Interferon-γ acutely augments inhibition of neocortical layer 5 pyramidal neurons

BACKGROUND: Interferon-γ (IFN-γ, a type II IFN) is present in the central nervous system (CNS) under various conditions. Evidence is emerging that, in addition to its immunological role, IFN-γ modulates neuronal morphology, function, and development in several brain regions. Previously, we have shown that raising levels of IFN-β (a type I IFN) lead to increased neuronal excitability of neocortical layer 5 pyramidal neurons. Because of shared non-canonical signaling pathways of both cytokines, we hypothesized a similar neocortical role of acutely applied IFN-γ. METHODS: We used semi-quantitative RT-PCR, immunoblotting, and immunohistochemistry to analyze neuronal expression of IFN-γ receptors and performed whole-cell patch-clamp recordings in layer 5 pyramidal neurons to investigate sub- and suprathreshold excitability, properties of hyperpolarization-activated cyclic nucleotide-gated current (Ih), and inhibitory neurotransmission under the influence of acutely applied IFN-γ. RESULTS: We show that IFN-γ receptors are present in the membrane of rat's neocortical layer 5 pyramidal neurons. As expected from this and the putative overlap in IFN type I and II alternative signaling pathways, IFN-γ diminished Ih, mirroring the effect of type I IFNs, suggesting a likewise activation of protein kinase C (PKC). In contrast, IFN-γ did neither alter subthreshold nor suprathreshold neuronal excitability, pointing to augmented inhibitory transmission by IFN-γ. Indeed, IFN-γ increased electrically evoked inhibitory postsynaptic currents (IPSCs) on neocortical layer 5 pyramidal neurons. Furthermore, amplitudes of spontaneous IPSCs and miniature IPSCs were elevated by IFN-γ, whereas their frequency remained unchanged. CONCLUSIONS: The expression of IFN-γ receptors on layer 5 neocortical pyramidal neurons together with the acute augmentation of inhibition in the neocortex by direct application of IFN-γ highlights an additional interaction between the CNS and immune system. Our results strengthen our understanding of the role of IFN-γ in neocortical neurotransmission and emphasize its impact beyond its immunological properties, particularly in the pathogenesis of neuropsychiatric disorders

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

about the protein kinase C mediated influence on suprathreshold excitability and the hyperpolarization-activated nonselective cation current (Ih)

Das Immun- und Nervensystem stehen in gegenseitiger Kommunikation. So wurde schon früh ein Zusammenhang zwischen Stress und einem geschwächten Immunsystem gefunden. Dass dieser Weg auch in entgegen gesetzter Richtung möglich ist, also die Beeinflussung des Nervensystems durch das Immunsystem, wurde bereits für Interleukine, die zur Gruppe der Cytokine gehören, gezeigt. Das, in meinem Fokus stehende, Typ I Interferon (IFN) gehört zur Gruppe der Cytokine und besitzt immunmodulatorische und antivirale Eigenschaften. Typ I IFN aktiviert über seinen Rezeptor verschiedene Signalwege, die in den klassischen Signalweg und in alternative Signalwege unterteilt werden. Da Typ I IFN auch direkt von Neuronen gebildet wird, lag die Vermutung nah, dass es neuromodulatorische Eigenschaften besitzt. Die Hinweise auf eine Typ I IFN induzierte Beeinflussung des hyperpolarisationsaktivierten unselektiven Kationenstroms (Ih) und dessen Einfluss auf die neuronalen Erregbarkeit, stellten die Basis meiner Arbeit dar. Der Ih ist beim Ruhemembranpotential (RMP) von Neuronen partiell aktiviert und spielt eine entscheidende Rolle bei der Kontrolle neuronaler Informationsverarbeitung. Aufgrund seiner Eigenschaften gilt er als einer der bestimmenden Faktoren der neuronalen Erregbarkeit. Ih wird über hyperpolarisationsaktivierte, von cyclischen Nukleotiden modulierte (HCN)-Kanäle geleitet. Diese werden von vier Untereinheiten (HCN1-4) gebildet und liegen als Homo- oder Heterotetramere im Gehirn vor. In den verschiedenen Arealen des Gehirns ist ihre Expression unterschiedlich stark ausgeprägt. In den von uns untersuchten neocortikalen Pyramidenzellen der Schicht 5 wird der Ih durch die Untereinheiten HCN1 und HCN2 vermittelt. HCN Kanäle werden durch verschiedene Modulatoren beeinflusst. Im Fokus meiner Arbeit steht seine Modulation durch Protein Kinase C (PKC), die auch bei alternativen Typ I IFN- Rezeptorsignalwegen eine Rolle spielt. Die Arbeit klärt die Modulation der unter- und überschwelligen neuronale Erregbarkeit durch das Typ I IFN IFN-β und PKC. PKC dient hypothetisch als Bindeglied zwischen IFN-Signalweg und multiplen neuronalen Ionenkanälen. Mein Fokus liegt auf den im Neocortex Ih mediierenden HCN Kanälen. Unsere Befunde basieren auf elektrophysiologischen ex vivo Messungen an neocortikalen Pyramidenzellen der Schicht 5 in Hirnschnitten von Maus- bzw. Ratten im whole cell Modus oder in vitro im Kulturmodell an Säugerzellen im whole cell und cell attached Modus mittels pharmakologischer Aktivierung oder Inhibition der neuronalen Typ I IFN Effekte. Zusätzlich nahmen wir in vivo Virusinfektionen vor oder analysierten das Maus- bzw. Rattengehirn elektroenzophalografische (EEG) nach direkter IFN-β Applikation. Alle Messungen erfolgten bei Typ I IFN Konzentrationen mit pathophysiologischer Relevanz. Die Wirkung verschiedener einzelner und kombinierter Ionenkanalmodulationen überprüften wir anhand eines in silico Computermodells. Des Weiteren untersuchten wir mittels Western blot und Immunohistochemie das Vorhandensein des Typ I IFN Rezeptors und die Aktivierung einiger seiner Signalwegsbestandteile. So konnten wir zeigen, dass Typ I IFN sowohl über eine virale Infektion als auch über die direkte Applikation den Ih reduziert und dass der Typ I IFN Effekt auf den Ih HCN1 spezifisch ist. Voraussetzung dafür ist ein intakter Typ I IFN- Rezeptorsignalweg. Für alternative Typ I IFN-Rezeptorsignalwege spielt PKC eine wichtige Rolle. Für PKC konnte von uns gezeigt werden, dass sie den HCN1 Kanal moduliert. Im Kulturmodell ist für den PKC mediierten Effekt auf den HCN1 Kanal eine intakte intrazelluläre Umgebung notwendig. Dabei wird der Ih durch die Aktivierung von PKC auf 60-70 % reduziert, wohingegen die vorherige Inhibition von PKC eine solche Reduktion verhindert. Nahezu gleiche Effektgrößen bei unterschiedlichen Säugerzellen-Kulturmodellen machen eine Speziesspezifität unwahrscheinlich. Eine Beeinflussbarkeit durch PKC ist den im in silico Modell modulierten Strömen gemein, die für die Nachahmung des ex vivo IFN Effekts auf die neuronale Erregbarkeit notwendig waren. Wodurch wir PKC als Verbindung zwischen Typ I IFN Signalkaskade und Ionenkanalmodulation schlussfolgern. In gleicher Weise verursachte eine PKC Aktivierung oder eine IFN-β Applikation in neocortikalen Pyramidenzellen der Schicht 5 eine Erhöhung der neuronalen Erregbarkeit. Dabei blieb jeweils die Aktionspotentialschwelle unverändert. Zusätzlich beobachteten wir eine reduzierte Nachhyperpolarisation (AHP) nach PKC Aktivierung wie auch nach Applikation von IFN-β. Übereinstimmend mit unseren Erkenntnissen reduziert eine IFN-β Applikation den M-Typ Kalium Strom (IM), der an der AHP beteiligt ist. Eine vorherige Inhibierung der PKC verhinderte die Wirkung von IFN-β auf die Erregbarkeit. Zusammenfassend bestätigen die Ergebnisse, dass IFN-β unter pathophysiologischen Bedingungen als Neuromodulator wirkt und zeigen erstmals dass diese Funktion durch PKC vermittelt wird. Dabei haben wir gezeigt, dass multiple Ionenkanäle, wie der HCN1 oder M-Typ Kalium Kanal, ihrer Wirkung unterliegen. Für die Vermittlung des Typ I IFN Effekts ist die PKC Aktivierung hinreichend und notwendig. Dadurch werden die Typ I IFN Signalkaskade und die Ionenkanäle miteinander verbunden. Daher sehen wir die Aktivierung von PKC als einheitlichen Mechanismus für das neuromodulatorische Potential von Typ I IFN in neocortikalen Neuronen an. Dadurch ist der Grundstein für eine Regulation neuronaler Antworten während einer Entzündung des zentralen Nervensystems gelegt. IFN-β konnte ebenfalls im gesunden ZNS nachgewiesen werden, was für IFN-β auch eine Rolle in der physiologischen Neuromodulation impliziert. Somit könnte die Neuromodulation durch IFN-β neben ihrer pathophysiologischen Relevanz auch unter physiologischen Bedingungen von großer Wichtigkeit sein.The immune and nervous system appear to communicate with each other. Early research uncovered that nervous structures influence the immune system, i. e. stress weakens the immune system. Further the immune system affects the nervous system for instance via interleukins, a class of cytokines. Here I focused on the cytokine type I Interferon (IFN) that has immunomodulatory and antiviral properties. It activates via its receptor different signal pathways, which were separated in the classical pathway and several alternative pathways. Because type I IFN is also directly produced by neurons, we assume additional neuromodulatory properties. Implications of a type I IFN induced influence on hyperpolarization-activated nonselective cation current (Ih) and its influence on neuronal excitability were the basis of my work. Ih is partially activated at resting membrane potential (RMP) of neurons and has an important role in the control of neuronal information processing. Because of its properties it is considered as one of the determinants of neuronal excitability. Ih is mediated by hyperpolarization-activated cyclic nucleotide- gated (HCN) channels, which are formed by 4 subunits (HCN1-4) and assembled as homo- or heterotetrameric complexes. Their expression pattern differs depending on the brain area. In neocortical layer 5 pyramidal neurons, which we used for our studies, Ih is mediated by HCN1 and HCN2 subunits. Different modulators influence the properties of HCN channels. I focused on its modulation via protein kinase C (PKC) that plays an important role in alternative IFN signaling pathways. This work clarifies the type I IFN, IFN-β and PKC mediated modulations of the sub- and suprathreshold neuronal excitability. Hypothetically, PKC links IFN signaling pathway and multiple neuronal ion channels. I focused on the Ih mediating HCN channels in the neocortex. Our findings are based on electrophysiological ex vivo whole cell recordings in neocortical layer 5 pyramidal neurons of mouse and rat brain as well as in vitro whole cell and cell attached recordings in mammalian cell culture systems with pharmacological activation or inhibition of the neuronal IFN effects. Additionally we elevated intracerebral type I IFNs by in vivo virus infection or analyzed mouse and rat brains activity via electroencephalography (EEG) after direct IFN application. We performed all measurements with pathophysiological relevant IFN concentrations. We tested the effect of different single and combined ion channel modulations in an in silico computer model. Furthermore we analyzed the presence of type I IFN receptors in neocortical layer 5 pyramidal neurons via immunohistochemistry and the activation of some components of its pathway via Western blotting. Therewith we could show that type I IFN reduced the Ih after virus infection and after a direct type I IFN application and that this type I IFN effect is HCN1 specific. Type I IFN effects depend on an intact type I IFN receptor signaling pathway. For alternative type I IFN receptor signaling pathways PKC plays an important role. Our findings demonstrate that PKC modulates HCN1 channels. For PKC mediated effects on HCN1 channels an intact intracellular environment of the cultured cells is necessary. PKC activation reduced Ih to 60-70 %, whereas a prior inhibition of the PKC prevents the PKC dependent Ih reduction. Because of similar magnitude of effects in different mammalian cell systems species specificity is unlikely. All currents necessary for mimicking the ex vivo IFN effects on neuronal excitability in silico are modulated by PKC. Therefore we reasoned that PKC links the type I IFN signaling cascade and ion channel modulations. An activation of PKC or an IFN-β application in neocortical layer 5 pyramidal neurons increased the neuronal excitability in the same manner. However, the threshold of the action potential remained constant. Additionally, after PKC activation or IFN-β application we observed a reduced after-hyperpolarisation (AHP). Consistent with our findings, we also observed a reduced M-type potassium current (IM) after IFN-β application. Prior inhibition of PKC prevents the type I IFN effects on neuronal excitability. In summary, these findings confirm the neuromodulatory effect of IFN-β under pathophysiological conditions and show for the first time that this effect is PKC mediated. Accordingly we could show that multiple ion channels, such as HCN1 or M-type potassium channel underlie this effect. For mediating the type I IFN effect, PKC activation is both sufficient and necessary and links IFN signaling cascade and ion channels. Therefore we propose PKC activation as unitary mechanism for the neuromodulatory potential of type I IFN in neocortical neurons. This represents the basis for regulation of neuronal response during an inflammation of the central nervous system (CNS). IFN-β was also detected in a healthy CNS implying a role in the physiological neuromodulation. Because of that neuromodulation via IFN-β may not only be pathophysiologically relevant, but also important under physiological conditions

Protein Kinase C Activation Inhibits Rat and Human Hyperpolarization Activated Cyclic Nucleotide Gated Channel (HCN)1 - Mediated Current in Mammalian Cells

Background/Aims: Hyperpolarization activated cyclic nucleotide gated 1 (HCN1) channels determine neuronal excitability in several brain regions. In contrast to HCN2 and HCN4, HCN1 is less sensitive to cAMP and the number of other known modulators is limited. One of those, the protein kinase C (PKC), showed opposing effects on mouse HCN1 channels expressed in Xenopus oocytes. Methods: In order to study PKC effects on HCN1 mediated currents in a mammalian environment we expressed rat HCN1 or human HCN1 in human embryonic kidney (HEK293) cells and rat HCN1 in murine neuroblastoma (N1E-115) cells. We recorded the resulting Ih before and during the application of the membrane permeable non-metabolizable PKC-activator 4βPMA in cell-attached mode of the patch-clamp technique, leaving the intracellular environment intact. Results: 4βPMA reduced maximal HCN1 mediated currents to about 60-70 % and slowed its activation, but left its voltage sensitivity unchanged. The effect was neither due to species-related differences nor restricted to HEK293 cells, because it was comparable for human and rat HCN1 in HEK293 and for rat HCN1 in N1E-115 cells. However, pre-treatment with the PKC blocker GF109203X abolished 4βPMA induced Ih changes. Disrupting the intracellular environment by recording in whole-cell mode drastically reduced the 4βPMA effect. Conclusion: PKC activation reduces and slows Ih in non-neuronal and neuronal mammalian cells transfected with rat or human HCN1 if the intracellular content remains intact

Protein kinase C activation mediates interferon-β-induced neuronal excitability changes in neocortical pyramidal neurons

Background Cytokines are key players in the interactions of the immune and nervous systems. Recently, we showed that such interplay is mediated by type I interferons (IFNs), which elevate the excitability of neocortical pyramidal neurons. A line of indirect evidence suggested that modulation of multiple ion channels underlies the effect. However, which currents are principally involved and how the IFN signaling cascade is linked to the respective ion channels remains elusive. Methods We tested several single and combined ionic current modulations using an in silico model of a neocortical layer 5 neuron. Subsequently we investigated resulting predictions by whole-cell patch-clamp recordings in layer 5 neurons of ex vivo neocortical rat brain slices pharmacologically reproducing or prohibiting neuronal IFN effects. Results The amount and type of modulation necessary to replicate IFN effects in silico suggested protein kinase C (PKC) activation as link between the type I IFN signaling and ion channel modulations. In line with this, PKC activation with 4β-phorbol 12-myristate 13-acetate (4β-PMA) or Bryostatin1 augmented the excitability of neocortical layer 5 neurons comparable to IFN-β in our ex vivo recordings. In detail, both PKC activators attenuated the rheobase and increased the input-output gain as well as the input resistance, thereby augmenting the neuronal excitability. Similar to IFN-β they also left the threshold of action potential generation unaffected. In further support of PKC mediating type I IFN effects, IFN-β, 4β-PMA and Bryostatin1 reduced the amplitude of post-train after-hyperpolarizations in a similar manner. In conjunction with this finding, IFN-β reduced M-currents, which contribute to after-hyperpolarizations and are modulated by PKC. Finally, blocking PKC activation with GF109203X at the catalytic site or calphostin C at the regulatory site prevented the main excitatory effects of IFN-β. Conclusion Multiple ion channel modulations underlie the neuromodulatory effect of type I IFNs. PKC activation is both sufficient and necessary for mediating the effect, and links the IFN signaling cascade to the intrinsic ion channels. Therefore, we regard PKC activation as unitary mechanism for the neuromodulatory potential of type I IFNs in neocortical neurons

Use Dependent Attenuation of Rat HCN1-Mediated Ih in Intact HEK293 Cells

Background/Aims: Cationic currents (Ih) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. Methods: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated Ih in cell-attached or whole-cell mode of the patch-clamp technique. Results: Recurring activation of rHCN1 reduced and slowed Ih in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced Ih attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in Ih. Conclusion: Reduction of rHCN1 mediated Ih is use dependent and may involve the coupling of voltage sensor and pore

Regularity of a crisis: media framing of the 2015 transboundary haze issue in Indonesia, Singapore, and Malaysia

The problem of the haze caused by huge forest fires persists as an annual transboundary problem for Indonesia and the rest of Southeast Asia. In 2015, the problem was worse than ever before, affecting many countries in the Association of Southeast Asian Nations (ASEAN) and causing respiratory ailments for more than half a million Indonesians. This study explores the media framing of the haze problem in Indonesia from June to December 2015. Using Entman’s framing approach, it investigates how media outlets from Indonesia, Malaysia, and Singapore covered the crisis. Articles from six online media outlets published in these three countries were analysed. Using cluster analysis, this research identified three frames as follows: (1) crisis frame, (2) immediate action frame, and (3) regular problem frame. The first cluster/frame consists of articles giving high salience to all problems and causes of the forest fires provoking the haze. In contrast, the second frame mostly ignores causes and problems and focuses almost exclusively on the need for immediate action. The third frame, which represents more than 60% of the articles, covers the haze problem as a regular issue without emphasizing prominently either the different aspects of the problem itself or its causes and solutions. Further results show that the media in Singapore and Malaysia used the crisis frame more often than the media in Indonesia

Interferon-γacutely augments inhibition of neocortical layer 5 pyramidal neurons

Background Interferon-γ (IFN-γ, a type II IFN) is present in the central nervous system (CNS) under various conditions. Evidence is emerging that, in addition to its immunological role, IFN-γ modulates neuronal morphology, function, and development in several brain regions. Previously, we have shown that raising levels of IFN-β (a type I IFN) lead to increased neuronal excitability of neocortical layer 5 pyramidal neurons. Because of shared non-canonical signaling pathways of both cytokines, we hypothesized a similar neocortical role of acutely applied IFN-γ. Methods We used semi-quantitative RT-PCR, immunoblotting, and immunohistochemistry to analyze neuronal expression of IFN-γ receptors and performed whole-cell patch-clamp recordings in layer 5 pyramidal neurons to investigate sub- and suprathreshold excitability, properties of hyperpolarization-activated cyclic nucleotide-gated current (Ih), and inhibitory neurotransmission under the influence of acutely applied IFN-γ. Results We show that IFN-γ receptors are present in the membrane of rat’s neocortical layer 5 pyramidal neurons. As expected from this and the putative overlap in IFN type I and II alternative signaling pathways, IFN-γ diminished Ih, mirroring the effect of type I IFNs, suggesting a likewise activation of protein kinase C (PKC). In contrast, IFN-γ did neither alter subthreshold nor suprathreshold neuronal excitability, pointing to augmented inhibitory transmission by IFN-γ. Indeed, IFN-γ increased electrically evoked inhibitory postsynaptic currents (IPSCs) on neocortical layer 5 pyramidal neurons. Furthermore, amplitudes of spontaneous IPSCs and miniature IPSCs were elevated by IFN-γ, whereas their frequency remained unchanged. Conclusions The expression of IFN-γ receptors on layer 5 neocortical pyramidal neurons together with the acute augmentation of inhibition in the neocortex by direct application of IFN-γ highlights an additional interaction between the CNS and immune system. Our results strengthen our understanding of the role of IFN-γ in neocortical neurotransmission and emphasize its impact beyond its immunological properties, particularly in the pathogenesis of neuropsychiatric disorders