Drosophila as a Model for MECP2 Gain of Function in Neurons

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model

Die Rolle von Ionen-Strömen für intrazelluläres Calcium-Signaling, intrinsische Erregbarkeit und Dendritenwachstum

Titelblatt und Inhaltsverzeichnis Publication, Statement, Contributions General Introduction Chapter 1 Chapter 3: Figure Legends and Figures SummaryThe actions of neurons can alter the output of neural circuits and is, therefore, the basis of animal behavior. In order to fulfill their tasks the key players have to be well equipped with cellular properties like ion channels. To investigate intrinsic properties underlying behavior, isolated insect neurons have proven a good choice. Neuronal calcium acts as a charge carrier during information processing and as an intracellular messenger. Ca2+ signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron. This study demonstrates a novel calcium signaling mechanism by simultaneous patch clamping and Ca2+ imaging from isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which leads to calcium release from intracellular stores via PLC and IP3 receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels. To investigate the role of intrinsic properties for behavior, the genetic model system Drosophila melanogaster has been used in various studies. The Drosophila MN5 is a well described central neuron innervating the dorsolongitudinal flight muscle that is involved in the escape response pathway. We investigated in situ whole cell K+ currents in MN5 with different genetic background. In wildtype MN5 we describe 4 different K+ currents, 1) 4-AP sensitive A-type current, 2) 4-AP sensitive, Ca2+ dependent A-type current, 3) TEA-sensitive delayed rectifier (DR) current, 4) TEA-sensitive Ca2+ dependent DR current. The A-type current is probably mediated by the expression of Shaker, since in eag Shaker double knock down, the A-type current is markedly reduced and shifts in activation voltage occur. The targeted expression of EKO, a modulated Shaker channel that activates at more negative potentials and does not inactivate, also leads to shifts in activation voltage but does not reduce A-type current amplitude. The characterization of ion currents of identified neurons is necessary for further analysis of ion channel function regarding behavioral, developmental and morphological aspects. Dendrites are the fundamental determinant of neuronal wiring. Intrinsic neuronal activity as well as synaptic activity impinging on dendritic trees can have profound effects on dendritic structure, but the underlying mechanisms are not fully understood. This study uses the genetic model system Drosophila to test the effects of altered intrinsic excitability on postembryonic dendritic shape development of the MN5. We show that targeted dominant negative knock down of K+ channel subunits in specific motoneurons allows for selectively increasing the intrinsic excitability of these motoneurons, whereas targeted expression of EKO decreases intrinsic excitability in vivo. Increased excitability causes increased dendritic branch formation whereas decreased excitability causes increased dendritic branch elongation. Therefore, dendritic branching and dendritic segment elongation are controlled by separate mechanism which can be selectively addressed in vivo by different manipulations of a neuron`s intrinsic excitability.Das Verhalten von Neuronen beeinflusst den Output neuronaler Netzwerke und ist damit die Grundlage vielfältiger verschiedener Verhaltensweisen von Tieren. Um diese Aufgabe bewerkstelligen zu können, müssen die beteiligten Neurone gut mit zellulären Eigenschaften wie Ionenkanälen ausgestattet sein. Für die Analyse intrinsischer Eigenschaften haben sich isolierte Insekten-Neurone als sehr nützlich erwiesen. Ca2+ Signale sind enorm wichtig für das Funktionieren und die Informationsweiterleitung eines Neurons. Gesteuerte Regulation dieser Ca2+ Dynamik wird durch die verschiedensten Ca2+ Signalwege erreicht. Hier beschreiben wir einen neuen Ca2+ Signalweg durch simultanes Patch Clamping und Ca2+ Imaging isolierter Neurone. Diese Neurone besitzen einen Spannungssensor, der Ca2+ unabhängig ein G-Protein aktiviert, was über PLC Aktivität zur IP3 Synthese führt. Die Aktivierung von IP3 Rezeptoren durch IP3 führt zur Ca2+ Ausschüttung aus internen Speichern. Dieser Mechanismus ist unabhängig von extern appliziertem Ca2+. Die Rolle intrinsischer Eigenschaften für Verhalten und Entwicklung kann man sehr gut an dem genetischen Modellorganismus Drosophila melanogaster untersuchen. Das MN5 in Drosophila ist ein gut untersuchtes zentrales Motoneuron, das den dorsolongitudinalen Flugmuskel innerviert. Wir untersuchten Whole Cell K+ Ströme im MN5 mit verschiedenem genetischen Hintergrund in situ. Im Wildtyp MN5 beschreiben wir vier verschiedene K+ Ströme: 1) A-Typ K+ Strom, 4-AP sensitiv, 2) A-Typ K+ Strom, 4-AP sensitiv, Ca2+ abhängig, 3) Delayed Rectifier (DR) K+ Strom, TEA- sensitiv, 4) DR K+ Strom, TEA-sensitiv, Ca2+ abhängig. Der A-Typ K+ Strom ist wahrscheinlich durch Shaker codiert, da der A-Typ K+ Strom im eag Shaker Doppel Knock Down stark reduziert und das Aktivierungspotential verschoben ist. Die gezielte Expression von EKO, ein modifizierter Shaker K+ Kanal, der bei negativeren Potentialen aktiviert und nicht inaktiviert, war auch das Aktivierungspotential verschoben, die Strom-Amplitude blieb unverändert. Die Charakterisierung der Ionenströme identifizierter Neurone in situ ist notwendig, um die Rolle von Ionenströmen auf Verhalten, Entwicklung und Morphologie untersuchen zu können. Dendriten leiten neuronale Signale weiter. Intrinsische neuronale Aktivität kann ebenso erhebliche Effekte auf die dendritische Struktur haben wie synaptische Aktivität. Hier nutzen wir das genetische Modellsystem Drosophila, um zu untersuchen, ob veränderte intrinsische Aktivität postembryonale Effekte auf die dendritische Struktur des MN5 hat. Wir zeigen, dass ein gezielter genetischer Knock Down bestimmter K+ Kanäle die intrinsische Erregbarkeit des MN5 in vivo erhöht, wohingegen Expression von EKO die Erregbarkeit stark verringert. Erhöhte Erregbarkeit führt zu einer Erhöhung der Anzahl dendritischer Verzweigungen, verringerte Erregbarkeit führt zu einer Verlängerung der Dendriten-Segmente. Verzweigung von Dendriten und Verlängerung der Segmente werden durch unterschiedliche Mechanismen kontrolliert, die man in vivo separat durch Manipulation der intrinsichen Erregbarkeit eines Neurons untersuchen kann

Different functions of two putative Drosophila α2δ subunits in the same identified motoneurons

Abstract Voltage gated calcium channels (VGCCs) regulate neuronal excitability and translate activity into calcium dependent signaling. The α1 subunit of high voltage activated (HVA) VGCCs associates with α2δ accessory subunits, which may affect calcium channel biophysical properties, cell surface expression, localization and transport and are thus important players in calcium-dependent signaling. In vertebrates, the functions of the different combinations of the four α2δ and the seven HVA α1 subunits are incompletely understood, in particular with respect to partially redundant or separate functions in neurons. This study capitalizes on the relatively simpler situation in the Drosophila genetic model containing two neuronal putative α2δ subunits, straightjacket and CG4587, and one Cav1 and Cav2 homolog each, both with well-described functions in different compartments of identified motoneurons. Straightjacket is required for normal Cav1 and Cav2 current amplitudes and correct Cav2 channel function in all neuronal compartments. By contrast, CG4587 does not affect Cav1 or Cav2 current amplitudes or presynaptic function, but is required for correct Cav2 channel allocation to the axonal versus the dendritic domain. We suggest that the two different putative α2δ subunits are required in the same neurons to regulate different functions of VGCCs

Tyramine actions on Drosophila flight behavior are affected by a glial dehydrogenase/reductase

The biogenic amines OA and TA modulate insect motor behavior in an antagonistic manner. OA generally enhances locomotor behaviors such as Drosophila larval crawling and flight, whereas TA decreases locomotor activity. However, the mechanisms and cellular targets of TA modulation of locomotor activity are incompletely understood. This study combines immunocytochemistry, genetics, and flight behavioral assays in the Drosophila model system to test the role of a candidate enzyme for TA catabolism, named nazgul (Naz), in flight motor behavioral control. We hypothesize that the dehydrogenase/reductases Naz represents a critical step in TA catabolism. Immunocytochemistry reveals that Naz is localized in a subset of Repo positive glial cells with cell bodies along the motor neuropil borders and numerous positive Naz arborizations extending into the synaptic flight motor neuropil. RNAi knock-down of Naz in Repo positive glial cells reduces Naz protein level below detection level by Western blotting. The resulting consequence is a reduction in flight durations, thus mimicking known motor behavioral phenotypes as resulting from increased TA levels. In accord with the interpretation that reduced TA degradation by Naz results in increased TA levels in the flight motor neuropil, the motor behavioral phenotype can be rescued by blocking TA receptors. Our findings indicate that TA modulates flight motor behavior by acting on central circuitry and that TA is normally taken up from the central motor neuropil by Repo-positive glial cells, desaminated, and further degraded by Naz

Tyramine Actions on Drosophila Flight Behavior Are Affected by a Glial Dehydrogenase/Reductase

The biogenic amines octopamine (OA) and tyramine (TA) modulate insect motor behavior in an antagonistic manner. OA generally enhances locomotor behaviors such as Drosophila larval crawling and flight, whereas TA decreases locomotor activity. However, the mechanisms and cellular targets of TA modulation of locomotor activity are incompletely understood. This study combines immunocytochemistry, genetics and flight behavioral assays in the Drosophila model system to test the role of a candidate enzyme for TA catabolism, named Nazgul (Naz), in flight motor behavioral control. We hypothesize that the dehydrogenase/reductase Naz represents a critical step in TA catabolism. Immunocytochemistry reveals that Naz is localized to a subset of Repo positive glial cells with cell bodies along the motor neuropil borders and numerous positive Naz arborizations extending into the synaptic flight motor neuropil. RNAi knock down of Naz in Repo positive glial cells reduces Naz protein level below detection level by Western blotting. The resulting consequence is a reduction in flight durations, thus mimicking known motor behavioral phenotypes as resulting from increased TA levels. In accord with the interpretation that reduced TA degradation by Naz results in increased TA levels in the flight motor neuropil, the motor behavioral phenotype can be rescued by blocking TA receptors. Our findings indicate that TA modulates flight motor behavior by acting on central circuitry and that TA is normally taken up from the central motor neuropil by Repo-positive glial cells, desaminated and further degraded by Naz

Intra-neuronal competition for synaptic partners conserves the amount of dendritic building material

Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, we show that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca2+ influx through Dα7nAChRs or through LVA channels following GABAAR-mediated depolarizations

Expanding the neuron’s calcium signaling repertoire: intracellular calcium release via voltageinduced PLC and IP3R activation

Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels

Shaker and Shal Mediate Transient Calcium-Independent Potassium Current in a Drosophila Flight Motoneuron

Ionic currents underlie the firing patterns, excitability, and synaptic integration of neurons. Despite complete sequence information in multiple species, our knowledge about ion channel function in central neurons remains incomplete. This study analyzes the potassium currents of an identified Drosophila flight motoneuron, MN5, in situ. MN5 exhibits four different potassium currents, two fast-activating transient ones and two sustained ones, one of each is calcium activated. Pharmacological and genetic manipulations unravel the specific contributions of Shaker and Shal to the calcium independent transient A-type potassium currents. α-dendrotoxin (Shaker specific) and phrixotoxin-2 (Shal specific) block different portions of the transient calcium independent A-type potassium current. Following targeted expression of a Shaker dominant negative transgene in MN5, the remaining A-type potassium current is α-dendrotoxin insensitive. In Shal RNAi knock down the remaining A-type potassium current is phrixotoxin-2 insensitive. Additionally, barium blocks calcium-activated potassium currents but also a large portion of phrixotoxin-2-sensitive A-type currents. Targeted knock down of Shaker or Shal channels each cause identical reduction in total potassium current amplitude as acute application of α-dendrotoxin or phrixotoxin-2, respectively. This shows that the knock downs do not cause upregulation of potassium channels underlying other A-type channels during development. Immunocytochemistry and targeted expression of modified GFP-tagged Shaker channels with intact targeting sequence in MN5 indicate predominant axonal localization. These data can now be used to investigate the roles of Shaker and Shal for motoneuron intrinsic properties, synaptic integration, and spiking output during behavior by targeted genetic manipulations