CURRENT POSITION: Research Group Leader, Principal Investigator in Molecular Neurobiology at the Donders Centre for Neuroscience, Department of Cognitive Neuroscience, Radboud University Nijmegen Medical Centre, The Netherlands email: a.aschrafi@cns.umcn.nl Phone: +31(0)24-36-14242 (office); -10559 (lab)
EDUCATION: • Ph.D. in Molecular Pharmacology, 2002 (magna cum laude) Institute of Pharmacology with Professor Dr. med. Guenther Schmalzing, J.W. Goethe-University, Frankfurt/Main, Germany • Master of Science in Medical Biotechnology, 1997, De Montfort University, Leicester, UK • Bachelor of Science in Biotechnology and Chemical Engineering, 1996, University of Applied Sciences Aachen, Germany • Abitur, 1991, Käthe-Kollwitz-Gymnasium, Wesseling, Germany
Abstract: BackgroundMicroRNAs (miRNAs) are non-coding gene transcripts involved in post-transcriptional regulation of genes. Recent studies identified miRNAs as important regulators of learning and memory in model organisms. So far, no mutations in specific miRNA genes have been associated with impaired cognitive functions.Methods and resultsIn three sibs and two unrelated patients with intellectual disability (ID), overlapping 1p21.3 deletions were detected by genome-wide array analysis. The shortest region of overlap included dihydropyrimidine dehydrogenase (DPYD) and microRNA 137 (MIR137). DPYD is involved in autosomal recessive dihydropyrimidine dehydrogenase deficiency. Hemizygous DPYD deletions were previously suggested to contribute to a phenotype with autism spectrum disorder and speech delay. Interestingly, the mature microRNA transcript microRNA-137 (miR-137) was recently shown to be involved in modulating neurogenesis in adult murine neuronal stem cells. Therefore, this study investigated the possible involvement of MIR137 in the 1p21.3-deletion phenotype. The patients displayed a significantly decreased expression of both precursor and mature miR-137 levels, as well as significantly increased expression of the validated downstream targets microphthalmia-associated transcription factor (MITF) and Enhancer of Zeste, Drosophila, Homologue 2 (EZH2), and the newly identified target Kruppel-like factor 4 (KLF4). The study also demonstrated significant enrichment of miR-137 at the synapses of cortical and hippocampal neurons, suggesting a role of miR-137 in regulating local synaptic protein synthesis machinery.ConclusionsThis study showed that dosage effects of MIR137 are associated with 1p21.3 microdeletions and may therefore contribute to the ID phenotype in patients with deletions harbouring this miRNA. A local effect at the synapse might be responsible.
Abstract: microRNAs (miRNAs) constitute a novel class of small, noncoding RNAs that act as negative post-transcriptional regulators of gene expression. Although the nervous system is a prominent site of miRNA expression, little is known about the spatial expression profiles of miRNAs in neurons. Here, we employed compartmentalized Campenot cell culture chambers to obtain a pure axonal RNA fraction of superior cervical ganglia (SCG) neurons, and determined the miRNA expression levels in these subcellular structural domains by microarray analysis and by real-time reverse-transcription polymerase chain reaction. The data revealed stable expression of a number of mature miRNAs that were enriched in the axons and presynaptic nerve terminals. Among the 130 miRNAs identified in the axon, miR-15b, miR-16, miR-204, and miR-221 were found to be highly abundant in distal axons as compared with the cell bodies of primary sympathetic neurons. Moreover, a number of miRNAs encoded by a common primary transcript (pri-miRNA) were differentially expressed in the distal axons, suggesting that there is a differential subcellular transport of miRNAs derived from the same coding region of the genome. Taken together, the data provide an important resource for future studies on the regulation of axonal protein synthesis and the role played by miRNAs in the maintenance of axonal structure and function as well as neuronal growth and development.
Abstract: Trafficking and local translation of axonal mRNAs play a critical role in the development and function of this neuronal subcellular structural domain. In this report, we studied cytochrome c oxidase subunit IV (COXIV) mRNA trafficking into distal axons of primary superior cervical ganglia (SCG) neurons, and provided evidence that axonal trafficking and mitochondrial association of the mRNA are mediated by an element located in a 38bp-long, hairpin-loop forming region within the 3'UTR of the transcript. Our results also suggest that suppression of local translation of COXIV mRNA results in significant attenuation of axonal elongation. Taken together, the results provide the first evidence for the existence of a cis-acting axonal transport element within a nuclear-encoding mitochondrial gene, and demonstrate the importance of the axonal trafficking and local translation of nuclear-encoded mitochondrial mRNAs in axonal growth. Published by Elsevier Inc.
Abstract: MicroRNAs (miRs) are evolutionarily conserved, noncoding RNA molecules of approximately 21 nt that regulate the expression of genes that are involved in various biological processes, such as cell proliferation and differentiation. Previously, we reported the presence of a heterogeneous population of mRNAs present in the axons and nerve terminals of primary sympathetic neurons to include the nuclear-encoded mitochondrial mRNA coding for COXIV. Sequence analysis of the 3'UTR of this mRNA revealed the presence of a putative binding site for miR-338, a brain-specific microRNA. Transfection of precursor miR-338 into the axons of primary sympathetic neurons decreases COXIV mRNA and protein levels and results in a decrease in mitochondrial activity, as measured by the reduction of ATP levels. Conversely, the transfection of synthetic anti-miR oligonucleotides that inhibit miR-338 increases COXIV levels, and results in a significant increase in oxidative phosphorylation and also norepinephrine uptake in the axons. Our results point to a molecular mechanism by which this microRNA participates in the regulation of axonal respiration and function by modulating the levels of COXIV, a protein which plays a key role in the assembly of the mitochondrial cytochrome c oxidase complex IV.
Abstract: A diverse set of mRNA-binding proteins (BPs) regulate local translation in neurons. However, little is known about the role(s) played by a family of cold-inducible, glycine-rich mRNA-BPs. Unlike neuronal mRNA-BPs characterized thus far, these proteins are induced by hypothermia and are comprised of one RNA recognition motif and an adjacent arginine- and glycine-rich domain. We studied the expression and function of the RNA-binding motif protein 3 (RBM3), a member of this family, in neurons. RBM3 was expressed in multiple brain regions, with the highest levels in cerebellum and olfactory bulb. In dissociated neurons, RBM3 was observed in nuclei and in a heterogeneous population of granules within dendrites. In sucrose gradient assays, RBM3 cofractionated with heavy mRNA granules and multiple components of the translation machinery. Two alternatively spliced RBM3 isoforms that differed by a single arginine residue were identified in neurons; both were post-translationally modified. The variant lacking the spliced arginine exhibited a higher dendritic localization and was the only isoform present in astrocytes. When overexpressed in neuronal cell lines, RBM3 isoforms-enhanced global translation, the formation of active polysomes, and the activation of initiation factors. These data suggest that RBM3 plays a distinctive role in enhancing translation in neurons.
Abstract: TSPY (testis-specific protein, Y-encoded) is a member of the greater SET/NAP family of molecules with various functions, e.g., in chromatin remodeling, regulation of gene expression, and has been implicated to play a role in the malignant development of gonadoblastoma, testicular and prostate cancer. Here we demonstrate that the C-terminus has a functional role for the nucleo-cytoplasmatic shuttling of the TSPY protein. Using various combinations of in vitro mutagenesis and enhanced green fluorescent protein reporter gene-expression experiments we were able to show that while the deletion of C-terminus leads to a decreased stability and enhanced degradation of the protein, the selective mutation of a C-terminal CK2 phosphorylation site (T300) prevents the TSPY protein from entering the nucleus. We conclude that phosphorylation of the (T300) residue is a necessary and functional prerequisite for TSPY's transport into the nucleus reminding of comparable data from a related Drosophila molecule, NAP1.
Abstract: Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.
Abstract: Fragile X syndrome results from the transcriptional silencing of a gene, Fmr1, that codes for an mRNA-binding protein (fragile X mental retardation protein, FMRP) present in neuronal dendrites. FMRP can act as a translational suppressor, and its own translation in dendrites is regulated by group I metabotropic glutamate receptors (mGluRs). Multiple lines of evidence suggest that mGluR-induced translation is exaggerated in Fragile X syndrome because of a lack of translational inhibition normally provided by FMRP. We characterized the role of FMRP in the regulation of mRNA granules, which sediment as a heavy peak after polysomes on sucrose gradients. In WT mouse brain, FMRP distributed with polysomes and granules. EM and biochemical analyses suggested that the granule fraction itself contained clusters of polysomes. In Fmr1 knockout brain, we observed a significant decrease in the amount of mRNA granules relative to WT mice. This difference appeared to be due to a role of FMRP in regulating the activation of granules during mGluR-induced translation; in vivo administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine increased granule content in Fmr1 knockout mouse brain to levels comparable with those seen in WT brain. In accord with a role of mGluR5 in the regulation of ongoing translation in vivo, we observed that the phosphorylation of several initiation factors in response to application of the mGluR1/5 agonist S-3,5-dihydroxyphenylglycine in vitro was blocked by methyl-6-(phenylethynyl)pyridine. Together, these data suggest that although large, polysome-containing granules can form in the absence of FMRP, their use in response to mGluR-induced translation is exaggerated.
Abstract: The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.
Abstract: Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.
Abstract: Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.
Abstract: Ceramide is a lipid second messenger produced by sphingolipid metabolism in cells exposed to a limited number of agonists and in turn triggers important cell responses including protein kinase C (PKC)-alpha activation. Using a fusion protein comprising bovine PKCalpha and the green fluorescent protein (GFP), we transfected human embryonic kidney (HEK) cells and investigated to which subcellular compartment ceramide triggers PKCalpha redistribution. Stimulation of HEK cells with exogenous C16-ceramide or bacterial sphingomyelinase (bSMase), which leads to increased endogenous ceramide formation, evokes a translocation of PKCalpha to the Golgi compartment. By using deletion mutants of PKCalpha lacking distinct domains in the regulatory region, it is shown that the Ca(2+)-dependent lipid binding C2 domain, but not one of the C1 domains is essentially required for the ceramide-triggered translocation of PKCalpha to the Golgi complex. In contrast, the C2 domain is not required for phorbol ester (TPA) binding and translocation of PKCalpha to the plasma membrane. In addition, evidence is provided that TPA requires only one of the two C1 subdomains to trigger translocation to the plasma membrane.In summary, our data provide evidence that ceramide either directly or indirectly interacts with the Ca(2+)-dependent lipid binding C2 domain of PKCalpha and thereby induces translocation of the enzyme to the Golgi compartment.
Abstract: NTPDase1 and NTPDase2 are two related plasma membrane-located enzymes involved in the extracellular degradation of nucleoside 5'-tri- and -diphosphates. They differ regarding their hydrolysis ratios for ATP and ADP. Both enzymes have a predicted transmembrane domain close to the N- and C-terminus, respectively, connected by an extensive extracellular domain that carries the active site. We expressed the rat-derived enzymes in Xenopus laevis oocytes and analyzed their quarternary structure. As revealed by application of blue native PAGE and a comparison of glutaraldehyde cross-linking, native NTPDase1 and NTPDase2 occur in oligomeric form. Oligomer formation of the cell surface-located pool of the enzymes was verified by surface iodination. The two enzymes differed in oligomeric structure and in oligomer complex stability. NTPDase1 preferentially occurred as a dimer that could be dissociated into monomeric forms in the presence of Coomassie Brilliant blue G-250 and dithiothreitol whereas NTPDase2 revealed higher oligomeric forms up to tetramers, largely resistant to dithiothreitol. Our results further suggest that the enzymes exist in varying oligomeric states. In contrast to NTPDase1, substrate specificity of NTPDase2 was altered with prolonged expression time, resulting in a decrease in the ATPase/ADPase activity ratio from 10 : 1 to 2.5 : 1. This was accompanied by a transition into a higher oligomeric state. Our results suggest that despite close sequence identity, NTPDase1 and NTPDase2 differ in oligomeric structure. Dynamic alterations in oligomeric state may induce changes in substrate preference and thus influence the pattern of extracellular nucleotide degradation in situ.
Abstract: Ceramide levels are strongly increased by stimulation of renal mesangial cells with nitric oxide (NO). This effect was shown previously to be due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. In this study we show that the NO-triggered inhibition of neutral ceramidase activity is paralleled by a down-regulation at the protein level. A complete loss of neutral ceramidase protein is obtained after 24 h of stimulation. Whereas the selective proteasome inhibitor lactacystin blocked NO-evoked ceramidase degradation, several caspase inhibitors were ineffective. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and also by the physiological PKC activators platelet-derived growth factor-BB (PDGF), angiotensin II and ATP, resulting in a normalization of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using (32)P(i)-labeled mesangial cells revealed that TPA, PDGF, angiotensin II, and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad spectrum PKC inhibitor Ro-31 8220 but not by CGP 41251, which has a preferential action on Ca(2+)-dependent isoforms, thus suggesting the involvement of a Ca(2+)-independent PKC isoform. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells show that particularly the PKC-delta isoform and to a lesser extent the PKC-alpha isoform are efficient in directly phosphorylating neutral ceramidase. In summary, our data show that NO is able to induce degradation of neutral ceramidase, thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-delta isoenzyme, which can directly phosphorylate and thereby prevent neutral ceramidase degradation. These novel regulatory interactions will provide therapeutically valuable information to target neutral ceramidase stability and subsequent ceramide accumulation.
Abstract: Na(+)/Cl(-)-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [(35)S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na(+)/Cl(-)-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.
Abstract: P2X(1) receptor subunits assemble in the ER of Xenopus oocytes to homotrimers that appear as ATP-gated cation channels at the cell surface. Here we address the extent to which N-glycosylation contributes to assembly, surface appearance, and ligand recognition of P2X(1) receptors. SDS-polyacrylamide gel electrophoresis (PAGE) analysis of glycan minus mutants carrying Gln instead of Asn at five individual NXT/S sequons reveals that Asn(284) remains unused because of a proline in the +4 position. The four other sites (Asn(153), Asn(184), Asn(210), and Asn(300)) carry N-glycans, but solely Asn(300) located only eight residues upstream of the predicted reentry loop of P2X(1) acquires complex-type carbohydrates. Like parent P2X(1), glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents reveals that elimination of Asn(153) or Asn(210) diminishes or increases functional expression levels, respectively. In addition, elimination of Asn(210) causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X(1) receptors is severely impaired or abolished, respectively. We conclude that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X(1) receptors and that individual N-glycans possess marked positional effects on expression levels (Asn(154), Asn(210)) and ATP potency (Asn(210)).
Abstract: Axons and presynaptic nerve terminals of both invertebrate and mammalian SCG neurons contain a heterogeneous population of nuclear-encoded mitochondrial mRNAs and a local cytosolic protein synthetic system. Nearly one quarter of the total protein synthesized in these structural/functional domains of the neuron is destined for mitochondria. Acute inhibition of axonal protein synthesis markedly reduces the functional activity of mitochondria. The blockade of axonal protein into mitochondria had similar effects on the organelle's functional activity. In addition to mitochondrial mRNAs, SCG axons contain approximately 200 different microRNAs (miRs), short, noncoding RNA molecules involved in the posttranscriptional regulation of gene expression. One of these miRs (miR-338) targets cytochrome c oxidase IV (COXIV) mRNA. This nuclear-encoded mRNA codes for a protein that plays a key role in the assembly of the mitochondrial enzyme complex IV and oxidative phosphorylation. Over-expression of miR-338 in the axon markedly decreases COXIV expression, mitochondrial functional activity, and the uptake of neurotransmitter into the axon. Conversely, the inhibition of endogeneous miR-338 levels in the axon significantly increased mitochondrial activity and norepinephrine uptake into the axon. The silencing of COXIV expression in the axon using short, inhibitory RNAs (siRNAs) yielded similar results, a finding that indicated that the effects of miR-338 on mitochondrial activity and axon function were mediated, at least in part, through local COXIV mRNA translation. Taken together, recent findings establish that proteins requisite for mitochondrial activity are synthesized locally in the axon and nerve terminal, and call attention to the intimacy of the relationship that has evolved between the distant cellular domains of the neuron and its energy generating systems.
