Abrogation of LRRK2 dependent Rab10 phosphorylation with TLR4 activation and alterations in evoked cytokine release in immune cells

LRRK2 protein is expressed prominently in immune cells, cell types whose contribution to LRRK2-associated genetic Parkinson's disease (PD) is increasingly being recognised. We investigated the effect of inflammatory stimuli using RAW264.7 murine macrophage cells as model systems. A detailed time course of TLR2 and TLR4 stimulation was investigated through measuring LRRK2 phosphorylation at its specific phospho-sites, and Rab8 and Rab10 phosphorylation together with cytokine release following treatment with LPS and zymosan. LRRK2 phosphorylation at Ser935, Ser955 and Ser973 was increased significantly over untreated conditions at 4-24h in both WT-LRRK2 and T1348N-LRRK2 cell lines to similar extents although levels of Ser910 phosphorylation were maintained at higher levels throughout. Importantly we demonstrate that LPS stimulation significantly decreased phospho-Rab10 but not phospho-Rab8 levels over 4-24h in both WT-LRRK2 and T1348N-LRRK2 cell lines. The dephosphorylation of Rab10 was not attributed to its specific phosphatase, PPM1H as the levels remained unaltered with LPS treatment. MAPK phosphorylation occurred prior to LRRK2 phosphorylation which was validated by blocking TLR4 and TLR2 receptors with TAK242 or Sparstolonin B respectively. A significant decrease in basal level of TNFα release was noted in both T1348N-LRRK2 and KO-LRRK2 cell lines at 48h compared to WT-LRRK2 cell line, however LPS and zymosan treatment did not cause any significant alteration in the TNFα and IL-6 release between the three cell lines. In contrast, LPS and zymosan caused significantly lower IL-10 release in T1348N-LRRK2 and KO-LRRK2 cell lines. A significant decrease in phospho-Rab10 levels was also confirmed in human IPS-derived macrophages with TLR4 activation. Our data demonstrates for the first time that LRRK2-dependent Rab10 phosphorylation is modulated by LPS stimulation, and that cytokine release may be influenced by the status of LRRK2. These data provide further insights into the function of LRRK2 in immune response, and has relevance for understanding cellular dysfunctions when developing LRRK2-based inhibitors for clinical treatment

Amyloid β peptides are differentially vulnerable to preanalytical surface exposure, an effect incompletely mitigated by the use of ratios

INTRODUCTION: We tested the hypothesis that the amyloid β (Aβ) peptide ratios are more stable than Aβ42 alone when biofluids are exposed to two preanalytical conditions known to modify measurable Aβ concentration. METHODS: Human cerebrospinal fluid (CSF) and culture media (CM) from human cortical neurons were exposed to a series of volumes and polypropylene surfaces. Aβ42, Aβ40, and Aβ38 peptide concentrations were measured using a multiplexed electrochemiluminescence immunoassay. Data were analyzed using mixed models in R. RESULTS: Decrease of measurable Aβ peptide concentrations was exaggerated in longer peptides, affecting the Aβ42:Aβ40 and Aβ42:Aβ38 ratios. However, the effect size of surface treatment was reduced in Aβ peptide ratios versus Aβ42 alone. For Aβ42:Aβ40, the effect was reduced by approximately 50% (volume) and 75% (transfer) as compared to Aβ42 alone. DISCUSSION: Use of Aβ ratios, in conjunction with concentrations, may mitigate confounding factors and assist the clinical diagnostic process for Alzheimer's disease

Amyloid precursor protein processing in human neurons with an allelic series of the PSEN1 intron 4 deletion mutation and total presenilin-1 knockout

Mutations in presenilin-1 (PSEN1), encoding the catalytic subunit of the amyloid precursor protein-processing enzyme γ-secretase, cause familial Alzheimer’s disease. However, the mechanism of disease is yet to be fully understood and it remains contentious whether mutations exert their effects predominantly through gain or loss of function. To address this question, we generated an isogenic allelic series for the PSEN1 mutation intron 4 deletion; represented by control, heterozygous and homozygous mutant induced pluripotent stem cells in addition to a presenilin-1 knockout line. Induced pluripotent stem cell-derived cortical neurons reveal reduced, yet detectable amyloid-beta levels in the presenilin-1 knockout line, and a mutant gene dosage-dependent defect in amyloid precursor protein processing in PSEN1 intron 4 deletion lines, consistent with reduced processivity of γ-secretase. The different effects of presenilin-1 knockout and the PSEN1 intron 4 deletion mutation on amyloid precursor protein-C99 fragment accumulation, nicastrin maturation and amyloid-beta peptide generation support distinct consequences of familial Alzheimer’s disease-associated mutations and knockout of presenilin-1 on the function of γ-secretase

Human myeloid progenitor glucocorticoid receptor activation causes genomic instability, type 1 IFN- response pathway activation and senescence in differentiated microglia; an early life stress model

This is the final version. Available on open access from Wiley via the DOI in this recordData availability statement: Accession codes: Gene Expression Omnibus GSE73721 (Zhang et al., 2016), GSE99074 (Galatro et al., 2017), dbGaP: phs001373.v2.p2 (Gosselin et al., 2017), SYNAPSE: syn3219045, syn11468526 (Olah et al., 2018).Our RNA-Seq data is available on request.One form of early life stress, prenatal exposure to glucocorticoids (GCs), confers a higher risk of psychiatric and neurodevelopmental disorders in later life. Increasingly, the importance of microglia in these disorders is recognized. Studies on GCs exposure during microglial development have been limited, and there are few, if any, human studies. We established an in vitro model of ELS by continuous pre-exposure of human iPS-microglia to GCs during primitive hematopoiesis (the critical stage of iPS-microglial differentiation) and then examined how this exposure affected the microglial phenotype as they differentiated and matured to microglia, using RNA-seq analyses and functional assays. The iPS-microglia predominantly expressed glucocorticoid receptors over mineralocorticoid receptors, and in particular, the GR-α splice variant. Chronic GCs exposure during primitive hematopoiesis was able to recapitulate in vivo ELS effects. Thus, pre-exposure to prolonged GCs resulted in increased type I interferon signaling, the presence of Cyclic GMP-AMP synthase-positive (cGAS) micronuclei, cellular senescence and reduced proliferation in the matured iPS-microglia. The findings from this in vitro ELS model have ramifications for the responses of microglia in the pathogenesis of GC- mediated ELS-associated disorders such as schizophrenia, attention-deficit hyperactivity disorder and autism spectrum disorder.Alzheimer’s SocietyAlzheimer's Research UKNational Institute for Health and Care Research (NIHR

Patient-physician interaction in general practice and health inequalities in a multidisciplinary study: design, methods and feasibility in the French INTERMEDE study

<p>Abstract</p> <p>Background</p> <p>The way in which patients and their doctors interact is a potentially important factor in optimal communication during consultations as well as treatment, compliance and follow-up care. The aim of this multidisciplinary study is to use both qualitative and quantitative methods to explore the 'black box' that is the interaction between the two parties during a general practice consultation, and to identify factors therein that may contribute to producing health inequalities. This paper outlines the original multidisciplinary methodology used, and the feasibility of this type of study.</p> <p>Methods and design</p> <p>The study design combines methodologies on two separate samples in two phases. Firstly, a qualitative phase collected ethnographical and sociological data during consultation, followed by in-depth interviews with both patients and doctors independently. Secondly, a quantitative phase on a different sample of patients and physicians collected data via several questionnaires given to patients and doctors consisting of specific 'mirrored' questions asked post-consultation, as well as collecting information on patient and physician characteristics.</p> <p>Discussion</p> <p>The design and methodology used in this study were both successfully implemented, and readily accepted by doctors and patients alike. This type of multidisciplinary study shows great potential in providing further knowledge into the role of patient/physician interaction and its influence on maintaining or producing health inequalities. The next challenge in this study will be implementing the multidisciplinary approach during the data analysis.</p

Elevated 4R-tau in astrocytes from asymptomatic carriers of the MAPT 10+16 intronic mutation

The microtubule-associated protein tau gene (MAPT) 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers which, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau transcript and protein ratios. These elevated levels of 4R-tau in astrocytes implicate glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of pre-clinical tauopathies

Familial Alzheimer's Disease Mutations in PSEN1 Lead to Premature Human Stem Cell Neurogenesis

Mutations in presenilin 1 (PSEN1) or presenilin 2 (PSEN2), the catalytic subunit of γ-secretase, cause familial Alzheimer’s disease (fAD). We hypothesized that mutations in PSEN1 reduce Notch signaling and alter neurogenesis. Expression data from developmental and adult neurogenesis show relative enrichment of Notch and γ-secretase expression in stem cells, whereas expression of APP and β-secretase is enriched in neurons. We observe premature neurogenesis in fAD iPSCs harboring PSEN1 mutations using two orthogonal systems: cortical differentiation in 2D and cerebral organoid generation in 3D. This is partly driven by reduced Notch signaling. We extend these studies to adult hippocampal neurogenesis in mutation-confirmed postmortem tissue. fAD cases show mutation-specific effects and a trend toward reduced abundance of newborn neurons, supporting a premature aging phenotype. Altogether, these results support altered neurogenesis as a result of fAD mutations and suggest that neural stem cell biology is affected in aging and disease

Plasma amyloid-β ratios in autosomal dominant Alzheimer's disease: the influence of genotype.

In-vitro studies of autosomal dominant Alzheimer's disease implicate longer amyloid-beta peptides in disease pathogenesis, however less is known about the behaviour of these mutations in-vivo. In this cross-sectional cohort study, we used liquid chromatography-tandem mass spectrometry to analyse 66 plasma samples from individuals who were at-risk of inheriting a mutation or were symptomatic. We tested for differences in amyloid-beta42:38, 42:40 and 38:40 ratios between presenilin1 and amyloid precursor protein carriers. We examined the relationship between plasma and in-vitro models of amyloid-beta processing and tested for associations with parental age at onset. 39 participants were mutation carriers (28 presenilin1 and 11 amyloid precursor protein). Age- and sex-adjusted models showed marked differences in plasma amyloid-beta between genotypes: higher amyloid-beta42:38 in presenilin1 versus amyloid precursor protein (p < 0.001) and non-carriers (p < 0.001); higher amyloid-beta38:40 in amyloid precursor protein versus presenilin1 (p < 0.001) and non-carriers (p < 0.001); while amyloid-beta42:40 was higher in both mutation groups compared to non-carriers (both p < 0.001). Amyloid-beta profiles were reasonably consistent in plasma and cell lines. Within presenilin1, models demonstrated associations between amyloid-beta42:38, 42:40 and 38:40 ratios and parental age at onset. In-vivo differences in amyloid-beta processing between presenilin1 and amyloid precursor protein carriers provide insights into disease pathophysiology, which can inform therapy development

Diagnosis of acute myeloid leukemia according to the WHO classification in the Japan Adult Leukemia Study Group AML-97 protocol

We reviewed and categorized 638 of 809 patients who were registered in the Japan Adult Leukemia Study Group acute myeloid leukemia (AML)-97 protocol using morphological means. Patients with the M3 subtype were excluded from the study group. According to the WHO classification, 171 patients (26.8%) had AML with recurrent genetic abnormalities, 133 (20.8%) had AML with multilineage dysplasia (MLD), 331 (51.9%) had AML not otherwise categorized, and 3 (0.5%) had acute leukemia of ambiguous lineage. The platelet count was higher and the rate of myeloperoxidase (MPO)-positive blasts was lower in AML with MLD than in the other WHO categories. The outcome was significantly better in patients with high (≥50%) than with low (<50%) ratios of MPO-positive blasts (P < 0.01). The 5-year survival rates for patients with favorable, intermediate, and adverse karyotypes were 63.4, 39.1, and 0.0%, respectively, and 35.5% for those with 11q23 abnormalities (P < 0.0001). Overall survival (OS) did not significantly differ between nine patients with t(9;11) and 23 with other 11q23 abnormalities (P = 0.22). Our results confirmed that the cytogenetic profile, MLD phenotype, and MPO-positivity of blasts are associated with survival in patients with AML, and showed that each category had the characteristics of the WHO classification such as incidence, clinical features, and OS

Inflammatory myofibroblastic tumor of epididymis: a case report and review of literature

Background Epididymal inflammatory myofibroblastic tumor, also known by various other synonyms is a rare benign disease. Only eight cases have been reported to date. The most common presentation is a scrotal mass of variable duration. For a scrotal mass it is difficult to distinguish a benign or malignant etiology, in addition to the origin whether from testis or epididymis. As a result the definitive diagnosis can only be established by surgical exploration. Case presentation We report the ninth case of epididymal IMT who based on clinical and radiological findings underwent radical orchidectomy, with the histology suggestive of inflammatory myofibroblastic tumor. At 4 years follow up the patient is free of disease recurrence. Conclusion IMT though rare should be considered in the differential diagnosis of epididymal mass. Clinically it is often difficult to distinguish the origin of mass and even though the disease has benign nature and course it is crucial to counsel patients for orchidectomy as definitive diagnosis is established on surgical exploration