Glycine Receptor Complex Analysis Using Immunoprecipitation-Blue Native Gel Electrophoresis-Mass Spectrometry.

The pentameric glycine receptor (GlyR), comprising the α1 and β subunits, is a major inhibitory ionotropic receptor in brainstem and spinal cord. GlyRs interact with gephyrin (GPHN), a scaffold protein that anchors the GlyR in the plasma membrane and enables it to form clusters in glycinergic postsynapses. Using an interaction proteomics approach, we provide evidence of the ArfGEFs IQ motif and Sec7 domain 3 (IQSEC3) and IQ motif and Sec7 domain 2 (IQSEC2) as two novel synaptic proteins interacting with GlyR complexes. When the affinity‐isolated GlyR complexes were fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1β‐GPHN appeared as the most abundant complex with a molecular weight of approximately 1 MDa, and GlyR α1β‐GPHN‐IQSEC3 as a minor protein complex of approximately 1.2 MDa. A third GlyR α1β‐GPHN‐IQSEC2 complex existed at the lowest amount with a mass similar to the IQSEC3‐containing complex. Using yeast two‐hybrid we demonstrate that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N‐terminal of the IQSEC3 IQ‐like domain. Our data provide direct evidence of the interaction of IQSEC3 with GlyR‐GPHN complexes, underscoring a potential role of these ArfGEFs in the function of glycinergic synapses

Combined cellomics and proteomics analysis reveals shared neuronal morphology and molecular pathway phenotypes for multiple schizophrenia risk genes

An enigma in studies of neuropsychiatric disorders is how to translate polygenic risk into disease biology. For schizophrenia, where > 145 significant GWAS loci have been identified and only a few genes directly implicated, addressing this issue is a particular challenge. We used a combined cellomics and proteomics approach to show that polygenic risk can be disentangled by searching for shared neuronal morphology and cellular pathway phenotypes of candidate schizophrenia risk genes. We first performed an automated high-content cellular screen to characterize neuronal morphology phenotypes of 41 candidate schizophrenia risk genes. The transcription factors Tcf4 and Tbr1 and the RNA topoisomerase Top3b shared a neuronal phenotype marked by an early and progressive reduction in synapse numbers upon knockdown in mouse primary neuronal cultures. Proteomics analysis subsequently showed that these three genes converge onto the syntaxin-mediated neurotransmitter release pathway, which was previously implicated in schizophrenia, but for which genetic evidence was weak. We show that dysregulation of multiple proteins in this pathway may be due to the combined effects of schizophrenia risk genes Tcf4, Tbr1, and Top3b. Together, our data provide new biological functions for schizophrenia risk genes and support the idea that polygenic risk is the result of multiple small impacts on common neuronal signaling pathways

Age-Dependent Hippocampal Proteomics in the APP/PS1 Alzheimer Mouse Model: A Comparative Analysis with Classical SWATH/DIA and directDIA Approaches

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the human population, for which there is currently no cure. The cause of AD is unknown; however, the toxic effects of amyloid-β (Aβ) are believed to play a role in its onset. To investigate this, we examined changes in global protein levels in a hippocampal synaptosome fraction of the Amyloid Precursor Protein swe/Presenelin 1 dE9 (APP/PS1) mouse model of AD at 6 and 12 months of age (moa). Data independent acquisition (DIA), or Sequential Window Acquisition of all THeoretical fragment-ion (SWATH), was used for a quantitative label-free proteomics analysis. We first assessed the usefulness of a recently improved directDIA workflow as an alternative to conventional DIA data analysis using a project-specific spectral library. Subsequently, we applied directDIA to the 6- and 12-moa APP/PS1 datasets and applied the Mass Spectrometry Downstream Analysis Pipeline (MS-DAP) for differential expression analysis and candidate discovery. We observed most regulation at 12-moa, in particular of proteins involved in Aβ homeostasis and microglial-dependent processes, like synaptic pruning and the immune response, such as APOE, CLU and C1QA-C. All proteomics data are available via ProteomeXchange with identifier PXD025777

Analytical workflow for rapid screening and purification of bioactives from venom proteomes

Animal venoms are important sources for finding new pharmaceutical lead molecules. We used an analytical platform for initial rapid screening and identification of bioactive compounds from these venoms followed by fast and straightforward LC-MS only guided purification to obtain bioactives for further chemical and biological studies. The analytical platform consists of a nano-LC separation coupled post-column to high-resolution mass spectrometry and parallel on-line bioaffinity profiling for the acetylcholine binding protein (AChBP) in a chip based fluorescent enhancement based bioassay. AChBP is a stable structural homologue of the extracellular ligand binding domain of the α7-nicotinic acetylcholine receptor (α7-nAChR). This receptor is an extensively studied medicinal target, previously associated with epilepsy, Alzheimer's, schizophrenia and anxiety. The workflow is demonstrated with the venom of the Naja mossambica mossambica. Two medium affinity AChBP ligands were found. After subsequent LC-MS guided purification of the respective venom peptides, the purified peptides were sequenced and confirmed as Cytotoxin 1 and 2. These peptides were not reported before to have affinity for the AChBP. The purified peptides can be used for further biological studies. © 2013 Elsevier Ltd. All rights reserved

Interaction proteomics of canonical Caspr2 (CNTNAP2) reveals the presence of two Caspr2 isoforms with overlapping interactomes

Autism is a human developmental brain disorder characterized by impaired social interaction and communication. Contactin-associated protein-like 2 (Caspr2, CNTNAP2) is a known genetic risk factor of autism. However, how this protein might contribute to pathology is unclear. In this study, we demonstrate that Caspr2 is abundantly present in lipid raft and in the synaptic membrane but is highly depleted in the postsynaptic density. The Caspr2 protein level in hippocampus is present at a constant level during synapse formation and myelination from P0 to P84. Interaction proteomics revealed the interactors of Caspr2, including CNTN2, KCNAs, members of the ADAM family (ADAM22, ADAM23 and ADAM11), members of LGI family and MAGUKs (DLGs and MPPs). Interestingly, a short form of Caspr2 was detected, which lacks most of the extracellular domains, however, is still associated with ADAM22 and to a lesser extent LGI1 and Kv1 channels. The comprehensive Caspr2 interactome revealed here might aid in understanding the molecular mechanisms underlying autism. This article is part of a Special Issue titled Neuroproteomics: Applications in Neuroscience and Neurology

Correlation profiling of brain sub-cellular proteomes reveals co-assembly of synaptic proteins and subcellular distribution

textabstractProtein correlation profiling might assist in defining co-assembled proteins and subcellular distribution. Here, we quantified the proteomes of five biochemically isolated mouse brain cellular sub-fractions, with emphasis on synaptic compartments, from three brain regions, hippocampus, cortex and cerebellum. We demonstrated the expected co-fractionation of canonical synaptic proteins belonging to the same functional groups. The enrichment profiles also suggested the presence of many novel pre- and post-synaptic proteins. Using super-resolution microscopy on primary neuronal culture we confirmed the postsynaptic localization of PLEKHA5 and ADGRA1. We further detected profound brain region specific differences in the extent of enrichment for some functionally associated proteins. This is exemplified by different AMPA receptor subunits and substantial differences in sub-fraction distribution of their potential interactors, which implicated the differences of AMPA receptor complex compositions. This resource aids the identification of proteins partners and subcellular distribution of synaptic proteins

Progression and recovery of Parkinsonism in a chronic progressive MPTP-induction model in the marmoset without persistent molecular and cellular damage

Chronic exposure to low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in marmoset monkeys was used to model the prodromal stage of Parkinson's disease (PD), and to investigate mechanisms underlying disease progression and recovery. Marmosets were subcutaneously injected with MPTP for a period of 12. weeks, 0.5. mg/kg once per week, and clinical signs of Parkinsonism, motor- and non-motor behaviors were recorded before, during and after exposure. In addition, postmortem immunohistochemistry and proteomics analysis were performed. MPTP-induced parkinsonian clinical symptoms increased in severity during exposure, and recovered after MPTP administration was ended. Postmortem analyses, after the recovery period, revealed no alteration of the number and sizes of tyrosine hydroxylase (TH)-positive dopamine (DA) neurons in the substantia nigra. Also levels of TH in putamen and caudate nucleus were unaltered, no differences were observed in DA, serotonin or nor-adrenalin levels in the caudate nucleus, and proteomics analysis revealed no global changes in protein expression in these brain areas between treatment groups. Our findings indicate that parkinsonian symptoms can occur without detectable damage at the cellular or molecular level. Moreover, we show that parkinsonian symptoms may be reversible when diagnosed and treated early

A lipidomic analysis approach to evaluate the response to cholesterol-lowering food intake

Plant sterols (PS) are well known to reduce serum levels of total cholesterol and LDL-cholesterol. Lipidomics potentially provides detailed information on a wide range of individual serum lipid metabolites, which may further add to our understanding of the biological effects of PS. In this study, lipidomics analysis was applied to serum samples from a placebo-controlled, parallel human intervention study (n = 97) of 4-week consumption of two PS-enriched, yoghurt drinks differing in fat content (based on 0.1% vs. 1.5% dairy fat). A comprehensive data analysis strategy was developed and implemented to assess and compare effects of two different PS-treatments and placebo treatment. The combination of univariate and multivariate data analysis approaches allowed to show significant effects of PS intake on the serum lipidome, and helped to distinguish them from fat content and non-specific effects. The PS-enriched 0.1% dairy fat yoghurt drink had a stronger impact on the lipidome than the 1.5% dairy fat yoghurt drink, despite similar LDL-cholesterol lowering effects. The PS-enriched 0.1% dairy fat yoghurt drink reduced levels of several sphingomyelins which correlated well with the reduction in LDL-cholesterol and can be explained by co-localization of sphingomyelins and cholesterol on the surface of LDL lipoprotein. Statistically significant reductions in serum levels of two lysophosphatidylcholines (LPC(16:1), LPC(20:1)) and cholesteryl arachidonate may suggest reduced inflammation and atherogenic potential. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0384-2) contains supplementary material, which is available to authorized users

Lipidomics of familial longevity

Molecular Epidemiolog

A lipidomic analysis approach to evaluate the response to cholesterol-lowering food intake

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