Quantitative proteomics suggests decrease in the secretogranin‐1 cerebrospinal fluid levels during the disease course of multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with unknown cause. Proteins with different abundance in the cerebrospinal fluid (CSF) from relapsing‐remitting MS (RRMS) patients and neurological controls could give novel insight to the MS pathogenesis and be used to improve diagnosis, predict prognosis and disease course, and guide in therapy decisions. We combined iTRAQ labeling and Orbitrap mass spectrometry to discover proteins with different CSF abundance between six RRMS patients and 18 neurological disease controls. From 777 quantified proteins seven were selected as biomarker candidates, namely chitinase‐3‐like protein 1, secretogranin‐1 (Sg1), cerebellin‐1, neuroserpin, cell surface glycoprotein MUC18, testican‐2 and glutamate receptor 4. An independent sample set of 13 early‐MS patients, 13 RRMS patients and 13 neurological controls was used in a multiple reaction monitoring verification study. We found the intracellular calcium binding protein Sg1 to be increased in early‐MS patients compared to RRMS and neurological controls. Sg1 should be included in further studies to elucidate its role in the early phases of MS pathogenesis and its potential as a biomarker for this disease.     

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.     

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.     

Oxidative Stress Induces Caveolin 1 Degradation and Impairs Caveolae Functions in Skeletal Muscle Cells

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H₂O₂) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H₂O₂ treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H₂O₂ treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H₂O₂ treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H₂O₂ decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.     

A Biochemical Approach to Understand the Pathogenesis of Advanced Pulmonary Arterial Hypertension: Metabolomic Profiles of Arginine,Sphingosine-1-Phosphate,and Heme of Human Lung

Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent precapillary pulmonary hypertension (PH), leading to progressive right heart failure and premature death. The pathological mechanisms underlying this condition remain elusive. Analysis of global metabolomics from lung tissue of patients with PAH (n = 8) and control lung tissue (n = 8) leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted arginine pathways with increased Nitric oxide (NO) and decreased arginine. Our results also showed specific metabolic pathways and genetic profiles with increased Sphingosine-1-phosphate (S1P) metabolites as well as increased Heme metabolites with altered oxidative pathways in the advanced stage of the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling in severe pulmonary hypertension. Profiling metabolomic alterations of the PAH lung has provided a new understanding of the pathogenic mechanisms of PAH, which benefits therapeutic targeting to specific metabolic pathways involved in the progression of PAH.     

Childhood Learning Disabilities and Atypical Dementia: A Retrospective Chart Review

Objective

To further our understanding of the association between self-reported childhood learning disabilities (LDs) and atypical dementia phenotypes (Atypical Dementia), including logopenic primary progressive aphasia (L-PPA), Posterior Cortical Atrophy (PCA), and Dysexecutive-type Alzheimer’s Disease (AD).

Methods

This retrospective case series analysis of 678 comprehensive neuropsychological assessments compared rates of self-reported LD between dementia patients diagnosed with Typical AD and those diagnosed with Atypical Dementia. 105 cases with neuroimaging or CSF data available and at least one neurology follow-up were identified as having been diagnosed by the neuropsychologist with any form of neurodegenerative dementia. These cases were subject to a consensus diagnostic process among three dementia experts using validated clinical criteria for AD and PPA. LD was considered Probable if two or more statements consistent with prior LD were documented within the Social & Developmental History of the initial neuropsychological evaluation.

Results

85 subjects (Typical AD n=68, Atypical AD n=17) were included in the final analysis. In logistic regression models adjusted for age, gender, handedness, education and symptom duration, patients with Probable LD, compared to patients without Probable LD, were significantly more likely to be diagnosed with Atypical Dementia vs. Typical AD (OR 13.1, 95% CI 1.3-128.4). All three of the L-PPA cases reporting a childhood LD endorsed childhood difficulty with language. By contrast, both PCA cases reporting Probable childhood LD endorsed difficulty with attention and/or math.

Conclusions

In people who develop dementia, childhood LD may predispose to atypical phenotypes. Future studies are required to confirm whether atypical neurodevelopment predisposes to regional-specific neuropathology in AD and other dementias.     

Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

Objective

In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis.

Methods

In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFL_MANUAL), a protocol in which images were manually selected for automated analysis (CNFL_{SEMI-AUTOMATED}), and one in which selection and analysis were performed electronically (CNFL_{FULLY-AUTOMATED}). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols.

Results

Mean CNFL_Manual was 16.7±4.0, 13.9±4.2 mm/mm² for non-diabetes controls and diabetes participants, while CNFL_{Semi-Automated} was 10.2±3.3, 8.6±3.0 mm/mm² and CNFL_{Fully-Automated} was 12.5±2.8, 10.9 ± 2.9 mm/mm². Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFL_{Fully-Automated} compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFL_{Semi-Automated} and CNFL_{Fully-Automated} underestimated CNFL_Manual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively.

Conclusions

Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.