Download 3 PhD-‐students in cellular neuroscience

 The Center for Neurogenomics and Cognitive Research in Amsterdam (see www.cncr.nl) participates in the Neuroscience Graduate School Amsterdam-­‐Rotterdam (www.ONWAR.nl) and is seeking applications for 3 PhD-­‐students in cellular neuroscience (cell biology, neurophysiology, life cell imaging) Department of Functional Genomics, VU/VUmc Amsterdam Project 1: The principles of vesicle docking in neurons and neurosecretory cells Project 2: Presynaptic modulation of synaptic transmission Project 3: The effect of genetic variation in schizophrenia on cellular trafficking and synaptic transmission Projects: These projects are part of a large EU-­‐funded project on cellular trafficking (ERC Advanced grant, ERC-­‐ADG-­‐
322966) and a international collaboration between the US, Sweden and Amsterdam on functional analysis of genetic variation in schizophrenia (SUN-­‐project). The PhD-­‐students will be fully integrated in these projects. Background and aim: Communication between neurons in the brain depends on the secretion of chemical messengers from synaptic and dense core vesicles and the trafficking of these vesicles to and within the synapse. Several genes involved in these processes are now firmly implicated in brain disorders like epilepsy, mental retardation and schizophrenia. The aim of the projects is to unravel the mechanisms of vesicle trafficking and secretion in neurons and neurosecretory cells and to analyze how genetic variation associated with these disorders affect these processes. Description of work: All projects use cultured rodent and human neurons and modern genome editing to delete candidate genes or introduce/correct disease-­‐relevant variation. As main analysis tools you will primarily use electronmicroscopy and life cell imaging, incl. TIRF (project 1), patch clamp electrophysiology and fluorescence imaging (project 2) and cellular trafficking assays (project 3). All projects will test their main findings in more integrated models (brain slices and in vivo). You will be part of international research networks and will be able to exploit a variety of other analysis methods available within the networks. You will be appointed in Amsterdam (project 1 and 2) or in Stockholm (project 3) and work primarily in Amsterdam with regular visits to the participating labs. The PhD-­‐
students will be trained on site and in specialized courses on campus. Prof. Matthijs Verhage is the main supervisor for all projects. In addition, you will receive support from senior staff: Dr. Jan van Weering (project 1), Dr. Marieke Meijer (project 2) and Dr. Ruud Toonen (project 3). Suitable candidates: We are looking for candidates that hold, or will soon hold, a master degree in (Medical) Biology, Biophysics or Physics, preferably with hands-­‐on experience in (neuronal) cell culture, electronmicroscopy, cellular imaging and/or patch clamp physiology, and a strong motivation to pursue a career in science. Applications and more info: Please send CV and cover letter to Els Borghols at [email protected] with ‘CNCR-­‐PhD-­‐56-­‐2015’ and the project number(s) in the subject line. Deadline: June 15, 2015. Start date: no later than November 1, 2015. More info via www.cncr.nl or from Dr. Jan van Weering (project 1, [email protected]), Dr. Marieke Meijer (project 2, [email protected]) or Dr. Ruud Toonen (project 3, [email protected]). 2
Project 1: The principles of vesicle docking in neurons and neurosecretory cells The stable docking of secretory vesicles prior to release is a unique feature of regulated secretion of chemical signals and one of the decisive factors in the velocity of chemical communication. We have previously identified core components of the docking machinery and how they work together to dock secretory vesicles1. However, how the docking machinery is positioned at the appropriate cellular location, for instance in the active zone of the nerve terminal, remains unknown. In this project, we aim to investigate the link between the docking machinery and (a) the sub-­‐membrane cytoskeleton and (b) lipid micro-­‐domains in the plasma membrane. Using available knock-­‐out mouse models, we will investigate alterations in cytoskeleton, lipid micro-­‐domains and docking using electronmicroscopy, immunocytochemistry and functional assays (e.g. TIRF microscopy, capacitance measurements, amperometry) in neurons and neuroendocrine cells. We will identify links between the docking machinery and the cytoskeleton/lipid micro-­‐domains, modify these using modern gene editing techniques (CRISPR/Cas9) and analyze the consequences with the same methods. Project 2: Presynaptic modulation of synaptic transmission Synaptic transmission in the brain is modified by previous experience and external modulation by chemical signals. Modification of presynaptic secretion is an important way to modify transmission. Presynaptic mechanisms modify both the amount of synaptic vesicles available for secretion and their likeliness to be secreted. The aim of this project is to identify and characterize the molecular factors that account for these two mechanisms to regulate secretion in nerve terminals. In this project, two factors will be central which have recently been implicated in human disease (schizophrenia, autism, mental retardation, epilepsy): (1) Munc18-­‐1 and its kinase Dyrk1a, and (2) tomosyn-­‐1 and -­‐2. All these 4 genes have been inactivated in mice. In addition, genetic variation associated with schizophrenia will be introduced in mouse and human neurons using genome editing methods. In all these cases, synaptic transmission will be analyzed in cultured neurons using patch clamp, complemented with optical recordings to directly assess presynaptic functions. Some recent papers on these approaches from our lab are found below2. Project 3: The effect of genetic variation in schizophrenia on cellular trafficking and synaptic transmission Several exon variants and single gene CNVs are now firmly associated with schizophrenia and also many single nucleotide polymorphisms outside coding regions. We have previously identified synaptic gene networks where many such risk factors accumulate3. Using two reduced and highly standardized model systems, we aim to systematically analyze the synaptic effects of different exonic variants and single gene CMVs. In this project, the focus will be on presynaptic function. As model system we will exploit autaptic cultures of mouse cortex and human iPS-­‐cell derived neurons, also from patients. We will genetically modify these systems using multiple lentiviral infections. We will exploit knockout and rescue methodology to generate models for schizophrenia, introducing mutations and gene deletions observed in patients and prioritized by the project management. In addition, we will over-­‐express or knockdown synaptic genes predicted to be expressed at higher/lower rate in schizophrenia cases, and overexpress or knockdown multiple genes in relevant gene networks containing driver foci. We will establish the cellular consequences of these modifications for synaptic function using patch clamp physiology and life cell imaging, again in a highly standardized manner, yielding roughly 50 functional parameters which together assist in predicting the synaptic consequences of genetic variation associated with schizophrenia. 1
de Wit et al. (2009) Cell 138:p935-­‐46; Verhage & Sørensen (2008) Traffic 9:p1414-­‐24 Meijer et al. (2012) EMBO J. 31:p2156-­‐68; Groffen et al. (2010) Science 327:p1614-­‐8 3
Ripke et al. (2013) Nature Genetics 45:p1150-­‐9; Lips et al. (2012) Mol. Psychiatry 17:p996-­‐1006.
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