Blue Native PAGE-Antibody Shift in Conjunction with Mass Spectrometry to Reveal Protein Subcomplexes: Detection of a Cerebellar α1/α6-Subunits Containing γ-Aminobutyric Acid Type A Receptor Subtype.

The pentameric γ-Aminobutyric acid type A receptors (GABAARs) are ligand-gated ion channels that mediate the majority of inhibitory neurotransmission in the brain. In the cerebellum, the two main receptor subtypes are the 2α1/2ß/γ and 2α6/2ß/δ subunits. In the present study, an interaction proteomics workflow was used to reveal additional subtypes that contain both α1 and α6 subunits. Immunoprecipitation of the α6 subunit from mouse brain cerebellar extract co-purified the α1 subunit. In line with this, pre-incubation of the cerebellar extract with anti-α6 antibodies and analysis by blue native gel electrophoresis mass-shifted part of the α1 complexes, indicative of the existence of an α1α6-containing receptor. Subsequent mass spectrometry of the blue native gel showed the α1α6-containing receptor subtype to exist in two main forms, i.e., with or without Neuroligin-2. Immunocytochemistry on a cerebellar granule cell culture revealed co-localization of α6 and α1 in post-synaptic puncta that apposed the presynaptic marker protein Vesicular GABA transporter, indicative of the presence of this synaptic GABAAR subtype.

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.

The proteome of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer's disease.

Granulovacuolar degeneration (GVD) is a common feature in Alzheimer's disease (AD). The occurrence of GVD is closely associated with that of neurofibrillary tangles (NFTs) and GVD is even considered to be a pre-NFT stage in the disease process of AD. Currently, the composition of GVD bodies, the mechanisms associated with GVD and how GVD exactly relates to NFTs is not well understood. By combining immunohistochemistry (IHC) and laser microdissection (LMD) we isolated neurons with GVD and those bearing tangles separately from human post-mortem AD hippocampus (n = 12) using their typical markers casein kinase (CK)1δ and phosphorylated tau (AT8). Control neurons were isolated from cognitively healthy cases (n = 12). 3000 neurons per sample were used for proteome analysis by label free LC-MS/MS. In total 2596 proteins were quantified across samples and a significant change in abundance of 115 proteins in GVD and 197 in tangle bearing neurons was observed compared to control neurons. With IHC the presence of PPIA, TOMM34, HSP70, CHMP1A, TPPP and VXN was confirmed in GVD containing neurons. We found multiple proteins localizing specifically to the GVD bodies, with VXN and TOMM34 being the most prominent new protein markers for GVD bodies. In general, protein groups related to protein folding, proteasomal function, the endolysosomal pathway, microtubule and cytoskeletal related function, RNA processing and glycolysis were found to be changed in GVD neurons. In addition to these protein groups, tangle bearing neurons show a decrease in ribosomal proteins, as well as in various proteins related to protein folding. This study, for the first time, provides a comprehensive human based quantitative assessment of protein abundances in GVD and tangle bearing neurons. In line with previous functional data showing that tau pathology induces GVD, our data support the model that GVD is part of a pre-NFT stage representing a phase in which proteostasis and cellular homeostasis is disrupted. Elucidating the molecular mechanisms and cellular processes affected in GVD and its relation to the presence of tau pathology is highly relevant for the identification of new drug targets for therapy.

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

The pentameric glycine receptor (GlyR), comprising the α1 and ß 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, 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 is provided. When the affinity-isolated GlyR complexes are fractionated by blue native gel electrophoresis and characterized by mass spectrometry, GlyR α1ß-GPHN appears as the most abundant complex with a molecular weight of ≈1 MDa, and GlyR α1ß-GPHN-IQSEC3 as a minor protein complex of ≈1.2 MDa. A third GlyR α1ß-GPHN-IQSEC2 complex exists at the lowest amount with a mass similar to the IQSEC3 containing complex. Using yeast two-hybrid it is demonstrated that IQSEC3 interacts with the GlyR complex by binding to the GPHN G domain at the N-terminal of the IQSEC3 IQ-like domain. The 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.

A Fast and Economical Sample Preparation Protocol for Interaction Proteomics Analysis.

A simple and fast immunoprecipitation (IP) protocol is designed with the sample preparation incorporated, applicable to both low and high throughput. This new protocol combines two procedures based on magnetic beads in 96-well plate format. Protein complexes are captured by antibodies and magnetic beads conjugated with protein A. Proteins are washed and on-bead digested by using Single-Pot solid-phase sample preparation (SP3). The whole IP-SP3 approach can be completed in one day, which is considerably faster compared to the classical approach. No major quantitative differences are found between SP3 and FASP (filter-aided sample preparation) or a longer incubation protocol. Taken together, the IP-SP3 protocol is a fast and economical approach easily applicable for large-scale protein interactome analysis.

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.

Hippocampal extracellular matrix levels and stochasticity in synaptic protein expression increase with age and are associated with age-dependent cognitive decline.

Age-related cognitive decline is a serious health concern in our aging society. Decreased cognitive function observed during healthy brain aging is most likely caused by changes in brain connectivity and synaptic dysfunction in particular brain regions. Here we show that aged C57BL/6J wild-type mice have hippocampus-dependent spatial memory impairments. To identify the molecular mechanisms that are relevant to these memory deficits, we investigated the temporal profile of mouse hippocampal synaptic proteome changes at 20, 40, 50, 60, 70, 80, 90, and 100 weeks of age. Extracellular matrix proteins were the only group of proteins that showed robust and progressive up-regulation over time. This was confirmed by immunoblotting and histochemical analysis, which indicated that the increased levels of hippocampal extracellular matrix might limit synaptic plasticity as a potential cause of age-related cognitive decline. In addition, we observed that stochasticity in synaptic protein expression increased with age, in particular for proteins that were previously linked with various neurodegenerative diseases, whereas low variance in expression was observed for proteins that play a basal role in neuronal function and synaptic neurotransmission. Together, our findings show that both specific changes and increased variance in synaptic protein expression are associated with aging and may underlie reduced synaptic plasticity and impaired cognitive performance in old age.

Expression and Interaction Proteomics of GluA1- and GluA3-Subunit-Containing AMPARs Reveal Distinct Protein Composition.

The AMPA glutamate receptor (AMPAR) is the major type of synaptic excitatory ionotropic receptor in the brain. AMPARs have four different subunits, GluA1-4 (each encoded by different genes, Gria1, Gria2, Gria3 and Gria4), that can form distinct tetrameric assemblies. The most abundant AMPAR subtypes in the hippocampus are GluA1/2 and GluA2/3 heterotetramers. Each subtype contributes differentially to mechanisms of synaptic plasticity, which may be in part caused by how these receptors are regulated by specific associated proteins. A broad range of AMPAR interacting proteins have been identified, including the well-studied transmembrane AMPA receptor regulatory proteins TARP-γ2 (also known as Stargazin) and TARP-γ8, Cornichon homolog 2 (CNIH-2) and many others. Several interactors were shown to affect biogenesis, AMPAR trafficking, and channel properties, alone or in distinct assemblies, and several revealed preferred binding to specific AMPAR subunits. To date, a systematic specific interactome analysis of the major GluA1/2 and GluA2/3 AMPAR subtypes separately is lacking. To reveal interactors belonging to specific AMPAR subcomplexes, we performed both expression and interaction proteomics on hippocampi of wildtype and Gria1- or Gria3 knock-out mice. Whereas GluA1/2 receptors co-purified TARP-γ8, synapse differentiation-induced protein 4 (SynDIG4, also known as Prrt1) and CNIH-2 with highest abundances, GluA2/3 receptors revealed strongest co-purification of CNIH-2, TARP-γ2, and Noelin1 (or Olfactomedin-1). Further analysis revealed that TARP-γ8-SynDIG4 interact directly and co-assemble into an AMPAR subcomplex especially at synaptic sites. Together, these data provide a framework for further functional analysis into AMPAR subtype specific pathways in health and disease.

Distinct cell type-specific protein signatures in GRN and MAPT genetic subtypes of frontotemporal dementia.

Frontotemporal dementia is characterized by progressive atrophy of frontal and/or temporal cortices at an early age of onset. The disorder shows considerable clinical, pathological, and genetic heterogeneity. Here we investigated the proteomic signatures of frontal and temporal cortex from brains with frontotemporal dementia due to GRN and MAPT mutations to identify the key cell types and molecular pathways in their pathophysiology. We compared patients with mutations in the GRN gene (n = 9) or with mutations in the MAPT gene (n = 13) with non-demented controls (n = 11). Using quantitative proteomic analysis on laser-dissected tissues we identified brain region-specific protein signatures for both genetic subtypes. Using published single cell RNA expression data resources we deduced the involvement of major brain cell types in driving these different protein signatures. Subsequent gene ontology analysis identified distinct genetic subtype- and cell type-specific biological processes. For the GRN subtype, we observed a distinct role for immune processes related to endothelial cells and for mitochondrial dysregulation in neurons. For the MAPT subtype, we observed distinct involvement of dysregulated RNA processing, oligodendrocyte dysfunction, and axonal impairments. Comparison with an in-house protein signature of Alzheimer's disease brains indicated that the observed alterations in RNA processing and oligodendrocyte function are distinct for the frontotemporal dementia MAPT subtype. Taken together, our results indicate the involvement of different brain cell types and biological mechanisms in genetic subtypes of frontotemporal dementia. Furthermore, we demonstrate that comparison of proteomic profiles of different disease entities can separate general neurodegenerative processes from disease-specific pathways, which may aid the development of disease subtype-specific treatment strategies.

Systematic assessment of variability in the proteome of iPSC derivatives.

The use of induced pluripotent stem cells (iPSC) to model human complex diseases is gaining popularity as it allows investigation of human cells that are otherwise sparsely available. However, due to its laborious and cost intensive nature, iPSC research is often plagued by limited sample size and putative large variability between clones, decreasing statistical power for detecting experimental effects. Here, we investigate the source and magnitude of variability in the proteome of parallel differentiated astrocytes using mass spectrometry. We compare three possible sources of variability: inter-donor variability, inter- and intra-clonal variability, at different stages of maturation. We show that the interclonal variability is significantly smaller than the inter-donor variability, and that including more donors has a much larger influence on statistical power than adding more clones per donor. Our results provide insight into the sources of variability at protein level between iPSC samples derived in parallel and will aid in optimizing iPSC studies.

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-ß (Aß) 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 Aß 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.

Torpor enhances synaptic strength and restores memory performance in a mouse model of Alzheimer's disease.

Hibernation induces neurodegeneration-like changes in the brain, which are completely reversed upon arousal. Hibernation-induced plasticity may therefore be of great relevance for the treatment of neurodegenerative diseases, but remains largely unexplored. Here we show that a single torpor and arousal sequence in mice does not induce dendrite retraction and synapse loss as observed in seasonal hibernators. Instead, it increases hippocampal long-term potentiation and contextual fear memory. This is accompanied by increased levels of key postsynaptic proteins and mitochondrial complex I and IV proteins, indicating mitochondrial reactivation and enhanced synaptic plasticity upon arousal. Interestingly, a single torpor and arousal sequence was also sufficient to restore contextual fear memory in an APP/PS1 mouse model of Alzheimer's disease. Our study demonstrates that torpor in mice evokes an exceptional state of hippocampal plasticity and that naturally occurring plasticity mechanisms during torpor provide an opportunity to identify unique druggable targets for the treatment of cognitive impairment.

Transcriptome and proteome profiling of neural stem cells from the human subventricular zone in Parkinson's disease.

It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson's disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271+ NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271+ cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271+ NSCs to the SVZ homogenate from control donors and then compared CD271+ cells to CD11b+ microglia. These results confirmed their neural stem cell identity. Finally we compared controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our data suggest that NSCs may transit into a primed-quiescent state, that is in an "alert" non-proliferative phase in PD. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also show that the NSCs from SVZ undergo significant changes at both transcriptome and proteome level following PD.

Comparative Hippocampal Synaptic Proteomes of Rodents and Primates: Differences in Neuroplasticity-Related Proteins.

Key to the human brain's unique capacities are a myriad of neural cell types, specialized molecular expression signatures, and complex patterns of neuronal connectivity. Neurons in the human brain communicate via well over a quadrillion synapses. Their specific contribution might be key to the dynamic activity patterns that underlie primate-specific cognitive function. Recently, functional differences were described in transmission capabilities of human and rat synapses. To test whether unique expression signatures of synaptic proteins are at the basis of this, we performed a quantitative analysis of the hippocampal synaptic proteome of four mammalian species, two primates, human and marmoset, and two rodents, rat and mouse. Abundance differences down to 1.15-fold at an FDR-corrected p-value of 0.005 were reliably detected using SWATH mass spectrometry. The high measurement accuracy of SWATH allowed the detection of a large group of differentially expressed proteins between individual species and rodent vs. primate. Differentially expressed proteins between rodent and primate were found highly enriched for plasticity-related proteins.

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

Protein 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.

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.

Lipidomics of familial longevity.

Middle-aged offspring of nonagenarians, as compared to their spouses (controls), show a favorable lipid metabolism marked by larger LDL particle size in men and lower total triglyceride levels in women. To investigate which specific lipids associate with familial longevity, we explore the plasma lipidome by measuring 128 lipid species using liquid chromatography coupled to mass spectrometry in 1526 offspring of nonagenarians (59 years ± 6.6) and 675 (59 years ± 7.4) controls from the Leiden Longevity Study. In men, no significant differences were observed between offspring and controls. In women, however, 19 lipid species associated with familial longevity. Female offspring showed higher levels of ether phosphocholine (PC) and sphingomyelin (SM) species (3.5-8.7%) and lower levels of phosphoethanolamine PE (38:6) and long-chain triglycerides (TG) (9.4-12.4%). The association with familial longevity of two ether PC and four SM species was independent of total triglyceride levels. In addition, the longevity-associated lipid profile was characterized by a higher ratio of monounsaturated (MUFA) over polyunsaturated (PUFA) lipid species, suggesting that female offspring have a plasma lipidome less prone to oxidative stress. Ether PC and SM species were identified as novel longevity markers in females, independent of total triglycerides levels. Several longevity-associated lipids correlated with a lower risk of hypertension and diabetes in the Leiden Longevity Study cohort. This sex-specific lipid signature marks familial longevity and may suggest a plasma lipidome with a better antioxidant capacity, lower lipid peroxidation and inflammatory precursors, and an efficient beta-oxidation function.

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.