Annual Report of Activities CNC 2008 - Center for Neuroscience and ...

Glutamatergic synapses | Head: Ana Luisa CarvalhoObjectivesWe are interested in understanding the connectionsbetween nerve cells in the brain, and how they aremodified with experience. The ability of synapses tochange their strength is thought to be the cellularcorrelate of learning and memory, and synapticdysfunction is correlated with several neurodegenerativediseases. We focus on excitatory glutamatergic synapses,and study their regulation from a cellular and molecularbiology viewpoint. We use a combination of primaryneuronal cultures, molecular cell biology andbiochemistry to address these questions.1. Proteomic analysis of the interactome of AMPAtypeglutamate receptors (AMPAR). AMPARbinding partners regulate their synaptic targeting. Weidentified several novel interactors for AMPARsubunits in a proteomic screening, and are currentlyaddressing their function in regulating AMPARs.2. Regulation of the mRNA stability of the mRNAfor GluR1 AMPAR subunit. We found evidence forpost‐transcriptional regulation of the mRNA levels forGluR1, and are addressing its mechanisms.3. Mechanisms of synaptic traffic of NMDAreceptors (NMDAR). NMDARs play a role in theinduction of synaptic plasticity. We use culturedneurons from knock‐out mice for NMDARsubunits to understand subunit‐specific rules thatgovern synaptic targeting of NMDARs.4. Regulation of glutamatergic transmission byghrelin in the hippocampus. Ghrelin is anappetite‐stimulating hormone which was shown toenhance memory processes and synaptic plasticityin the hippocampus. We are characterizing thesubcellular localization of ghrelin receptors in thehippocampus, and evaluating how ghrelinreceptors affect glutamatergic transmission.5. Synaptic dysfunction in a mouse model ofMachado‐Joseph disease (MJD). MJD is aspinocerebellar ataxia caused by a glutamineexpansion in ataxin‐3, an ubiquitin protease. Weare using a transgenic mouse model of MJD toaddress the role of excitotoxicity and synapticdysfunction in the pathogenesis of this disease.Main AchievementsProteins that interact with AMPA‐type glutamatereceptors (AMPAR) are crucial for appropriatetargeting of receptors to synapses (Santos et al. 2009).We have performed a proteomic screening forbinding partners of Ca 2+ ‐permeable AMPARs. Afterre‐isolating known AMPAR interactors, we identifiednovel interactors, such as motor proteins and proteinsof the neuronal RNA granules. Moreover, we isolatedthe cell adhesion molecule Contactin associatedprotein 1 (Caspr1). Functional analyses revealed thatCaspr1 affects the cell surface and synaptic expressionof AMPAR subunits in cultured hippocampalneurons (Manuscripts in preparation).NMDA receptors (NMDAR) are the coincidencedetector in the induction of synaptic plasticity. We areaddressing the mechanism of synaptic accumulationof NMDARs, and found that the NR2B subunit ofNMDARs is necessary to establish the synapticscaffold for NMDARs, since synaptic clusters ofNMDARs are dramatically reduced in primaryhippocampal neuronal cultures from NR2B knock‐outmice. Engineered subunits of the NMDAR complexare being reintroduced into neuron cultures fromknockout mice, to dissect molecular signals involvedin NMDAR trafficking.The number of dendritic spines, where excitatorysynapses are located, is regulated by molecules thatorganize their actin cytoskeleton, e.g. cortactin. Weare studying how cortactin acetylation influences itsrole in the morphogenesis of spines, and found thatoverexpression of the acetylation and deacetylationmimetics of cortactin changes the density of synapsesin hippocampal neurons in culture (Fig.1).Fig.1. Hippocampal neurons in culture transfected at 7 DIVwith GFP and cortactin, and labeled at 15 DIV for a marker ofexcitatory synapses (PSD95), and a somatodendritic marker(MAP2). Expression of cortactin changes the density ofsynapses in cultured hippocampal neurons.Another interest in the group is to study dysfunctionof glutamatergic transmission in pathology. We areinterested in ataxin‐3, the polyQ‐containing proteaseinvolved in Machado‐Joseph disease (MJD). We havecharacterized its nucleocytoplasmic shuttling activity(Macedo‐Ribeiro et al. 2009), and will now test thehypothesis that there are alterations in glutamatergicsynapses in MJD.22