Regulated expression of Down Syndrome cell adhesion molecule controls precise synaptic targeting

Neurons are organized in functional circuits to allow an organism to perform complex behaviors. The mechanisms that dictate self-assembly of hard-wired neuronal circuits during development are still poorly understood. How are neurons able to distinguish correct from incorrect synaptic targets when they are faced with thousands of possible partners? To address this question, my dissertation explores the molecular mechanisms that regulate synaptic specificity in a hard-wired neuronal circuit of the model organism Drosophila melanogaster. I describe a method that allows combined structural and functional analysis of axonal targeting in single identifiable sensory neurons. This method is used to demonstrate that elevated levels of Down Syndrome Cell Adhesion Molecule, due to gene triplication or due to loss of regulation by Fragile X Mental Retardation Protein, perturbs the fine-scale connectivity of single sensory neurons and results in impaired circuit function and behavioral response to sensory stimuli. Single cell analysis of identified sensory neurons also allows for determining the molecular wiring code of a neuron. Using an RNA interference screen for genes involved in axonal targeting, I identified Teneurin-m as a cell surface receptor required for proper axonal branch targeting. I characterized stereotyped miswiring that occurs due to loss of Teneurin-m expression and the behavioral consequences of these synaptic targeting errors. The experiments presented here support the model that tightly regulated expression of genetically-encoded wiring instructions in the form of cell surface molecules determine the precise connectivity of single neurons in hard-wired circuits. Advancing our knowledge of the basic mechanisms of neural circuit formation may help us understand how genetic variation contributes to altered neuronal connectivity in human neurodevelopmental disorders.Les neurones sont organisés en circuits fonctionnels pour permettre aux organismes d'effectuer des comportements complexes. Cependant, les mécanismes qui gouvernent l'assemblée des circuits neuronaux durant le développement sont à ce jour peu compris. Comment les neurones peuvent-ils faire la distinction entre une cible synaptique adéquate et inadéquate lorsqu'ils font face à des milliers de partenaires synaptiques potentiels? Pour répondre à cette question, ma dissertation explore les mécanismes moléculaires qui régulent la spécificité synaptique des circuits neuronaux de l'organisme modèle Drosophila melanogaster. J'y décris un modèle à l'aide de neurones sensoriels identifiés qui permet l'analyse combinée structurelle et fonctionnelle de l'embranchement axonal ciblé. Cette méthode est utilisée pour démontrer qu'un niveau élevé de Down Syndrome Cell Adhesion Molecule (Dscam), qui peut être le résultat de trois copies du gène Dscam ou de la perte de régulation par le Fragile X Mental Retardation Protein, perturbe la connectivité de fins embranchements neuronaux sensoriels et résulte en une déficience des circuits fonctionnels ainsi qu'en réponses comportementales non appropriées lors de la stimulation sensorielle. L'analyse combinée structurale et fonctionnelle de cellule identifiée permet aussi d'établir le code d'embranchement moléculaire de neurones. En utilisant de criblage par ARN interférent pour des gènes qui régulent un embranchement axonal ciblé, j'y identifie Teneurin-m comme un récepteur de surface cellulaire nécessaire pour l'embranchement axonal ciblé. J'y caractérise les défauts de connexion stéréotypés qui surviennent lors de la perte d'expression de Teneurin-m et les conséquences comportementales de ces erreurs de ciblage synaptiques. Les expériences présentées dans cette dissertation supportent un modèle qui régule de façon fiable l'expression d'instructions d'embranchement génétiquement encodées, c'est-à-dire que les molécules en surface des cellules déterminent la connectivité précise de neurones à l'intérieur des circuits d'embranchement. L'avancement de nos connaissances sur les mécanismes de base de formation des circuits neuronaux pourrait nous aider à comprendre comment la variation génétique contribue à la connectivité neuronale altérée lors de désordres humains neuro-développementaux

Quantification of Protein Levels in Single Living Cells

Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation. The fluorescence intensity is proportional to the number of molecules produced of the protein of interest and is used to determine the relative amount of protein within the cell. We use PQR to quantify protein expression of different genes using quantitative imaging, electrophysiology, and phenotype. We use genome editing to insert Protein Quantitation Reporters into endogenous genomic loci in three different genomes for quantitation of endogenous protein levels. The PQR technique will allow for a wide range of quantitative experiments examining gene-to-phenotype relationships with greater accuracy

Loss of Synapse Repressor MDGA1 Enhances Perisomatic Inhibition, Confers Resistance to Network Excitation, and Impairs Cognitive Function

Synaptopathies contributing to neurodevelopmental disorders are linked to mutations in synaptic organizing molecules, including postsynaptic neuroligins, presynaptic neurexins, and MDGAs, which regulate their interaction. The role of MDGA1 in suppressing inhibitory versus excitatory synapses is controversial based on in vitro studies. We show that genetic deletion of MDGA1 in vivo elevates hippocampal CA1 inhibitory, but not excitatory, synapse density and transmission. Furthermore, MDGA1 is selectively expressed by pyramidal neurons and regulates perisomatic, but not distal dendritic, inhibitory synapses. Mdga1−/− hippocampal networks demonstrate muted responses to neural excitation, and Mdga1−/− mice are resistant to induced seizures. Mdga1−/− mice further demonstrate compromised hippocampal long-term potentiation, consistent with observed deficits in spatial and context-dependent learning and memory. These results suggest that mutations in MDGA1 may contribute to cognitive deficits through altered synaptic transmission and plasticity by loss of suppression of inhibitory synapse development in a subcellular domain- and cell-type-selective manner

Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins.

(Neuron 95, 896–913; August 16, 2017) After publication, we noticed a number of minor errors within the main text and Figures 1 and 4 that escaped our attention during the proofreading of the manuscript. In the top left panel in Figure 1C, FnIII loop C'E is incorrectly labeled as CE. In the legend to Figure 3A, the buried surface area of Site II is incorrectly stated as 859 Å , while the correct value is 1,000 Å . In Figure 4B, residue Glu294 (E294) is incorrectly labeled as Asp294 (D294). This error is also present in the main text paragraph “MDGA and NRX Share Binding Interfaces on NL.” The errors have no effect on any of the conclusions in the paper, and the main text and Figures 1 and 4 have now been corrected online. The authors apologize for any confusion the errors may have caused. [formula presented] [formula presented]. 7 2

Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins

Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.status: publishe