospho-tau, and amyloid-beta) by immunoassays in the cerebrospinal fluid (CSF) of 293 iNPH patients from two independent cohorts. To compare the prevalence of LB copathology between iNPH participants and a control group representative of the general population, we searched for a-synuclein seeding activity in 89 age-matched individuals who died of Creutzfeldt-Jakob disease (CJD). Finally, in one of the iNPH cohorts, we also measured the CSF levels of neurofilament light chain protein (NfL) and evaluated the association between all CSF biomarkers, baseline clinical features, and surgery outcome at 6 months.Results: Sixty (20.5%) iNPH patients showed alpha-synuclein seeding activity with no significant difference between cohorts. In contrast, the prevalence observed in CJD was only 6.7% (p= 0.002). Overall, 24.0% of iNPH participants showed an amyloid-positive (A+) status, indicating a brain co-pathology related to A beta deposition. At baseline, in the Italian cohort, a-synuclein RT-QuIC positivity was associated with higher scores on axial and upper limb rigidity (p=0.003 and p =0.011, respectively) and lower MMSEc scores (p =0.003). A+ patients showed lower scores on the MMSEc (p =0.037) than A- patients. Higher NfL levels were also associated with lower scores on the MMSEc (rho = -0.213; p= 0.021). There were no significant associations between CSF biomarkers and surgical outcome at 6 months (i.e. responders defined by decrease of 1 point on the mRankin scale).Conclusions: Prevalent LB- and AD-related neurodegenerative pathologies affect a significant proportion of iNPH patients and contribute to cognitive decline (both) and motor impairment (only LB pathology) but do not significantly influence the surgical outcome at 6 months. Their effect on the clinical benefit after surgery over a more extended period remains to be determined

The quail genome:insights into social behaviour, seasonal biology and infectious disease response

Background: The Japanese quail (Coturnix japonica) is a popular domestic poultry species and an increasingly significant model species in avian developmental, behavioural and disease research. Results: We have produced a high-quality quail genome sequence, spanning 0.93 Gb assigned to 33 chromosomes. In terms of contiguity, assembly statistics, gene content and chromosomal organisation, the quail genome shows high similarity to the chicken genome. We demonstrate the utility of this genome through three diverse applications. First, we identify selection signatures and candidate genes associated with social behaviour in the quail genome, an important agricultural and domestication trait. Second, we investigate the effects and interaction of photoperiod and temperature on the transcriptome of the quail medial basal hypothalamus, revealing key mechanisms of photoperiodism. Finally, we investigate the response of quail to H5N1 influenza infection. In quail lung, many critical immune genes and pathways were downregulated after H5N1 infection, and this may be key to the susceptibility of quail to H5N1. Conclusions: We have produced a high-quality genome of the quail which will facilitate further studies into diverse research questions using the quail as a model avian species

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD