Temporal lobe epilepsy (TLE) can develop from alterations in hippocampal structure and circuit characteristics, and can be modeled in mice by administration of kainic acid (KA). Adult neurogenesis in the dentate gyrus (DG) contributes to hippocampal functions and has been reported to contribute to the development of TLE. Some of the phenotypical changes include neural stem and precursor cells (NPSC) apoptosis, shortly after their birth, before they produce hippocampal neurons. Here we explored these early phenotypical changes in the DG 3 days after a systemic injection of KA inducing status epilepticus (KA-SE), in mice. We performed a multi-omics experimental setup and analyzed DG tissue samples using proteomics, transcriptomics and microRNA profiling techniques, detecting the expression of 2327 proteins, 13401 mRNAs and 311 microRNAs. We here present a description of how these data were obtained and make them available for further analysis and validation. Our data may help to further identify and characterize molecular mechanisms involved in the alterations induced shortly after KA-SE in the mouse DG.

Adult neurogenesis continuously contributes new neurons to hippocampal circuits and the programmed death of a subset of immature cells provides a primary mechanism controlling this contribution. Epileptic seizures induce strong structural changes in the hippocampus, including the induction of adult neurogenesis, changes in gene expression and mitochondrial dysfunction, which may all contribute to epileptogenesis. However, a possible interplay between this factors remains largely unexplored. Here, we investigated gene expression changes in the hippocampal dentate gyrus shortly after prolonged seizures induced by kainic acid, focusing on mitochondrial functions. Using comparative proteomics, we identified networks of proteins differentially expressed shortly after seizure induction, including members of the BCL2 family and other mitochondrial proteins. Within these networks, we report for the first time that the atypical BCL2 protein BCL2L13 controls caspase-3 activity and cytochrome C release in neural stem/progenitor cells. Furthermore, we identify BCL2L13 as a novel target of the cooperative action of microRNA-124 and microRNA-137, both upregulated shortly after seizure induction. This cooperative microRNA-mediated fine-tuning of BCL2L13 expression controls casp3 activity, favoring non-apoptotic caspase-3 functions in NSPC exposed to KA and thereby may contribute to the early neurogenic response to epileptic seizures in the dentate gyrus.

The locus ceruleus is among the earliest affected brain regions in Parkinson’s disease (PD) showing Lewy body pathology and neuronal loss. To improve our understanding of the pathogenesis of PD, we performed the first proteomic analysis ever of post-mortem locus ceruleus tissue of six pathologically confirmed PD patients, and six ageand gendermatched non-neurological controls. In total 2495 proteins were identified, of which 87 proteins were differentially expressed in the locus ceruleus of PD patients compared to controls. The majority of these differentially expressed proteins are known to be involved in processes that have been implicated in the pathogenesis of PD previously, including mitochondrial dysfunction, oxidative stress, protein misfolding, cytoskeleton dysregulation and inflammation. Several individual proteins were identified that have hitherto not been associated with PD, such as regucalcin, which plays a role in maintaining intracellular calcium homeostasis, and isoform 1 of kinectin, which is involved in transport of cellular components along microtubules. In addition, pathway analysis suggests a pathogenetic role for aminoacyl-tRNA-biosynthesis. These findings indicate that the proteome of the locus ceruleus of PD patients and non-neurological controls provides data that are relevant to the pathogenesis of PD, reflecting both known and potentially novel pathogenetic pathways. Karin d. van dijk1,2, Henk W. Berendse3, Benjamin drukarch1, silvina a. Fratantoni3, Thang V. Pham3, sander r. Piersma3, evelien Huisman1, John J.P. Brevé1, Henk J. Groenewegen1, Connie r. Jimenez3*, Wilma d.J. van de Berg3* 1Department of Anatomy and Neurosciences, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands, 2Department of Neurology, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands, 3OncoProteomics Laboratory of the Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. *Shared senior authorship Brain Pathology 2012; 22(4):485-98

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring.

Cerebrospinal fluid (CSF) contains peptides and proteins important for brain physiology and potentially also relevant to brain pathology. Therefore, CSF provides an attractive source for biomarker discovery in brain and neurological diseases. CSF proteomics provides an analytical challenge as similar to 80% of proteins originate from serum, and removal of these major proteins is necessary to study brain-derived proteins that are present at low concentrations. In this book chapter, we describe a CSF sample pretreatment method to allow for in-depth and comparative analysis of the CSF proteome. To this end, we provide a protocol for batch-mode abundant protein depletion using the multiple affinity removal system MARS cartridge (Agilent) as well as for subsequent protein concentration using ultrafiltration with a 3 kDa molecular weight cutoff (MWCO) spin filter (Millipore). In addition, our experience with this depletion method coupled to 1D page/LC-MS/MS as well as other depletion and protein fractionation methods for CSF analysis is discussed.

The locus ceruleus is among the earliest affected brain regions in Parkinson's disease (PD) showing Lewy body pathology and neuronal loss. To improve our understanding of the pathogenesis of PD, we performed the first proteomic analysis ever of post-mortem locus ceruleus tissue of six pathologically confirmed PD patients, and six age- and gender-matched non-neurological controls. In total 2495 proteins were identified, of which 87 proteins were differentially expressed in the locus ceruleus of PD patients compared with controls. The majority of these differentially expressed proteins are known to be involved in processes that have been implicated in the pathogenesis of PD previously, including mitochondrial dysfunction, oxidative stress, protein misfolding, cytoskeleton dysregulation and inflammation. Several individual proteins were identified that have hitherto not been associated with PD, such as regucalcin, which plays a role in maintaining intracellular calcium homeostasis, and isoform 1 of kinectin, which is involved in transport of cellular components along microtubules. In addition, pathway analysis suggests a pathogenetic role for aminoacyl-tRNA-biosynthesis. These findings indicate that the proteome of the locus ceruleus of PD patients and non-neurological controls provides data that are relevant to the pathogenesis of PD, reflecting both known and potentially novel pathogenetic pathways.

Alzheimer's disease (AD) is clinically diagnosed with relative certainty, but only in advanced stages of the disease. Although the analysis of amyloid beta, tau and phosphorylated tau protein (pTau) in cerebrospinal fluid (CSF) can increase the accuracy of the diagnosis for AD, these biomarkers have limited value for early diagnosis in individual patients and prognosis. CSF provides an attractive source for biomarker discovery in brain and neurological diseases. However, CSF proteomics provides an analytical challenge as approximately 80% of proteins originate from serum, and removal of these major proteins is necessary to identify brain-derived biomarkers for mild cognitive impairment (MCI) and AD that are present at low concentrations. The aims of this study are: 1. to establish a robust, reproducible tandem mass spectrometry-based workflow for CSF proteomics. 2. comparative analysis of CSF samples to identify biomarkers for risk of MCI-AD progression and early detection of AD. The label-free proteomics workflow is based on abundant protein depletion coupled to 1D gel electrophoresis and nanoLC-MS/MS and employs spectral counting for protein quantitation. Proteomics was performed on 20 patient CSF samples (5 neurological controls, 5 MCI stable, 5 MCI converting to AD within 2 years of follow-up and 5 AD). Our proteomics method shows good reproducibility of protein identification and quantitation (Fratantoni, Piersma and Jimenez, Prot. Clin. Applic. 2010). CSF proteomics yielded a dataset of > 1000 CSF proteins including several candidate biomarkers for conversion of stable MCI to MCI-AD as well as for AD. Data integration with an independent CSF discovery set applying a similar workflow to 20 independent CSF samples (in collaboration with Dr. T. Muller and Prof. K. Marcus, MPC, Bochum) yielded ∼30 common candidate markers elevated in the CSF of MCI patients that in follow-up developed AD. The most promising ones include brain plasma membrane derived proteins, neuroplasticity and neurodevelopment related proteins underscoring the sensitivity of the approach. We have identified a set of promising candidate CSF biomarkers for identifying MCI patients at risk of developing AD. Candidates will be followed up in a larger series of CSF samples using targeted mass spectrometry based on multiple reaction monitoring and immunological assays.

An important neuropathological feature of brain injury and neuroinflammation, including Multiple Sclerosis (MS), is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that mediate scar formation. Tissue Transglutaminase (TG2) is a multifunctional enzyme with a ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can induce TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-ECM interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. TG2 on the surface of astrocytes interacts with fibronectin and is involved in astrocytic adhesion to fibronectin. TG2 is an essential factor in stimulating focal adhesion formation which is necessary for interaction of astrocytes with the ECM. We conclude that astrocyte-derived surface TG2 contributes to the interaction between astrocytes and fibronectin, thereby regulating ECM remodeling and possibly glial scarring.

PURPOSE:For biomarker discovery in cerebrospinal fluid (CSF), removal of major serum proteins is advantageous as more CSF proteins including brain-derived proteins can be identified. Our goal was to create a reproducible discovery workflow with acceptable throughput that can identify 500-1000 CSF proteins in small volumes of CSF.EXPERIMENTAL DESIGN:In this study, we compared the performance of two multi-affinity depletion methods in spin filter format: MARS Human 14 and Seppro-IgY-14. To this end, we analyzed depleted and bound CSF fractions isolated from 0.5 mL aliquots of the same CSF sample (n=3 per depletion method) by label-free GeLC-MS/MS-based proteomics and normalized spectral counting.RESULTS:The whole CSF dataset contained 884 proteins identified at high confidence. Depletion spin filter performance was assessed in terms of sensitivity and reproducibility of the CSF analysis. MARS and IgY-14 spin filters yielded comparable reproducibility of protein identification (71-74%) and quantification (CV 17-18%) but a significant difference in the total number of identified CSF proteins (767 and 703 proteins, respectively).CONCLUSIONS AND CLINICAL RELEVANCE:The MARS filter compared to IgY-14 filter provides a CSF analysis with enhanced proteome coverage. We anticipate that this enhanced sensitivity will facilitate biomarker discovery in early stages of cancer or neurological disease.

Deficiency of cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1(P3H1) has been reported in autosomal-recessive lethal or severe osteogenesis imperfecta (OI). CRTAP, P3H1, and cyclophilin B (CyPB) form an intracellular collagen-modifying complex that 3-hydroxylates proline at position 986 (P986) in the alpha1 chains of collagen type I. This 3-prolyl hydroxylation is decreased in patients with CRTAP and P3H1 deficiency. It was suspected that mutations in the PPIB gene encoding CyPB would also cause OI with decreased collagen 3-prolyl hydroxylation. To our knowledge we present the first two families with recessive OI caused by PPIB gene mutations. The clinical phenotype is compatible with OI Sillence type II-B/III as seen with COL1A1/2, CRTAP, and LEPRE1 mutations. The percentage of 3-hydroxylated P986 residues in patients with PPIB mutations is decreased in comparison to normal, but it is higher than in patients with CRTAP and LEPRE1 mutations. This result and the fact that CyPB is demonstrable independent of CRTAP and P3H1, along with reported decreased 3-prolyl hydroxylation due to deficiency of CRTAP lacking the catalytic hydroxylation domain and the known function of CyPB as a cis-trans isomerase, suggest that recessive OI is caused by a dysfunctional P3H1/CRTAP/CyPB complex rather than by the lack of 3-prolyl hydroxylation of a single proline residue in the alpha1 chains of collagen type I.

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.

G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.

In this study, we replaced the basic amine function of the known histamine H(3) receptor agonists imbutamine or immepip with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human H(3) receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed an increased selectivity for the human H(3) receptor over the human H(4) receptor, but none of the compounds in this series possess increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-imidazol-4-yl)-pentan-1-ol, is the most potent histamine H(3) receptor agonist (pK(i) = 8.0 and pEC(50) = 8.1) with a 320-fold selectivity at the human H(3) receptor over the human H(4) receptor.