Translational Aspects of the Novel Object Recognition Task in Rats Abstinent Following Sub-Chronic Treatment with Phencyclidine (PCP): Effects of Modafinil and Relevance to Cognitive Deficits in Schizophrenia

Phencyclidine (PCP) induces a behavioral syndrome in rodents that bears remarkable similarities to some of the core symptoms observed in schizophrenic patients, among those cognitive deficits. The successful alleviation of cognitive impairments associated with schizophrenia (CIAS) has become a major focus of research efforts as they remain largely untreated. The aim of the present study was to investigate the effects of selected antipsychotic and cognition enhancing drugs, namely haloperidol, risperidone, donepezil, and modafinil in an animal model widely used in preclinical schizophrenia research. To this end, the novel object recognition (NOR) task was applied to rats abstinent following sub-chronic treatment with PCP. Rats were administered either PCP (5 mg/kg, i.p.) or vehicle twice a day for 7 days, followed by a 7-day washout period, before testing in NOR. Upon testing, vehicle-treated rats successfully discriminated between novel and familiar objects, an effect abolished in rats that had previously been exposed to PCP treatment. Acute treatment with modafinil (64 mg/kg, p.o.) ameliorated the PCP-induced deficit in novel object exploration, whereas haloperidol (0.1 mg/kg, s.c.), risperidone (0.2 mg/kg, i.p.), and donepezil (3 mg/kg, p.o.) were without significant effect. Given the negligible efficacy of haloperidol and risperidone, and the contradictory data with donepezil to treat CIAS in the clinic, together with the promising preliminary pro-cognitive effects of modafinil in certain subsets of schizophrenic patients, the sub-chronic PCP–NOR abstinence paradigm may represent an attractive option for the identification of potential novel treatments for CIAS

Comparison of quantitative trait loci methods:Total expression and allelic imbalance method in brain RNA-seq

BackgroundOf the 108 Schizophrenia (SZ) risk-loci discovered through genome-wide association studies (GWAS), 96 are not altering the sequence of any protein. Evidence linking non-coding risk-SNPs and genes may be established using expression quantitative trait loci (eQTL). However, other approaches such allelic expression quantitative trait loci (aeQTL) also may be of use.MethodsWe applied both the eQTL and aeQTL analysis to a biobank of deeply sequenced RNA from 680 dorso-lateral pre-frontal cortex (DLPFC) samples. For each of 340 genes proximal to the SZ risk-SNPs, we asked how much SNP-genotype affected total expression (eQTL), as well as how much the expression ratio between the two alleles differed from 1:1 as a consequence of the risk-SNP genotype (aeQTL).ResultsWe analyzed overlap with comparable eQTL-findings: 16 of the 30 risk-SNPs known to have gene-level eQTL also had gene-level aeQTL effects. 6 of 21 risk-SNPs with known splice-eQTL had exon-aeQTL effects. 12 novel potential risk genes were identified with the aeQTL approach, while 55 tested SNP-pairs were found as eQTL but not aeQTL. Of the tested 108 loci we could find at least one gene to be associated with 21 of the risk-SNPs using gene-level aeQTL, and with an additional 18 risk-SNPs using exon-level aeQTL.ConclusionOur results suggest that the aeQTL strategy complements the eQTL approach to susceptibility gene identification

Cell Lineage Specific Distribution of H3K27 Trimethylation Accumulation in an In Vitro Model for Human Implantation

Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ∼25% of the trophectoderm cells and in ∼7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion

CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice

Neuroinflammation and microglial activation are significant processes in Alzheimer’s disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer’s disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer’s disease and other tau-mediated neurodegenerative diseases

Inflammatory biomarkers in Alzheimer's disease plasma

Introduction:Plasma biomarkers for Alzheimer’s disease (AD) diagnosis/stratification are a“Holy Grail” of AD research and intensively sought; however, there are no well-established plasmamarkers.Methods:A hypothesis-led plasma biomarker search was conducted in the context of internationalmulticenter studies. The discovery phase measured 53 inflammatory proteins in elderly control (CTL;259), mild cognitive impairment (MCI; 199), and AD (262) subjects from AddNeuroMed.Results:Ten analytes showed significant intergroup differences. Logistic regression identified five(FB, FH, sCR1, MCP-1, eotaxin-1) that, age/APOε4 adjusted, optimally differentiated AD andCTL (AUC: 0.79), and three (sCR1, MCP-1, eotaxin-1) that optimally differentiated AD and MCI(AUC: 0.74). These models replicated in an independent cohort (EMIF; AUC 0.81 and 0.67). Twoanalytes (FB, FH) plus age predicted MCI progression to AD (AUC: 0.71).Discussion:Plasma markers of inflammation and complement dysregulation support diagnosis andoutcome prediction in AD and MCI. Further replication is needed before clinical translatio

Functional characterization of the immediate early gene Arg3.1

Titelblatt und Inhaltsverzeichnis 1 1\. Zusammenfassung 6 2\. Einleitung 10 3\. Ergebnisse 27 4\. Diskussion 76 5\. Material 104 6\. Methoden 113 7\. Literatur und Anhang 135Die Fähigkeit zur Ausbildung aktivitätsabhängiger Veränderungen in ihrer synaptischen Verschaltung ist eine der bemerkenswertesten Eigenschaften von Nervenzellen. So können kurze Episoden synaptischer Aktivität zu einer lang anhaltenden Potenzierung oder Schwächung der neuronalen Konnektivität führen. Diese synaptische Plastizität liegt physiologischen Prozessen zugrunde, wie dem Lernen und Speichern von Informationen, während Defizite in der Plastizität im Zusammenhang mit neurodegenerativen Erkrankungen diskutiert werden. Um Veränderungen in der synaptischen Stärke langfristig zu stabilisieren, wird in Nervenzellen die Transkription einer Reihe von Genen aktiviert. Die Translation der spezifischen mRNAs führt dann zu Veränderungen in der molekularen Zusammensetzung und der morphologische Struktur der stimulierten Neuronen. Inhibitoren der Transkription und Translation verhindern demzufolge die Ausbildung lang anhaltender synaptischer Plastizität sowie die Bildung eines Langzeitgedächtnisses. Eine der zentralen Fragestellungen innerhalb der Neurowissenschaften beschäftigt sich daher mit der Identifizierung aktivitätsregulierter Gene und der Charakterisierung ihrer Funktion bei den Prozessen der synaptischen Plastizität. Arg3.1 stellt ein aktivitätsreguliertes Gen dar, das eine besondere Stellung innerhalb dieser Gruppe von Genen einnimmt, da Arg3.1 mRNA und Protein dendritisch lokalisiert sind. Diese Eigenschaft ermöglicht die lokale Translation von Arg3.1 an der Synapse und deutet auf eine Beteiligung von Arg3.1 bei den synapsenspezifischen Veränderungen nach neuronaler Aktivität hin. In dieser Arbeit wurden biochemische, molekularbiologische, elektrophysiologische und verhaltensbiologische Analysen durchgeführt, um einen Einblick in die subzelluläre Lokalisierung von Arg3.1 und dessen Rolle bei den Prozessen der synaptischen Plastizität sowie der Gedächtnisbildung zu gewinnen. So konnte das Arg3.1 Protein selektiv im Bereich aktivierter Synapsen detektiert werden, wo es bis unmittelbar an die postsynaptische Membran transportiert wird. Dort liegt es als zentraler Bestandteil eines komplexen Proteinnetzwerkes, der postsynaptischen Dichte (PSD), vor. Biochemische Affinitätsstudien zeigten, dass Arg3.1 innerhalb der PSD über das Strukturmolekül PSD-95 mit dem NMDA- Rezeptor assoziiert ist, dessen Aktivierung für die Induktion und Aufrechterhaltung synaptischer Plastizität erforderlich ist. Aufgrund des Expressionsmusters von Arg3.1 findet diese Einbindung in die PSD mit einer geringen zeitlichen Verzögerung im Anschluss an eine plastizitätsinduzierende Stimulation statt. Diese Beobachtungen unterstützen die Annahme einer Beteiligung des Arg3.1 Genproduktes bei der Ausbildung lang anhaltender Veränderungen in der synaptischen Effizienz. Mit Hilfe einer Nullmutations- Mauslinie für Arg3.1 wurde diese Hypothese überprüft. Die Deletion von Arg3.1 führt zu einem Verlust der späten Phase der Langzeit-Potenzierung (LTP), einem zellulären Modell für bestimmte Formen des Lernens. Diesem Verlust entsprechen auf der Verhaltensebene Defizite der Arg3.1 knockout-Mäuse bei einer Reihe von kognitiven Aufgaben, die das langzeitige Abspeichern von Informationen verlangen. Dabei sind sowohl explizite Gedächtnisformen, wie das räumliche Lernen im Morris water maze oder die kontextabhängige Furchtkonditionierung, als auch implizite Gedächtnisformen, wie die tonabhängige Furchtkonditionierung und die conditioned taste aversion, beeinträchtigt. Neben der Lokalisierung im Proteinnetzwerk aktivierter Synapsen kann Arg3.1 auch im Zellkern detektiert werden. Dies deutet auf eine Funktion von Arg3.1 in diesem Zellkompartiment, möglicherweise bei der Transkriptionskontrolle, hin. Eine auf der Affymetrix-Technologie basierende Genexpressionsanalyse zeigte, dass die Deletion von Arg3.1 tatsächlich Auswirkungen auf die Transkription von Genen hat, die im Gehirn von Wildtyp- aber nicht von Arg3.1 knockout-Tieren als Folge neuronaler Aktivität angeschaltet werden. So wurden mit SOCS3 und c-fos zwei Gene identifiziert, die an aktivitätsregulierten Signaltransduktionskaskaden beteiligt sind und in stimulierten Neuronen Arg3.1 defizienter Tiere differentiell reguliert werden. Demnach könnte Arg3.1 in unterschiedlichen zellulären Kompartimenten eine Funktion als Vermittler plastizitätsinduzierender Stimulation ausüben und Veränderungen initiieren, die zur Konsolidierung der synaptischen Verstärkung beitragen. Die in dieser Arbeit durchgeführten Experimente zeigen erstmals, dass Arg3.1 für die Ausbildung lang anhaltender Formen synaptischer Plastizität sowie für die Bildung eines Langzeitgedächtnisses unbedingt erforderlich ist.One of the most remarkable features of neurons is their capacity to undergo activity-dependent changes in order to adjust their synaptic strength. Brief episodes of synaptic activity can lead to either long lasting potentiation or depression of neuronal connectivity. This synaptic plasticity contributes to a variety of physiological processes, including learning and memory, whereas impairment of synaptic plasticity might contribute to the course of several neurodegenerative diseases. To stabilize changes in synaptic strength, the transcription of a variety of genes is activated in neurons. Subsequent translation of the specific mRNAs leads to modifications of the molecular composition and morphological structure of stimulated neurons. Consequently, inhibitors of transcription and translation lead to the impairment of robust synaptic plasticity and prevent the formation of long term memory. Much attention has therefore been focused on the identification and characterization of activity-regulated genes and their role in synaptic plasticity. Arg3.1 is an activity-regulated gene that is unique among these in that its mRNA and protein are dendritically located. These properties allow the local translation of Arg3.1 at the stimulated synapse and open the possibility of synapse-specific changes that neurons undergo after neuronal activity. Biochemical, molecular biological, electrophysiological and behavioural analyses were conducted in order to gain insight into the subcellular localization of Arg3.1 at the synapse and its role in the processes of synaptic plasticity and memory formation. Following neuronal stimulation Arg3.1 was detected selectively at activated synapses where it translocates to the postsynaptic membrane. Here it constitutes a central part of a highly organized protein network, the postsynaptic density (PSD). Affinity chromatography studies reveal an association of Arg3.1 and the NMDA- receptor within the PSD, in which the NMDA-receptor plays a pivotal role in both the induction and maintenance of synaptic plasticity. Due to the expression pattern of Arg3.1 the association with the NMDA-receptor, which is mediated by the scaffolding protein PSD-95, occurs with a slight delay following plasticity-inducing stimulation. These observations support the assumption that Arg3.1 is involved in the formation of long lasting changes in synaptic efficacy. To test this hypothesis a mouse line carrying a null mutation for Arg3.1 was analysed. Arg3.1 deficient animals show a complete loss of the late phase of long-term potentiation (LTP), a cellular model for certain forms of learning. This deficiency corresponds to deficits in several cognitive tasks which require the consolidation of newly formed memories. The deficits affect both explicit memory, as shown by spatial learning in the Morris water maze and context dependent fear conditioning, and implicit memory, such as cue dependent fear conditioning and conditioned taste aversion. Besides its localization in protein networks of recently activated synapses, Arg3.1 protein can also be detected in the nucleus. This suggests a role for Arg3.1 in this compartment, possibly in the regulation of transcription. A gene chip analysis based on Affymetrix technology revealed alterations in the transcription of genes that are activated following neuronal activity in brains of wildtype but not Arg3.1 knockout animals. Two genes, SOCS3 and c-fos, which are involved in activity-regulated signalling cascades, were identified to be differentially regulated in Arg3.1 knockout animals. Thus, Arg3.1 might function as a mediator of plasticity-induced stimulation in different cellular compartments and initiate changes that result in the consolidation of increased synaptic strength. The experiments performed in this work are the first to show that Arg3.1 is required for the formation of lasting synaptic plasticity and long term memory