A rare loss-of-function variant of ADAM17 is associated with late-onset familial Alzheimer disease.

Common variants of about 20 genes contributing to AD risk have so far been identified through genome-wide association studies (GWAS). However, there is still a large proportion of heritability that might be explained by rare but functionally important variants. One of the so far identified genes with rare AD causing variants is ADAM10. Using whole-genome sequencing we now identified a single rare nonsynonymous variant (SNV) rs142946965 [p.R215I] in ADAM17 co-segregating with an autosomal-dominant pattern of late-onset AD in one family. Subsequent genotyping and analysis of available whole-exome sequencing data of additional case/control samples from Germany, UK, and USA identified five variant carriers among AD patients only. The mutation inhibits pro-protein cleavage and the formation of the active enzyme, thus leading to loss-of-function of ADAM17 alpha-secretase. Further, we identified a strong negative correlation between ADAM17 and APP gene expression in human brain and present in vitro evidence that ADAM17 negatively controls the expression of APP. As a consequence, p.R215I mutation of ADAM17 leads to elevated Aß formation in vitro. Together our data supports a causative association of the identified ADAM17 variant in the pathogenesis of AD.

Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

SORLA regulates calpain-dependent degradation of synapsin.

INTRODUCTION: Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor in neurons and a major risk factor for Alzheimer disease. METHODS: Here, we performed global proteome analyses in the brain of SORLA-deficient mice followed by biochemical and histopathologic studies to identify novel neuronal pathways affected by receptor dysfunction. RESULTS: We demonstrate that the lack of SORLA results in accumulation of phosphorylated synapsins in cortex and hippocampus. We propose an underlying molecular mechanism by demonstrating that SORLA interacts with phosphorylated synapsins through 14-3-3 adaptor proteins to deliver synapsins to calpain-mediated proteolytic degradation. DISCUSSION: Our results suggest a novel function for SORLA which is in control of synapsin degradation, potentially impacting on synaptic vesicle endocytosis and/or exocytosis.

The pro-neurotrophin receptor sortilin is a major neuronal apolipoprotein E receptor for catabolism of amyloid-ß peptide in the brain.

Apolipoprotein E (APOE) is the major risk factor for sporadic Alzheimer's disease. Among other functions, APOE is proposed to sequester neurotoxic amyloid-ß (Aß) peptides in the brain, delivering them to cellular catabolism via neuronal APOE receptors. Still, the receptors involved in this process remain controversial. Here, we identified the pro-neurotrophin receptor sortilin as major endocytic pathway for clearance of APOE/Aß complexes in neurons. Sortilin binds APOE with high affinity. Lack of receptor expression in mice results in accumulation of APOE and of Aß in the brain and in aggravated plaque burden. Also, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aß complexes despite proper expression of other APOE receptors. Despite higher than normal brain APOE levels, sortilin-deficient animals display anomalies in brain lipid metabolism (e.g., accumulation of sulfatides) seen in APOE-deficient mice, indicating functional deficiency in cellular APOE uptake pathways. Together, our findings identified sortilin as an essential neuronal pathway for APOE-containing lipoproteins in vivo and suggest an intriguing link between Aß catabolism and pro-neurotrophin signaling converging on this receptor.

Isolation and Identification of Novel Taste-Modulating N2-Guanosine 5'-Monophosphate Derivatives Generated by Maillard-Type Reactions.

Several compounds with taste-modulating properties have been investigated, improving the taste impression without having a pronounced intrinsic taste. The best-known representatives of umami taste-modulating compounds are ribonucleotides and their derivatives. Especially the thio derivatives showed high taste-modulating potential in structure-activity relationship investigations. Therefore, this study focuses on the formation of guanosine 5'-monophosphate derivatives consisting of Maillard-type generated compounds like the aroma-active thiols (2-methyl-3-furanthiol, 3-mercapto-2-pentanone, 2-furfurylthiol) and formaldehyde to gain insights into the potential of combinations of taste and aroma-active compounds. One literature-known (N2-(furfurylthiomethyl)-guanosine 5'-monophosphate) and three new derivatives (N2-(2-methyl-1-furylthiomethyl)-guanosine 5'-monophosphate, N2-((5-hydroxymethyl)-2-methyl-1-furylthiomethyl)-guanosine 5'-monophosphate, N2-((2-pentanon-1-yl)thiomethyl)-guanosine 5'-monophosphate) were successfully produced using green natural deep eutectic solvents and isolated, and their structures were completely elucidated. Besides the intrinsic taste properties, the kokumi and umami taste-modulating effects of the four derivatives were evaluated via psychophysical investigations, ranging from 19 to 22 µmol/L.

Production of glycosylated soluble amyloid precursor protein alpha (sAPPalpha) in Leishmania tarentolae.

Soluble amyloid precursor protein alpha (sAPPalpha) is a cleavage product of the amyloid precursor protein (APP), the etiologic agent in Alzheimer's disease (AD). Reduced expression of sAPPalpha was previously found in the brains of AD patients, and it was suggested that sAPPalpha might counteract neurotoxic effects of Abeta, another APP cleavage product with enhanced abundance in Alzheimer's diseased brains. However, little is known about the biological functions of sAPPalpha. Thus, efficient production of this protein is a prerequisite for further studies. The unicellular eukaryotic parasite Leishmania tarentolae has recently emerged as a promising expression system for eukaryotic proteins due to its ability to posttranslationally modify proteins combined with easy cultivation and high protein yield. Interestingly, sAPPalpha produced in L. tarentolae was biologically active and glycosylated. In contrast to nonglycosylated sAPPalpha expressed in Eschericha coli, it also featured higher stability against enzymatic degradation. Detailed analysis of the glycosylation pattern of sAPPalpha produced in L. tarentolae by PGC-LC-ESI-MS/MS N-glycan analysis identified among eukaryotic species the highly conserved core pentasaccharide (Man3GlcNAc2) as being attached to Asn467 of sAPPalpha. Using oxonium ion scanning of CID-MS/MS spectra in combination with ETD fragmentation, we also identified two peptides (peptides 269-288 and 575-587) modified with N-acetyl hexosamine (HexNAc) residues. One of these O-glycosylation sites could be unambiguously assigned to Thr576 of sAPPalpha. This is the first time that O-glycosylation of a recombinant protein expressed in L. tarentolae has been demonstrated. Together, human sAPPalpha produced in L. tarentolae was N- and O-glycosylated on similar sites as described for mammalian-expressed sAPPalpha and showed similar biological activity. This demonstrates that L. tarentolae is a very suitable and simple to handle expression system for mammalian glycoproteins.

Generation and characterization of a Leishmania tarentolae strain for site-directed in vivo biotinylation of recombinant proteins.

Leishmania tarentolae is a non-human-pathogenic Leishmania species of growing interest in biotechnology, as it is well-suited for the expression of human recombinant proteins. For many applications it is desirable to express recombinant proteins with a tag allowing easy purification and detection. Hence, we adopted a scheme to express recombinant proteins with a His6-tag and, additionally, to site-specifically in vivo biotinylate them for detection. Biotinylation is a relatively rare modification of endogenous proteins that allows easy detection with negligible cross-reactivity. Here, we established a genetically engineered L. tarentolae strain constitutively expressing the codon-optimized biotin-protein ligase from Escherichia coli (BirA). We thoroughly analyzed the strain for functionality using 2-D polyacrylamide-gel electrophoresis (PAGE), mass spectrometry, and transmission electron microscopy (TEM). We could demonstrate that neither metabolic changes (growth rate) nor structural abnormalities (TEM) occurred. To our knowledge, we show the first 2-D PAGE analyses of L. tarentolae. Our results demonstrate the great benefit of the established L. tarentolae in vivo biotinylation strain for production of dual-tagged recombinant proteins. Additionally, 2-D PAGE and TEM results give insights into the biology of L. tarentolae, helping to better understand Leishmania species. Finally, we envisage that the system is transferable to human-pathogenic species.

A New Inert Natural Deep Eutectic Solvent (NADES) as a Reaction Medium for Food-Grade Maillard-Type Model Reactions.

Sustainability, low toxicity, and high solute potential are the fundamental reasons for focusing green chemistry on natural deep eutectic solvents (NADES). The application of NADES ranges from organic chemistry to the agricultural sector and the food industry. In the food industry, the desired food quality can be achieved by the extraction of small molecules, macromolecules, and even heavy metals. The compound yield in Maillard-type model reactions can also be increased using NADES. To extend the so-called "kitchen-type chemistry" field, an inert, food-grade NADES system based on sucrose/D-sorbitol was developed, characterized, and examined for its ability as a reaction medium by evaluating its temperature and pH stability. Reaction boundary conditions were determined at 100 °C for three hours with a pH range of 3.7-9.0. As proof of principle, two Maillard-type model reactions were implemented to generate the taste-modulating compounds N2-(1-carboxyethyl)guanosine 5'-monophosphate) (161.8 µmol/mmol) and N2-(furfuryl thiomethyl)guanosine 5'-monophosphate (95.7 µmol/g). Since the yields of both compounds are higher than their respective taste-modulating thresholds, the newly developed NADES is well-suited for these types of "kitchen-type chemistry" and, therefore, a potential solvent candidate for a wide range of applications in the food industry.

Toward High-Throughput Analysis of Aroma Compounds Using Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry: Screening of Key Food Odorants in Various Foods.

Recent studies show the immense capacities of the unified quantitation of aroma and taste compounds using liquid chromatography-mass spectrometry (LC-MS). The goal of this study was to highlight the broad application of this unified method. Thus, a stable isotope dilution analysis quantification method of the most important key food odorants in various food categories by LC-MS was developed. Using the well-known derivatization agent 3-nitrophenylhydrazine for carbonyl derivatization and a newly developed approach for alcohol and thiol derivatization, a method for the quantitation of 20 key food odorants was established. Intraday precision was determined to be ≤26%, and interday precision was between 24 and 31%. Limits of quantitation were determined between 0.014 and 283 µg/kg. The work shows that a wide array of aroma compounds can be analyzed accurately by LC-MS.

Presymptomatic alterations in energy metabolism and oxidative stress in the APP23 mouse model of Alzheimer disease.

Glucose hypometabolism is the earliest symptom observed in the brains of Alzheimer disease (AD) patients. In a former study, we analyzed the cortical proteome of the APP23 mouse model of AD at presymptomatic age (1 month) using a 2-D electrophoresis-based approach. Interestingly, long before amyloidosis can be observed in APP23 mice, proteins associated with energy metabolism were predominantly altered in transgenic as compared to wild-type mice indicating presymptomatic changes in energy metabolism. In the study presented here, we analyzed whether the observed changes were associated with oxidative stress and confirmed our previous findings in primary cortical neurons, which exhibited altered ADP/ATP levels if transgenic APP was expressed. Reactive oxygen species produced during energy metabolism have important roles in cell signaling and homeostasis as they modify proteins. We observed an overall up-regulation of protein oxidation status as shown by increased protein carbonylation in the cortex of presymptomatic APP23 mice. Interestingly, many carbonylated proteins, such as Vilip1 and Syntaxin were associated to synaptic plasticity. This demonstrates an important link between energy metabolism and synaptic function, which is altered in AD. In summary, we demonstrate that changes in cortical energy metabolism and increased protein oxidation precede the amyloidogenic phenotype in a mouse model for AD. These changes might contribute to synaptic failure observed in later disease stages, as synaptic transmission is particularly dependent on energy metabolism.

Kainate promotes alterations in neuronal RNA splicing machinery.

Kainate, a glutamate analogue, activates kainate and AMPA receptors inducing strong synaptic activation. Systemic kainate application to rodents results in seizures, neurodegeneration, and neuronal remodeling in the brain. It is therefore used to investigate molecular mechanisms responsible for these conditions. We analyzed proteome alterations in murine primary cortical neurons after 24 h of kainate treatment. Our 2-D gel based proteomics approach revealed 91 protein alterations, some already associated with kainate-induced pathology. In addition, we found a large number of proteins which have not previously been reported to be associated with kainate-induced pathology. Functional classification of altered proteins revealed that they predominantly participate in mRNA splicing and cytoskeleton remodeling.

A large number of protein expression changes occur early in life and precede phenotype onset in a mouse model for huntington disease.

Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

Aging in mouse brain is a cell/tissue-level phenomenon exacerbated by proteasome loss.

Biological aging is often described by its phenotypic effect on individuals. Still, its causes are more likely found on the molecular level. Biological organisms can be considered as reliability-engineered, robust systems and applying reliability theory to their basic nonaging components, proteins, could provide insight into the aging mechanism. Reliability theory suggests that aging is an obligatory trade-off in a fault-tolerant system such as the cell which is constructed based on redundancy design. Aging is the inevitable redundancy loss of functional system components, that is proteins, over time. In our study, we investigated mouse brain development, adulthood, and aging from embryonic day 10 to 100 weeks. We determined redundancy loss of different protein categories with age using reliability theory. We observed a near-linear decrease of protein redundancy during aging. Aging may therefore be a phenotypic manifestation of redundancy loss caused by nonfunctional protein accumulation. This is supported by a loss of proteasome system components faster than dictated by reliability theory. This loss is highly detrimental to biological self-renewal and seems to be a key contributor to aging and therefore could represent a major target for therapies for aging and age-related diseases.

PROTEOMER: A workflow-optimized laboratory information management system for 2-D electrophoresis-centered proteomics.

In recent years proteomics became increasingly important to functional genomics. Although a large amount of data is generated by high throughput large-scale techniques, a connection of these mostly heterogeneous data from different analytical platforms and of different experiments is limited. Data mining procedures and algorithms are often insufficient to extract meaningful results from large datasets and therefore limit the exploitation of the generated biological information. In our proteomic core facility, which almost exclusively focuses on 2-DE/MS-based proteomics, we developed a proteomic database custom tailored to our needs aiming at connecting MS protein identification information to 2-DE derived protein expression profiles. The tools developed should not only enable an automatic evaluation of single experiments, but also link multiple 2-DE experiments with MS-data on different levels and thereby helping to create a comprehensive network of our proteomics data. Therefore the key feature of our "PROTEOMER" database is its high cross-referencing capacity, enabling integration of a wide range of experimental data. To illustrate the workflow and utility of the system, two practical examples are provided to demonstrate that proper data cross-referencing can transform information into biological knowledge.

Transcriptome and proteome analysis of early embryonic mouse brain development.

Mouse embryonic brain development involves sequential differentiation of multipotent progenitors into neurons and glia cells. Using microarrays and large 2-DE, we investigated the mouse brain transcriptome and proteome of embryonic days 9.5, 11.5, and 13.5. During this developmental period, neural progenitor cells shift from proliferation to neuronal differentiation. As expected, we detected numerous expression changes between all time points investigated, but interestingly, the rate of alteration remained in a similar range within 2 days of development. Furthermore, up- and down-regulation of gene products was balanced at each time point which was also seen at embryonic days 16-18. We hypothesize that during embryonic development, the rate of gene expression alteration is rather constant due to limited cellular resources such as energy, space, and free water. A similar complexity in terms of expressed genes and proteins suggests that changes in relative concentrations rather than an increase in the number of gene products dominate cellular differentiation. In general, expression of metabolism and cell cycle related gene products was down-regulated when precursor cells switched from proliferation to neuronal differentiation (days 9.5-11.5), whereas neuron specific gene products were up-regulated. A detailed functional analysis revealed their implication in differentiation related processes such as rearrangement of the actin cytoskeleton as well as Notch- and Wnt-signaling pathways.

An approach to handling and interpretation of ambiguous data in transcriptome and proteome comparisons.

A major challenge towards a comprehensive analysis of biological systems is the integration of data from different "omics" sources and their interpretation at a functional level. Here we address this issue by analysing transcriptomic and proteomic datasets from mouse brain tissue at embryonic days 9.5 and 13.5. We observe a high concordance between transcripts and their corresponding proteins when they were compared at the level of expression ratios between embryonic stages. Absolute expression values show marginal correlation. We show in examples, that poor concordance between protein and transcript expression is in part explained by the fact, that single genes give rise to multiple transcripts and protein variants. The integration of transcriptomic and proteomic data therefore requires proper handling of such ambiguities. A closer inspection of such cases in our datasets suggests, that comparing gene expression at exon level instead of gene level could improve the comparability. To address the biological relevance of differences in expression profiles, literature-data mining and analysis of gene ontology terms are widely used. We show here, that this can be complemented by the inspection of physical properties of genes, transcripts, and proteins.

BrainProfileDB - a platform for integration of functional genomics data.

BrainProfileDB is a database system for integrating large sets of high throughput functional genomics data of the Human Brain Proteome Project (HBPP). Within HBPP (http://www.smp-proteomics.de/) the molecular pathology of neurodegenerative diseases is investigated, using complementary methods from transcriptomics, proteomics, toponomics and interaction measurements. Aim of the database system is to provide a broad spectrum of scientific users joined in the consortium with a practical integrated view on their data. Employing appropriate mapping techniques and levels of data representation the user is relieved from technical details of gene identification or experimental measurement technique.

Protein expression overlap: more important than which proteins change in expression?

In recent years, a large number of proteomics studies for various diseases were conducted, such as for cancer, cardiovascular and neurodegenerative disorders (NDs). The availability of huge data sets with a large number of differentially expressed proteins showed for the first time that not all protein changes between a diseased and a control state were specific. This review focuses on this protein expression overlap, specifically between NDs, and tries to investigate the possible reasons for this overlap by investigating 14 ND proteomics studies of Alzheimer's (six studies), Parkinson's (four studies) and Huntington's disease (three studies), as well as amyotrophic lateral sclerosis (one study). Studies were selected according to the availability of quantitative changes, number of (biological) repeats and numbers of proteins changed. The studies include investigations of human tissue and mouse, as well as cell culture, models. A change in metabolism-related proteins was found to be common among all disorders. These changes can be explained by alterations in key regulatory proteins, such as those involved in transcription. Since most NDs affect, at least initially, very specific areas of the brain, the location of the changes may be more important than the kind of protein alterations that occur, since they are very similar among NDs.

Rare ABCA7 variants in 2 German families with Alzheimer disease.

OBJECTIVE: The aim of this study was to identify variants associated with familial late-onset Alzheimer disease (AD) using whole-genome sequencing. METHODS: Several families with an autosomal dominant inheritance pattern of AD were analyzed by whole-genome sequencing. Variants were prioritized for rare, likely pathogenic variants in genes already known to be associated with AD and confirmed by Sanger sequencing using standard protocols. RESULTS: We identified 2 rare ABCA7 variants (rs143718918 and rs538591288) with varying penetrance in 2 independent German AD families, respectively. The single nucleotide variant (SNV) rs143718918 causes a missense mutation, and the deletion rs538591288 causes a frameshift mutation of ABCA7. Both variants have previously been reported in larger cohorts but with incomplete segregation information. ABCA7 is one of more than 20 AD risk loci that have so far been identified by genome-wide association studies, and both common and rare variants of ABCA7 have previously been described in different populations with higher frequencies in AD cases than in controls and varying penetrance. Furthermore, ABCA7 is known to be involved in several AD-relevant pathways. CONCLUSIONS: We conclude that both SNVs might contribute to the development of AD in the examined family members. Together with previous findings, our data confirm ABCA7 as one of the most relevant AD risk genes.

The miRNome of Alzheimer's disease: consistent downregulation of the miR-132/212 cluster.

MicroRNAs (miRNAs) are small noncoding RNA molecules, with essential functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs appear to regulate the development and function of the nervous system. Alterations of miRNA expression have been associated with Alzheimer's disease (AD). To characterize the AD miRNA signature, we examined genome-wide miRNA and mRNA expression patterns in the temporal cortex of AD and control samples. We validated our miRNA results by semiquantitative real-time polymerase chain reaction (PCR) in independent prefrontal cortex. Furthermore, we separated gray and white matter brain sections to identify the cellular origin of the altered miRNA expression. We observed genome-wide downregulation of hsa-miR-132-3p and hsa-miR-212-3p in AD with a stronger decrease in gray matter AD samples. We further identified 10 differently expressed transcripts achieving genome-wide levels of significance. Significantly deregulated miRNAs and mRNAs were correlated and examined for potential binding sites (in silico). This miRNome-wide study in AD provides supportive evidence and corroborates an important contribution of miR-132/212 and corresponding target mRNAs to the pathogenesis of AD.