10 research outputs found
Signaltransduktion des Neurotrophinrezeptors p75 durch die Zinkfingerproteine NRIF1 und NRIF2
Der Neurotrophinrezeptor p75 wurde als erstes Mitglied der Tumornekrosefaktor-Rezeptor-Superfamilie
kloniert. Da die trophischen Eigenschaften der Neurotrophine durch eine zweite
Klasse von Rezeptoren, die Trk-Rezeptor-Tyrosinkinasen, vermittelt werden, wurde p75
lange als deren âCorezeptorâ angesehen. Inzwischen gibt es viele Beweise fĂŒr eine eigen-stĂ€ndige
Signaltransduktion durch p75, die beispielsweise zur Aktivierung von NF-Îș B oder zu
programmiertem Zelltod fĂŒhren kann.
Zu Beginn dieser Arbeit war weitgehend unbekannt, wie der Neurotrophinrezeptor p75
apoptotische Signale im Inneren der Zelle weiterleitet. Unter Verwendung des Hefe-âTwo-Hybridâ-
Systems war im Vorfeld das Zinkfingerprotein NRIF1 (âNeurotrophin Receptor
Interacting Factorâ) als potentieller p75-Interaktionspartner identifiziert worden. Die wichtige
Rolle dieses Proteins als Vermittler von apoptotischen Signalen konnte spÀter durch gezielte
Deletion des nrif1-Gens in der Maus bestÀtigt werden: In nrif1-Nullmutanten wurde eine
Reduktion des embryonalen Zelltodes von Nervenzellen der Netzhaut nachgewiesen, deren
AusmaĂ dem einer p75- oder ngf (âNerve Growth Factorâ)-Deletion entsprach (Casademunt
et al., 1999).
In der vorliegenden Arbeit wurde ein zweites nrif-Gen (nrif2) kloniert, das zu ĂŒber 90% mit
nrif1 identisch ist. Beide Proteine besitzen typische Strukturmerkmale negativ regulatorisch
wirkender Transkriptionsfaktoren. Der carboxyterminale Abschnitt enthĂ€lt fĂŒnf Zinkfinger des
Cys2-His2-Typs, die eine Bindung an DNA vermitteln könnten, wÀhrend der aminoterminale
Bereich zwei KRAB-DomÀnen enthÀlt, die Transkriptionsrepressor-Module darstellen. Das
nrif2-Gen wird im 5â-untranslatierten Bereich differentiell gespleiĂt.
Durch Experimente mit rekombinanten Proteinen aus E. coli und Fibroblasten-Zellinien
wurde die vermutete Interaktion von NRIF und p75 biochemisch nachgewiesen. Die Ver-wendung
unterschiedlicher Deletionskonstrukte zeigte, daĂ fĂŒr die Wechselwirkung nur die
Juxtamembran-DomÀne des Rezeptors und ein carboxyterminaler Abschnitt von NRIF
(stromaufwÀrts der Zinkfinger) nötig sind. NRIF-Proteine können unter Bildung von Homo-
und Heterodimeren mit sich selbst interagieren.
Die Colokalisierung von NRIF1 und NRIF2 bzw. NRIF und p75 nach transienter Transfektion
von Fibroblasten-Zellinien bestÀtigte die biochemisch nachgewiesenen Interaktionen auch in
vivo.
Die Expression von NRIF in Zellinien neuronalen Ursprungs offenbarte eine mögliche
Funktion als Auslöser von Apoptose, welche unabhĂ€ngig von p75 allein durch Ăber-expression
dieser Zinkfingerproteine verursacht wurde. AuĂerdem war in NRIF-ĂŒberexprimierenden
Zellen der Einbau von BrdU, einem Thymidin-Analogon, das Zellen in
der S-Phase des Zellzyklus markiert, stark eingeschrĂ€nkt. Diese Funktionen von NRIF sindbesonders interessant, da in den letzten zwei Jahren weitere AdaptermolekĂŒle des Neuro-trophinrezeptors
p75 identifiziert wurden, die einen EinfluĂ auf Apoptose und/oder den
Ablauf des Zellzyklus besitzen.
p75 spielt insbesondere wÀhrend des programmierten Zelltodes in der Entwicklung des
Nervensystems eine wichtige Rolle und wird im Embryonalstadium am stÀrksten exprimiert.
Die Analyse der mRNA-Expression von nrif bestÀtigte, daà auch nrif1 und nrif2 wÀhrend der
Embryonalentwicklung deutlich höher exprimiert sind als in adulten MÀusen. Nrif-mRNA
wurde jedoch im Gegensatz zu p75-mRNA ubiquitÀr in allen untersuchten embryonalen
Geweben nachgewiesen.
In weiterfĂŒhrenden Experimenten wurden transgene MĂ€usen untersucht, in denen das nrif1-Gen
durch homologe Rekombination entfernt worden war. Diese MĂ€use sind in einem
kongenen Sv129- oder einem gemischten Sv129/BL6-Hintergrund lebensfÀhig und fertil,
sterben jedoch im BL6-Hintergrund ungefĂ€hr am zwölften Embryonaltag. Die Hypothese, daĂ
die nrif2-mRNA-Menge in ĂŒberlebenden Tieren erhöht sein könnte, wurde zuerst in neugebo-renen
Sv129-MÀusen durch semiquantitative RT-PCR-Analysen bestÀtigt. Eine vergleichen-de
Untersuchung 10,5 Tage alter Embryonen aus verschiedenen StÀmmen deutete auf die
Möglichkeit einer funktionellen Kompensation hin: WÀhrend in Nullmutanten des Sv129-
Stammes die nrif2-mRNA-Konzentration ungefÀhr doppelt so hoch war wie in Wildtyp-Tieren,
konnten BL6-Nullmutanten die nrif2-Menge nicht differentiell regulieren. Das Fehlen von
NRIF1 und die gleichzeitige UnfÀhigkeit einer ausgleichenden Hochregulation von NRIF2
könnten ein wichtiger Grund fĂŒr die embryonale LetalitĂ€t der nrif1 (-/-)-Embryonen im BL6-
Stamm sein. Eine genau ausgewogene Menge der Zinkfingerproteine NRIF1 und NRIF2
scheint demnach essentiell zu sein, damit wichtige Prozesse wie Zellzyklus und Apoptose
ungestört ablaufen können
d-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia
The N-methyl-d-aspartate receptor co-agonist d-serine is synthesized by serine racemase and degraded by d-amino acid oxidase. Both d-serine and its metabolizing enzymes are implicated in N-methyl-d-aspartate receptor hypofunction thought to occur in schizophrenia. We studied d-amino acid oxidase and serine racemase immunohistochemically in several brain regions and compared their immunoreactivity and their mRNA levels in the cerebellum and dorsolateral prefrontal cortex in schizophrenia. d-Amino acid oxidase immunoreactivity was abundant in glia, especially Bergmann glia, of the cerebellum, whereas in prefrontal cortex, hippocampus and substantia nigra, it was predominantly neuronal. Serine racemase was principally glial in all regions examined and demonstrated prominent white matter staining. In schizophrenia, d-amino acid oxidase mRNA was increased in the cerebellum, and as a trend for protein. Serine racemase was increased in schizophrenia in the dorsolateral prefrontal cortex but not in cerebellum, while serine racemase mRNA was unchanged in both regions. Administration of haloperidol to rats did not significantly affect serine racemase or d-amino acid oxidase levels. These findings establish the major cell types wherein serine racemase and d-amino acid oxidase are expressed in human brain and provide some support for aberrant d-serine metabolism in schizophrenia. However, they raise further questions as to the roles of d-amino acid oxidase and serine racemase in both physiological and pathophysiological processes in the brain
Interactions among genes in the ErbB-Neuregulin signalling network are associated with increased susceptibility to schizophrenia
<p>Abstract</p> <p>Background</p> <p>Evidence of genetic association between the NRG1 (Neuregulin-1) gene and schizophrenia is now well-documented. Furthermore, several recent reports suggest association between schizophrenia and single-nucleotide polymorphisms (SNPs) in ERBB4, one of the receptors for Neuregulin-1. In this study, we have extended the previously published associations by investigating the involvement of all eight genes from the ERBB and NRG families for association with schizophrenia.</p> <p>Methods</p> <p>Eight genes from the ERBB and NRG families were tested for association to schizophrenia using a collection of 396 cases and 1,342 blood bank controls ascertained from Aberdeen, UK. A total of 365 SNPs were tested. Association testing of both alleles and genotypes was carried out using the fast Fisher's Exact Test (FET). To understand better the nature of the associations, all pairs of SNPs separated by â„ 0.5 cM with at least nominal evidence of association (<it>P </it>< 0.10) were tested for evidence of pairwise interaction by logistic regression analysis.</p> <p>Results</p> <p>42 out of 365 tested SNPs in the eight genes from the ERBB and NRG gene families were significantly associated with schizophrenia (<it>P </it>< 0.05). Associated SNPs were located in ERBB4 and NRG1, confirming earlier reports. However, novel associations were also seen in NRG2, NRG3 and EGFR. In pairwise interaction tests, clear evidence of gene-gene interaction was detected for NRG1-NRG2, NRG1-NRG3 and EGFR-NRG2, and suggestive evidence was also seen for ERBB4-NRG1, ERBB4-NRG2, ERBB4-NRG3 and ERBB4-ERBB2. Evidence of intragenic interaction was seen for SNPs in ERBB4.</p> <p>Conclusion</p> <p>These new findings suggest that observed associations between NRG1 and schizophrenia may be mediated through functional interaction not just with ERBB4, but with other members of the NRG and ERBB families. There is evidence that genetic interaction among these loci may increase susceptibility to schizophrenia.</p
Investigation of G72 (DAOA) expression in the human brain
<p>Abstract</p> <p>Background</p> <p>Polymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions.</p> <p>Methods</p> <p>The expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth <it>in silico </it>analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability.</p> <p>Results</p> <p>Despite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed <it>in silico </it>analysis provides several lines of evidence that support the apparent low or absent expression of G72.</p> <p>Conclusion</p> <p>Our results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. <it>In silico </it>analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.</p
Binding affinity and capacity of putative adaptor-mediated sorting of a Type I membrane protein in Leishmania mexicana
The membrane-bound acid phosphatase (MBAP), a Type I membrane protein predominantly associated with endosomal/lysosomal structures of Leishmania mexicana promastigotes, contains motifs in its cytosolic COOH-terminal tail (-MEVWRRYMKFKNKQSEAIIV-COOH) akin to tyrosine- and di-leucine-based sorting signals in multicellular organisms. Here, we first show that the COOH-terminal residues IIV of MBAP, but not the Y-residue, are required for endosomal targeting, suggesting specific binding to an adaptor complex at the cell surface. We then determine whether specific binding can be saturated by analysing the efficiency of endosomal targeting for increasing numbers of MBAP molecules per cell. The ratio of the steady-state abundance of wild-type MBAP on the cell surface to MBAP on endosomes increases until the distribution is no longer different from that observed for a mutant MBAP which lacks the IIV-motif or for a glycosylphosphatidylinositol-anchored form, both of which are distributed according to bulk membrane flow. A quantitative analysis of these in vivo results indicates specific binding to a putative adaptor complex with an affinity of about 10-4M to 50,000 sorting sites on the cell surface
Interactions among genes in the ErbB-Neuregulin signalling network are associated with increased susceptibility to schizophrenia-0
<p><b>Copyright information:</b></p><p>Taken from "Interactions among genes in the ErbB-Neuregulin signalling network are associated with increased susceptibility to schizophrenia"</p><p>http://www.behavioralandbrainfunctions.com/content/3/1/31</p><p>Behavioral and Brain Functions 2007;3():31-31.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934910.</p><p></p>intervals. The region covered by the gene is indicated in the centre of the figure by a horizontal line, with an arrow head indicating the direction of transcription. A crossing vertical line indicates the position of each exon. At the base of the figure, LD between SNP loci is indicated by shading. White: = 0; black: = 1
Interactions among genes in the ErbB-Neuregulin signalling network are associated with increased susceptibility to schizophrenia-3
<p><b>Copyright information:</b></p><p>Taken from "Interactions among genes in the ErbB-Neuregulin signalling network are associated with increased susceptibility to schizophrenia"</p><p>http://www.behavioralandbrainfunctions.com/content/3/1/31</p><p>Behavioral and Brain Functions 2007;3():31-31.</p><p>Published online 28 Jun 2007</p><p>PMCID:PMC1934910.</p><p></p>action presented in that table for the gene combination in question