ABSTRACT
LINC-HELLP, showing chromosomal linkage with the pregnancy-specific HELLP syndrome in Dutch families, reduces differentiation from a proliferative to an invasive phenotype of first-trimester extravillous trophoblasts. Here we show that mutations in LINC-HELLP identified in HELLP families negatively affect this trophoblast differentiation either by inducing proliferation rate or by causing cell cycle exit as shown by a decrease in both proliferation and invasion. As LincRNAs predominantly function through interactions with proteins, we identified the directly interacting proteins using chromatin isolation by RNA purification followed by protein mass spectrometry. We found 22 proteins predominantly clustering in two functional networks, i.e. RNA splicing and the ribosome. YBX1, PCBP1, PCBP2, RPS6 and RPL7 were validated, and binding to these proteins was influenced by the HELLP mutations carried. Finally, we show that the LINC-HELLP transcript levels are significantly upregulated in plasma of women in their first trimester of pregnancy compared with non-pregnant women, whereas this upregulation seems absent in a pilot set of patients later developing pregnancy complications, indicative of its functional significance in vivo.
Subject(s)
HELLP Syndrome/genetics , Mutation , Pregnancy Trimester, First/genetics , RNA, Long Noncoding/genetics , Trophoblasts/cytology , Cell Cycle , Cell Differentiation , Cell Proliferation , Female , Gene Expression Regulation , HELLP Syndrome/blood , Humans , Pregnancy , Pregnancy Trimester, First/blood , Proteins/metabolism , RNA Splicing , RNA, Long Noncoding/blood , Ribosomes/metabolismABSTRACT
The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout-controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse.
Subject(s)
Cerebral Cortex/metabolism , Hippocampus/metabolism , Receptor, Metabotropic Glutamate 5/metabolism , Receptors, Metabotropic Glutamate/metabolism , Animals , HEK293 Cells , Humans , Immunoprecipitation , Mice , Receptor, Metabotropic Glutamate 5/analysis , Receptors, Metabotropic Glutamate/analysisABSTRACT
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.
Subject(s)
Hippocampus/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Proteomics , Animals , Humans , Mice , Protein Isoforms/metabolismABSTRACT
INTRODUCTION: We performed a comprehensive quantitative proteomics study on human hippocampus tissue involving all Braak stages to assess changes in protein abundance over the various stages of Alzheimer's disease (AD). METHODS: Hippocampal subareas CA1 and subiculum of 40 cases were isolated using laser capture microdissection and analyzed using mass spectrometry. Immunoblotting and immunohistochemistry were used for validation. RESULTS: Over the Braak stages, an altered expression was found for 372 proteins including changes in levels of extracellular matrix components, and in calcium-dependent signaling proteins. Early changes were observed in levels of proteins related to cytoskeletal dynamics and synaptic components including an increase in RIMS1 and GRIK4. Several synaptic proteins, such as BSN, LIN7A, DLG2, -3, and -4, exhibit an early-up, late-down expression pattern. DISCUSSION: This study provides new insight into AD-dependent changes in protein levels in the hippocampus during AD pathology, identifying potential novel therapeutic targets and biomarkers.
Subject(s)
Alzheimer Disease/pathology , CA1 Region, Hippocampal/metabolism , Proteome/metabolism , Aged , Aged, 80 and over , Extracellular Matrix Proteins/metabolism , Female , Humans , Laser Capture Microdissection , Male , Mass Spectrometry , Middle Aged , Nerve Tissue Proteins/metabolism , Proteomics , Psychiatric Status Rating Scales , Signal TransductionABSTRACT
Fast excitatory synaptic transmission in the brain is mediated by glutamate acting on postsynaptic AMPA receptors. Recent studies have revealed a substantial number of AMPA receptor auxiliary proteins, which potentially contribute to the regulation of AMPA receptor trafficking, subcellular receptor localization, and receptor gating properties. Here we examined the AMPA receptor interactomes from cortex, hippocampus, and cerebellum by comprehensive interaction proteomics. The study reveals that AMPA receptor auxiliary proteins are engaged in distinct brain region-specific AMPA receptors subcomplexes, which might underlie brain region-specific differential regulation of AMPA receptor properties. Depending on the brain region, an interacting protein can be involved in an AMPA and a non-AMPA receptor complex.
Subject(s)
Brain/metabolism , Multiprotein Complexes/metabolism , Proteomics/methods , Receptors, AMPA/metabolism , Animals , Blotting, Western , Carrier Proteins/metabolism , Cerebellum/metabolism , Cerebral Cortex/metabolism , Chromatography, Liquid , Hippocampus/metabolism , Immunoprecipitation , Mice, Inbred C57BL , Protein Binding , Protein Subunits/metabolism , Proteome/metabolism , Tandem Mass SpectrometryABSTRACT
During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood.
Subject(s)
Neuronal Plasticity , Proteome/metabolism , Synapses/metabolism , Visual Cortex/growth & development , Visual Cortex/physiology , Age Factors , Animals , Clathrin/genetics , Clathrin/metabolism , Cytoskeleton/genetics , Cytoskeleton/metabolism , Darkness , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Proteome/genetics , Sensory Deprivation , Synapses/genetics , Vision, Monocular/geneticsABSTRACT
Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.
Subject(s)
Aging/metabolism , DNA Repair , DNA-Binding Proteins/genetics , Endonucleases/genetics , Nerve Tissue Proteins/metabolism , Proteome/metabolism , Synapses/metabolism , Aging/genetics , Animals , Cells, Cultured , Cognition Disorders/metabolism , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , Gene Expression Regulation , Hippocampus/metabolism , Hippocampus/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/genetics , Neurons/metabolism , Neurons/physiology , Proteome/genetics , Synapses/pathologyABSTRACT
Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET(+)) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET(+) and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET(+)-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET(+) produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors.
Subject(s)
Acetylcholine/chemistry , Aplysia/chemistry , Carrier Proteins/chemistry , Cysteine/chemistry , Mutation, Missense , Acetylcholine/genetics , Acetylcholine/metabolism , Amino Acid Substitution , Animals , Aplysia/genetics , Aplysia/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Crystallography, X-Ray , Cysteine/genetics , Cysteine/metabolism , Protein Stability , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.
Subject(s)
Fragile X Syndrome/metabolism , Fragile X Syndrome/pathology , Hippocampus/physiopathology , Hippocampus/ultrastructure , Phenotype , Proteomics , Synapses/metabolism , Actins/metabolism , Animals , CA1 Region, Hippocampal/metabolism , CA1 Region, Hippocampal/pathology , CA1 Region, Hippocampal/physiopathology , CA1 Region, Hippocampal/ultrastructure , Cell Differentiation , Cytoskeleton/metabolism , Disease Models, Animal , Excitatory Postsynaptic Potentials/physiology , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Syndrome/physiopathology , Gene Knockout Techniques , Hippocampus/metabolism , Hippocampus/pathology , Mice , Neurites/metabolism , Neuronal Plasticity/physiology , Pseudopodia/metabolism , Synapses/pathology , Synaptic Vesicles/metabolism , Synaptic Vesicles/pathology , Tandem Mass SpectrometryABSTRACT
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.
Subject(s)
Proteomics , Receptors, AMPA , Animals , Mice , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Synapses/metabolism , Neuronal Plasticity/physiology , Hippocampus/metabolism , Mice, KnockoutABSTRACT
Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)-based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34-P78), with an over-representation of cell-membrane proteins during adolescent development (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44-P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed (14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non-stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development.
Subject(s)
Prefrontal Cortex , Proteomics/methods , Synapses , Adolescent , Adolescent Development , Adult , Age Factors , Animals , Cell Membrane/chemistry , Female , Humans , Male , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Prefrontal Cortex/growth & development , Prefrontal Cortex/ultrastructure , Rats , Rats, Wistar , Synapses/chemistry , Synapses/physiology , Synapses/ultrastructure , Synaptic Vesicles/chemistry , Synaptic Vesicles/metabolismABSTRACT
Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a beta-barrel pore in the outer membrane implicated in translocation of the upstream 'passenger' domain across the outer membrane. However, recent structural data suggest that the diameter of the beta-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the beta-domain was not inserted and folded as an integral beta-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Endopeptidases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Virulence Factors/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Biological Transport, Active , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cysteine/chemistry , Endopeptidases/chemistry , Endopeptidases/genetics , Escherichia coli/genetics , Escherichia coli/pathogenicity , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Genes, Bacterial , Models, Biological , Models, Molecular , Multiprotein Complexes , Mutagenesis, Site-Directed , Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/genetics , Peptidylprolyl Isomerase/metabolism , Protein Folding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Virulence Factors/chemistry , Virulence Factors/geneticsABSTRACT
Alzheimer's disease (AD) is characterized by amyloid beta (Aß) deposits as plaques in the parenchyma and in the walls of cortical and leptomeningeal blood vessels of the brain called cerebral amyloid angiopathy (CAA). It is suggested that CAA type-1, which refers to amyloid deposition in both capillaries and larger vessels, adds to the symptomatic manifestation of AD and correlates with disease severity. Currently, CAA cannot be diagnosed pre-mortem and disease mechanisms involved in CAA are elusive. To obtain insight in the disease mechanism of CAA and to identify marker proteins specifically associated with CAA we performed a laser dissection microscopy assisted mass spectrometry analysis of post-mortem human brain tissue of (I) AD cases with only amyloid deposits in the brain parenchyma and no vascular related amyloid, (II) AD cases with severe CAA type-1 and no or low numbers of parenchymal amyloid deposits and (III) cognitively healthy controls without amyloid deposits. By contrasting the quantitative proteomics data between the three groups, 29 potential CAA-selective proteins were identified. A selection of these proteins was analysed by immunoblotting and immunohistochemistry to confirm regulation and to determine protein localization and their relation to brain pathology. In addition, specificity of these markers in relation to other small vessel diseases including prion CAA, CADASIL, CARASAL and hypertension related small vessel disease was assessed using immunohistochemistry.Increased levels of clusterin (CLU), apolipoprotein E (APOE) and serum amyloid P-component (APCS) were observed in AD cases with CAA. In addition, we identified norrin (NDP) and collagen alpha-2(VI) (COL6A2) as highly selective markers that are clearly present in CAA yet virtually absent in relation to parenchymal amyloid plaque pathology. NDP showed the highest specificity to CAA when compared to other small vessel diseases. The specific changes in the proteome of CAA provide new insight in the pathogenesis and yields valuable selective biomarkers for the diagnosis of CAA.
Subject(s)
Alzheimer Disease/complications , Amyloid beta-Peptides/metabolism , Brain/pathology , Cerebral Amyloid Angiopathy/etiology , Cerebral Amyloid Angiopathy/metabolism , Proteomics , Aged , Aged, 80 and over , Apolipoproteins E/blood , Brain/metabolism , Cerebral Amyloid Angiopathy/blood , Clusterin/blood , Female , Humans , Laser Capture Microdissection , Male , Mass Spectrometry , Serum Amyloid P-Component/metabolism , Statistics, NonparametricABSTRACT
Patients with depression often suffer from cognitive impairments that contribute to disease burden. We used social defeat-induced persistent stress (SDPS) to induce a depressive-like state in rats and then studied long-lasting memory deficits in the absence of acute stressors in these animals. The SDPS rat model showed reduced short-term object location memory and maintenance of long-term potentiation (LTP) in CA1 pyramidal neurons of the dorsal hippocampus. SDPS animals displayed increased expression of synaptic chondroitin sulfate proteoglycans in the dorsal hippocampus. These effects were abrogated by a 3-week treatment with the antidepressant imipramine starting 8 weeks after the last defeat encounter. Next, we observed an increase in the number of perineuronal nets (PNNs) surrounding parvalbumin-expressing interneurons and a decrease in the frequency of inhibitory postsynaptic currents (IPSCs) in the hippocampal CA1 region in SDPS animals. In vivo breakdown of the hippocampus CA1 extracellular matrix by the enzyme chondroitinase ABC administered intracranially restored the number of PNNs, LTP maintenance, hippocampal inhibitory tone, and memory performance on the object place recognition test. Our data reveal a causal link between increased hippocampal extracellular matrix and the cognitive deficits associated with a chronic depressive-like state in rats exposed to SDPS.
Subject(s)
Cognitive Dysfunction/pathology , Depression/pathology , Extracellular Matrix/metabolism , Hippocampus/pathology , Animals , CA1 Region, Hippocampal/drug effects , CA1 Region, Hippocampal/pathology , CA1 Region, Hippocampal/physiopathology , Chondroitin ABC Lyase/metabolism , Chondroitin Sulfate Proteoglycans/metabolism , Chronic Disease , Cognitive Dysfunction/complications , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/physiopathology , Depression/complications , Depression/drug therapy , Depression/physiopathology , Hippocampus/drug effects , Hippocampus/physiopathology , Imipramine/pharmacology , Imipramine/therapeutic use , Interneurons/drug effects , Interneurons/pathology , Male , Memory/drug effects , Neural Inhibition/drug effects , Rats, Wistar , Social Behavior , Stress, Psychological/drug therapy , Stress, Psychological/etiology , Stress, Psychological/physiopathology , Synapses/drug effects , Synapses/metabolismABSTRACT
In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.
Subject(s)
Endothelial Cells/metabolism , Heat-Shock Proteins/metabolism , Neoplasm Proteins/metabolism , Ornithine/analogs & derivatives , Pyrimidines/metabolism , Pyruvaldehyde/metabolism , Cells, Cultured , Endothelial Cells/drug effects , Glucose/metabolism , Glutathione/analogs & derivatives , Glutathione/pharmacology , Glycation End Products, Advanced/biosynthesis , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/analysis , Humans , Lactoylglutathione Lyase/antagonists & inhibitors , Molecular Chaperones , Neoplasm Proteins/analysis , Ornithine/analysis , Ornithine/metabolism , Pyrimidines/analysisABSTRACT
The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.
Subject(s)
Epidermis/physiology , Microfilament Proteins/genetics , Proteomics , Ultraviolet Rays/adverse effects , Actin Depolymerizing Factors , Blotting, Western , Destrin , Dose-Response Relationship, Radiation , Epidermis/radiation effects , Gene Expression Regulation/physiology , Gene Expression Regulation/radiation effects , Humans , In Vitro Techniques , Microfilament Proteins/metabolism , Phosphorylation/radiation effects , SunlightABSTRACT
Tobacco smoking and nicotine exposure during adolescence interfere with prefrontal cortex (PFC) development and lead to cognitive impairments in later life. The molecular and cellular underpinnings of these consequences remain elusive. We found that adolescent nicotine exposure induced lasting attentional disturbances and reduced mGluR2 protein and function on presynaptic terminals of PFC glutamatergic synapses. Restoring mGluR2 activity in vivo by local infusion of a group II mGluR agonist in adult rats that received nicotine as adolescents rescued attentional disturbances.
Subject(s)
Attention Deficit Disorder with Hyperactivity/chemically induced , Attention Deficit Disorder with Hyperactivity/physiopathology , Cognition Disorders/chemically induced , Cognition Disorders/physiopathology , Synaptic Transmission/drug effects , Tobacco Use Disorder/physiopathology , Adolescent , Age Factors , Animals , Attention Deficit Disorder with Hyperactivity/diagnosis , Cognition Disorders/diagnosis , Disease Models, Animal , Humans , Rats , Synaptic Transmission/physiology , Tobacco Use Disorder/complications , Tobacco Use Disorder/diagnosisABSTRACT
MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.
Subject(s)
Apoptosis , Microtubule-Associated Proteins/metabolism , Mixed Function Oxygenases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Base Sequence , Blotting, Western , COS Cells , Chlorocebus aethiops , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , HEK293 Cells , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , L Cells , LIM Domain Proteins , Mice , Mice, Knockout , Microfilament Proteins , Microtubule-Associated Proteins/genetics , Mixed Function Oxygenases/genetics , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , RNA InterferenceABSTRACT
Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.
Subject(s)
Extracellular Matrix/metabolism , Heroin Dependence , Heroin/adverse effects , Narcotics/adverse effects , Prefrontal Cortex/pathology , Pyramidal Cells/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Chromatography, High Pressure Liquid/methods , Conditioning, Operant/drug effects , Cues , Drug Administration Schedule , Enzyme Inhibitors/pharmacology , Extracellular Matrix/classification , Gene Expression Regulation, Enzymologic/drug effects , Heroin Dependence/etiology , Heroin Dependence/metabolism , Heroin Dependence/pathology , Hydroxamic Acids/pharmacology , In Vitro Techniques , Male , Mass Spectrometry , Oligopeptides/pharmacology , Proteomics/methods , Rats , Rats, Wistar , Reinforcement Schedule , Self Administration/methods , Signal Transduction/drug effects , Synaptic Potentials/drug effectsABSTRACT
Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimer's disease (AD) and polyglutamine diseases such as Huntington's disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex. Moreover, by proteomic analysis, we identified expression changes in proteins involved in energy metabolism or organization of the cytoskeleton. These changes show a striking resemblance to the proteomic profiles of both AD brain and several AD mouse models. Moreover, UBB(+1) transgenic mice show a deficit in contextual memory in both water maze and fear conditioning paradigms. Although UBB(+1) partially inhibits the UPS in the cortex, these mice do not have an overt neurological phenotype. These mouse models do not replicate the full spectrum of AD-related changes, yet provide a tool to understand how the UPS is involved in AD pathological changes and in memory formation.