Ceramide Analog [\u3csup\u3e18\u3c/sup\u3eF]F-HPA-12 Detects Sphingolipid Disbalance in the Brain of Alzheimer’s Disease Transgenic Mice by Functioning as a Metabolic Probe

The metabolism of ceramides is deregulated in the brain of Alzheimer’s disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-β pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [18F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [18F]F-HPA-12 in the brain, independently from the APOE4 or APOE3 genetic background. FAD mice could be distinguished from littermate control animals with a sensitivity of 85.7% and a specificity of 87.5%, by gamma counter measurements. Metabolic analysis of [18F]F-HPA-12 in the brain suggested that the tracer is degraded less efficiently in the FAD mice. Furthermore, the radioactive signal registered in the hippocampus correlated with an increase of Cer d18:1/20:2 levels measured in the same brain region by mass spectrometry. Our data gives additional proof that ceramide metabolism is different in FAD mice compared to controls. Ceramide analogs like HPA-12 may function as metabolic probes to study ceramide disbalance in the brain

A retrospective multicenter study on clinical and serological parameters in patients with MuSK myasthenia gravis with and without general immunosuppression

Introduction: Muscle-specific kinase (MuSK)- myasthenia gravis (MG) is caused by pathogenic autoantibodies against MuSK that correlate with disease severity and are predominantly of the IgG4 subclass. The first-line treatment for MuSK-MG is general immunosuppression with corticosteroids, but the effect of treatment on IgG4 and MuSK IgG4 levels has not been studied. Methods: We analyzed the clinical data and sera from 52 MuSK-MG patients (45 female, 7 male, median age 49 (range 17–79) years) from Italy, the Netherlands, Greece and Belgium, and 43 AChR-MG patients (22 female, 21 male, median age 63 (range 2–82) years) from Italy, receiving different types of immunosuppression, and sera from 46 age- and sex-matched non-disease controls (with no diagnosed diseases, 38 female, 8 male, median age 51.5 (range 20–68) years) from the Netherlands. We analyzed the disease severity (assessed by MGFA or QMG score), and measured concentrations of MuSK IgG4, MuSK IgG, total IgG4 and total IgG in the sera by ELISA, RIA and nephelometry. Results: We observed that MuSK-MG patients showed a robust clinical improvement and reduction of MuSK IgG after therapy, and that MuSK IgG4 concentrations, but not total IgG4 concentrations, correlated with clinical severity. MuSK IgG and MuSK IgG4 concentrations were reduced after immunosuppression in 4/5 individuals with before-after data, but data from non-linked patient samples showed no difference. Total serum IgG4 levels were within the normal range, with IgG4 levels above threshold (1.35g/L) in 1/52 MuSK-MG, 2/43 AChR-MG patients and 1/45 non-disease controls. MuSK-MG patients improved within the first four years after disease onset, but no further clinical improvement or reduction of MuSK IgG4 were observed four years later, and only 14/52 (26.92%) patients in total, of which 13 (93.3%) received general immunosuppression, reached clinical remission. Discussion: We conclude that MuSK-MG patients improve clinically with general immunosuppression but may require further treatment to reach remission. Longitudinal testing of individual patients may be clinically more useful than single measurements of MuSK IgG4. No significant differences in the serum IgG4 concentrations and IgG4/IgG ratio between AChR- and MuSK-MG patients were found during follow-up. Further studies with larger patient and control cohorts are necessary to validate the findings

CERT\u3csub\u3eL\u3c/sub\u3e Reduces C16 Ceramide, Amyloid-β Levels, and Inflammation in a Model of Alzheimer’s Disease

BACKGROUND: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer\u27s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. METHODS: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. RESULTS: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. CONCLUSION: Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases

CERTL reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

Background: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion: Our results demonstrate a crucial role of CERTL in r

FTICR Mass spectrometry imaging at extreme mass resolving power using a dynamically harmonized ICR cell with 1ω or 2ω detection

MALDI mass spectrometry imaging (MALDI MSI) is a powerful analytical method providing the 2D localization of compounds from thin sections of typically but not exclusively biological samples. The dynamically harmonized ICR cell (ParaCell©) was recently introduced to achieve extreme spectral resolution capable to provide the isotopic fine structure of ions detected in complex samples. The latest improvement in ICR technology also includes 2ω detection which significantly reduces the transient time while preserving the nominal mass resolving power of the ICR cell. High-resolution MS images acquired on FT-ICR instruments equipped with 7T and 9.4T superconducting magnets and the dynamically harmonized ICR cell operating at suboptimal parameters, suffered severely from the pixel-to-pixel shifting of m/z peaks due to space-charge effects. The resulting profile average mass spectra have depreciated mass measurement accuracy and mass resolving power under the instrument specifications that affect the confidence level of the identified ions. Here we propose an analytical workflow based on the monitoring of the Total Ion Current to restrain the pixel-to-pixel m/z shift. Adjustment of the laser parameters is proposed to maintain high spectral resolution and mass accuracy measurement within the instrument specifications during MSI analyses. The optimized method has been successfully employed in replicates to perform high-quality MALDI MS images at resolving power (FWHM) above 1,000,000 in the lipid mass range across the whole image for superconducting magnets of 7T and 9.4T using 1 and 2ω detection. Our data also compare favorably with MALDI MSI experiments performed on higher magnetic field superconducting magnets, including the 21T MALDI FT-ICR prototype instrument of the NHMFL group at Tallahassee, Florida

Rapid Visualization of Chemically Related Compounds Using Kendrick Mass Defect as a Filter in Mass Spectrometry Imaging

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry dataset. The method enables an automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of family of homologous compounds from MSI images. We show that the KMD filtering, based on an in-house software, is suitable and robust for high resolution (full width at half-maximum, FWHM, at m/z 410 of 20 000) and very high-resolution (FWHM, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria co-cultures and brain tissue section </div

Rapid Visualization of Chemically Related Compounds Using Kendrick Mass Defect As a Filter in Mass Spectrometry Imaging

Kendrick mass defect (KMD) analysis is widely used for helping the detection and identification of chemically related compounds based on exact mass measurements. We report here the use of KMD as a criterion for filtering complex mass spectrometry data set. The method allow automated, easy and efficient data processing, enabling the reconstruction of 2D distributions of families of homologous compounds from MSI images. We show that KMD filtering, based on in-house software, is suitable and robust for high resolution (full width at half-maximum, fwhm, at m/z 410 of 20 000) and very high-resolution (fwhm, at m/z 410 of 160 000) MSI data. This method has been successfully applied to two different types of samples, bacteria cocultures, and brain tissue sections.RHIZOCLI

Alpha7 acetylcholine receptor autoantibodies are rare in sera of patients diagnosed with schizophrenia or bipolar disorder

The α7 acetylcholine receptor (AChR) has been linked with the onset of psychotic symptoms and we hypothesized therefore that it might also be an autoimmune target. Here, we describe a new radioimmunoassay (RIA) using iodine 125-labelled α-bungarotoxin and membrane extract from transfected HEK293 cells expressing human α7 AChR. This RIA was used to analyze sera pertaining to a cohort of 711 subjects, comprising 368 patients diagnosed with schizophrenia spectrum disorders, 140 with bipolar disorder, 58 individuals diagnosed of other mental disorders, and 118 healthy comparison subjects. We identified one patient whose serum tested positive although with very low levels (0.2 nM) for α7 AChR-specific antibodies by RIA. Three out of 711 sera contained antibodies against iodine 125-labelled α-bungarotoxin, because they precipitated with it in the absence of α7 AChR. This first evidence suggests that autoantibodies against α7 AChR are absent or very rare in these clinical groups.status: publishe