Cerebrospinal fluid proteomic profiling of individuals with mild cognitive impairment and suspected non-Alzheimer's disease pathophysiology

BACKGROUND Suspected non-Alzheimer's disease pathophysiology (SNAP) is a biomarker concept that encompasses individuals with neuronal injury but without amyloidosis. We aim to investigate the pathophysiology of SNAP, defined as abnormal tau without amyloidosis, in individuals with mild cognitive impairment (MCI) by cerebrospinal fluid (CSF) proteomics. METHODS Individuals were classified based on CSF amyloid beta (Aβ)1-42 (A) and phosphorylated tau (T), as cognitively normal A-T- (CN), MCI A-T+ (MCI-SNAP), and MCI A+T+ (MCI-AD). Proteomics analyses, Gene Ontology (GO), brain cell expression, and gene expression analyses in brain regions of interest were performed. RESULTS A total of 96 proteins were decreased in MCI-SNAP compared to CN and MCI-AD. These proteins were enriched for extracellular matrix (ECM), hemostasis, immune system, protein processing/degradation, lipids, and synapse. Fifty-one percent were enriched for expression in the choroid plexus. CONCLUSION The pathophysiology of MCI-SNAP (A-T+) is distinct from that of MCI-AD. Our findings highlight the need for a different treatment in MCI-SNAP compared to MCI-AD

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting

Cerebrospinal fluid tau levels are associated with abnormal neuronal plasticity markers in Alzheimer's disease

BACKGROUND Increased total tau (t-tau) in cerebrospinal fluid (CSF) is a key characteristic of Alzheimer's disease (AD) and is considered to result from neurodegeneration. T-tau levels, however, can be increased in very early disease stages, when neurodegeneration is limited, and can be normal in advanced disease stages. This suggests that t-tau levels may be driven by other mechanisms as well. Because tau pathophysiology is emerging as treatment target for AD, we aimed to clarify molecular processes associated with CSF t-tau levels. METHODS We performed a proteomic, genomic, and imaging study in 1380 individuals with AD, in the preclinical, prodromal, and mild dementia stage, and 380 controls from the Alzheimer's Disease Neuroimaging Initiative and EMIF-AD Multimodality Biomarker Discovery study. RESULTS We found that, relative to controls, AD individuals with increased t-tau had increased CSF concentrations of over 400 proteins enriched for neuronal plasticity processes. In contrast, AD individuals with normal t-tau had decreased levels of these plasticity proteins and showed increased concentrations of proteins indicative of blood-brain barrier and blood-CSF barrier dysfunction, relative to controls. The distinct proteomic profiles were already present in the preclinical AD stage and persisted in prodromal and dementia stages implying that they reflect disease traits rather than disease states. Dysregulated plasticity proteins were associated with SUZ12 and REST signaling, suggesting aberrant gene repression. GWAS analyses contrasting AD individuals with and without increased t-tau highlighted several genes involved in the regulation of gene expression. Targeted analyses of SNP rs9877502 in GMNC, associated with t-tau levels previously, correlated in individuals with AD with CSF concentrations of 591 plasticity associated proteins. The number of APOE-e4 alleles, however, was not associated with the concentration of plasticity related proteins. CONCLUSIONS CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and blood-brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF proteomic profiles

Plant Mol Biol

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms

The influence of the R47H triggering receptor expressed on myeloid cells 2 variant on microglial exosome profiles

Variants in the triggering receptor expressed on myeloid cells 2 gene are linked with an increased risk of dementia, in particular the R47H^{het} triggering receptor expressed on myeloid cells 2 variant is linked to late-onset Alzheimer’s disease. Using human induced pluripotent stem cells-derived microglia, we assessed whether variations in the dynamics of exosome secretion, including their components, from these cells might underlie some of this risk. We found exosome size was not altered between common variant controls and R47H^{het} variants, but the amount and constitution of exosomes secreted were different. Exosome quantities were rescued by incubation with an ATP donor or with lipids via a phosphatidylserine triggering receptor expressed on myeloid cells 2 ligand. Following a lipopolysaccharide or phagocytic cell stimulus, exosomes from common variant and R47H^{heht} microglia were found to contain cytokines, chemokines, APOE and triggering receptor expressed on myeloid cells 2. Differences were observed in the expression of CCL22, IL-1β and triggering receptor expressed on myeloid cells 2 between common variant and R47H^{het} derived exosomes. Furthermore unlike common variant-derived exosomes, R47H^{het} exosomes contained additional proteins linked to negative regulation of transcription and metabolic processes. Subsequent addition of exosomes to stressed neurones showed R47H^{het}derived exosomes to be less protective. These data have ramifications for the responses of microglia in Alzheimer’s disease and may point to further targets for therapeutic intervention

Proteome analysis of Arabidopsis thaliana by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry

In the present study we show results of a large-scale proteome analysis of the recently sequenced plant Arabidopsis thaliana. On the basis of a previously published sequential protein extraction protocol, we prepared protein extracts from eight different A. thaliana tissues (primary leaf, leaf, stem, silique, seedling, seed, root, and inflorescence) and analysed these by two-dimensional gel electrophoresis. A total of 6000 protein spots, from three of these tissues, namely primary leaf, silique and seedling, were excised and the contained proteins were analysed by matrix assisted laser desorption/ionisation time of flight mass spectrometry peptide mass fingerprinting. This resulted in the identification of the proteins contained in 2943 spots, which were found to be products of 663 different genes. In this report we present and discuss the methodological and biological results of our plant proteome analysis

Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a ∼200–500-µm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-µm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-µl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-µl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS. 1 2 Copyright © 2005 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48772/1/916_ftp.pd

Neuroligin-1 in brain and CSF of neurodegenerative disorders: investigation for synaptic biomarkers

Synaptic pathology is a central event in Alzheimer’s disease (AD) and other neurodegenerative conditions, and investigation of synaptic proteins can provide valuable tools to follow synaptic dysfunction and loss in these diseases. Neuroligin-1 (Nlgn1) is a postsynaptic cell adhesion protein, important for synapse stabilization and formation. Nlgn1 has been connected to cognitive disorders, and specifically to AD, as target of the synaptotoxic effect of amyloid-β (Aβ) oligomers and Aβ fibrils. To address changes in Nlgn1 expression in human brain, brain regions in different neurological disorders were examined by Western blot and mass spectrometry. Brain specimens from AD (n = 23), progressive supranuclear palsy (PSP, n = 11), corticobasal degeneration (CBD, n = 10), and Pick’s disease (PiD, n = 9) were included. Additionally, cerebrospinal fluid (CSF) samples of AD patients (n = 43) and non-demented controls (n = 42) were analysed. We found decreased levels of Nlgn1 in temporal and parietal cortex (~ 50–60% reductions) in AD brains compared with controls. In frontal grey matter the reduction was not seen for AD patients; however, in the same region, marked reduction was found for PiD (~ 77%), CBD (~ 66%) and to a lesser extent for PSP (~ 43%), which could clearly separate these tauopathies from controls. The Nlgn1 level was reduced in CSF from AD patients compared to controls, but with considerable overlap. The dramatic reduction of Nlgn1 seen in the brain extracts of tauopathies warrants further investigation regarding the potential use of Nlgn1 as a biomarker for these neurodegenerative diseases

Protein aggregate formation permits millennium-old brain preservation

Human proteins have not been reported to survive in free nature, at ambient temperature, for long periods. Particularly, the human brain rapidly dissolves after death due to auto-proteolysis and putrefaction. The here presented discovery of 2600-year-old brain proteins from a radiocarbon dated human brain provides new evidence for extraordinary long-term stability of non-amyloid protein aggregates. Immunoelectron microscopy confirmed the preservation of neurocytoarchitecture in the ancient brain, which appeared shrunken and compact compared to a modern brain. Resolution of intermediate filaments (IFs) from protein aggregates took 2–12 months. Immunoassays on micro-dissected brain tissue homogenates revealed the preservation of the known protein topography for grey and white matter for type III (glial fibrillary acidic protein, GFAP) and IV (neurofilaments, Nfs) IFs. Mass spectrometry data could be matched to a number of peptide sequences, notably for GFAP and Nfs. Preserved immunogenicity of the prehistoric human brain proteins was demonstrated by antibody generation (GFAP, Nfs, myelin basic protein). Unlike brain proteins, DNA was of poor quality preventing reliable sequencing. These long-term data from a unique ancient human brain demonstrate that aggregate formation permits for the preservation of brain proteins for millennia

Differential stimulation of pluripotent stem cell-derived human microglia leads to exosomal proteomic changes affecting neurons

Microglial exosomes are an emerging communication pathway, implicated in fulfilling homeostatic microglial functions and transmitting neurodegenerative signals. Gene variants of triggering receptor expressed on myeloid cells-2 (TREM2) are associated with an increased risk of developing dementia. We investigated the influence of the TREM2 Alzheimer’s disease risk variant, R47Hhet, on the microglial exosomal proteome consisting of 3019 proteins secreted from human iPS-derived microglia (iPS-Mg). Exosomal protein content changed according to how the iPS-Mg were stimulated. Thus lipopolysaccharide (LPS) induced microglial exosomes to contain more inflammatory signals, whilst stimulation with the TREM2 ligand phosphatidylserine (PS+) increased metabolic signals within the microglial exosomes. We tested the effect of these exosomes on neurons and found that the exosomal protein changes were functionally relevant and influenced downstream functions in both neurons and microglia. Exosomes from R47Hhet iPS-Mg contained disease-associated microglial (DAM) signature proteins and were less able to promote the outgrowth of neuronal processes and increase mitochondrial metabolism in neurons compared with exosomes from the common TREM2 variant iPS-Mg. Taken together, these data highlight the importance of microglial exosomes in fulfilling microglial functions. Additionally, variations in the exosomal proteome influenced by the R47Hhet TREM2 variant may underlie the increased risk of Alzheimer’s disease associated with this variant