Memories, thoughts and behavior are encoded in changes in the strength of synaptic communication. These changes constitute the cellular and molecular basis of information processing underlying brain function. However, the functional transformations used by synapses to encode and process information are still poorly understood. We are interested in the cellular and molecular mechanisms of synaptic function and plasticity, and in how synaptic dysfunction underlies neuropsychiatric and neurodegenerative disorders. We use a combination of techniques like primary cultures of dissociated neurons and brain slices, biochemistry, molecular and cellular biology, mouse molecular genetics, electrophysiology and behavior analysis to address the role of molecular players and of neuromodulatory processes that regulate neurotransmitter receptors and synaptic function and plasticity. This fundamental research has strong implications to cognitive disorders, since genetic variants in multiple synaptic proteins are linked to intellectual disability, schizophrenia and autism spectrum disorders, and age-associated neurodegenerative disorders are at their initial stages originated by defects in synaptic transmission. We focus on disease-related alterations in synaptic function, either genetic, triggered by experience or by antibodies produced by autoimmune synaptic encephalitis patients, to understand how synaptic dysfunction underlies disease pathogenesis. Our cellular and molecular studies and the animal models that we are generating can also contribute to the rational development of therapies for these diseases.
1 - Mechanisms that regulate synaptic transmission and synaptic plasticity
We are interested in understanding how synaptic physiology is regulated pre- and postsynaptically at the molecular level. We have addressed this question by studying mechanisms of subcellular targeting and clustering of neurotransmitter receptors at synapses, and have focused on glutamate receptor, the major excitatory receptors in the brain. We have identified proteins that directly or indirectly bind to glutamate receptors, for example the recently identified AMPA receptor associated proteins Caspr1 and Caspr2 (Santos et al., J Biol Chem 2012, Fernandes et al., Cerebral Cortex 2019). We uncovered how they regulate AMPA receptor function and synaptic scaling, and revealed that anti-CASPR2 antibodies from autoimmune synaptic encephalitis patients block synaptic transmission in the visual cortex by interfering with the synaptic role of Caspr2. We are currently further tackling how antibodies targeting different CASPR2 epitopes impact synaptic physiology.
Stargazin is a member of the transmembrane AMPA receptor regulatory protein family, which we found to be implicated in experience-dependent plasticity (Louros et al., Cell Rep 2014). More recently, we observed that stargazin phosphorylation mediates synaptic scaling (Louros et al., Front Mol Neurosci 2018). We are now interested in the regulation of neuronal excitability by stargazin, and in how disease-associated mutations in the stargazin-coding gene affect synapse and function, and result in altered behavior (see below).
Synaptic scaling depends on changes on protein expression. We found that miRNA-186-5p mediates synaptic scaling in the hippocampus, by targeting the GluA2 subunit of AMPA receptors and regulating AMPA receptor subunit composition (Silva et al., PNAS 2019). We are presently studying abnormal upregulation of miRNA-186-5p levels in the brain during chronic stress, and whether it is implicated in chronic stress-related alterations in synaptic transmission.
NMDA receptors are crucial players in the induction of synaptic plasticity. The subunit composition of NMDA receptors determines their functional properties and neuronal distribution. We have found that GluN2B-containing NMDA receptors determine the synapse composition throughout development (Ferreira et al., J Neurosci 2015), and characterized the nanostructural synaptic organization of GluN2A- and GluN2B-containing NMDA receptors (in collaboration with Laurent Groc, Kellermayer et al., Neuron 2018). We are currently investigating a RhoGAP that is anchored at the synapse by GluN2B-NMDA receptors (Schmidt et al., in preparation).
The group has an interest in how metabolic hormones regulate synaptic function. We have found that the orexigenic hormone ghrelin, which has cognitive enhancer properties, promotes the synaptic traffic of AMPA receptors and facilitates synaptic plasticity in the hippocampus (Ribeiro et al., PNAS 2014). Our recent work showed that the unusually high constitutive activity of the ghrelin receptor is required for hippocampal-dependent memory, and regulates synaptic function and plasticity (Ribeiro et al., accepted for publication in Science Signaling).
A line of research lead by Paulo Pinheiro is focused on investigating the role of alternate SNAP isoforms, namely SNAP-29 and SNAP-47, in the different phases leading to the fusion of vesicles with the plasma membrane. We also study how these proteins may interact/compete with the dominant isoforms, and with the calcium sensor proteins for release triggering, and how that affects the dynamics of exocytosis. On the other hand, these proteins have been implicated in synaptic plasticity processes and genetic studies found links to human cognitive disorders, namely schizophrenia. We are evaluating genetically modified mice lacking their expression for behavior and synaptic alterations.
2 - Synaptic dysfunction in brain diseases
Understanding synaptic dysfunction underlying brain diseases may reveal crucial disease pathways. We have characterized for the first time synaptic alterations triggered by polyQ-expanded ataxin-3, which causes Spinocerebellar Ataxia type 3 (SCA3), and found that they are prevented by ataxin-3 phosphorylation in a newly described phosphorylation site (Matos et al., J Cell Biol 2016).
We are interested in understanding how disease-associated human mutations in genes encoding for synaptic proteins impact synaptic function. We have focused on mutation sin the CACNG2 gene encoding stargazin and found that knock-in mice harboring an intellectual disability-variant of the CACNG2 gene reproduce alterations in cognitive and social behavior reminiscent of the clinical symptoms found in patients. Morphological and electrophysiological analyses revealed that stargazin knock-in mice present abnormalities in neuronal morphology and synaptic function in the hippocampus, which constitute a potential disease mechanism (Caldeira, Inácio et al., in preparation).
Ongoing Research Projects
MStar - Potassium channel dysfunction in models of neurodevelopmental disorders (2020-2023), Iniciativa Ibérica de Investigación y Innovación Biomedica, "la Caixa" Foundation/FCT.
Coordination of Syn2Psy-Synaptic Dysfunction in Neuropsychiatric Disorders (2019-2023), H2020-MSCA-ITN-2018 ‘European Training Network’ 813986 - consortium project funded by the European Commission.
Cognitive disorders as defect in synaptic function: insight from human mutations in the CACNG2 gene (2018-2021), FCT, Portugal.
Understanding the pathogenesis in CASPR2-antibody autoimmune synaptic encephalitis (2018-2021), FCT, Portugal.
Alternate SNAPs in neurotransmitter release: from molecular function to neurocognitive dysfunction (2018-2021), FCT, Portugal.
Ann Marie Craig
Brain Research Centre, University of British Columbia, Vancouver, Canada
Carlos N Pato
University of Southern California, Los Angeles, USA
Harvard Medical School, Boston, USA
University of Dundee, Dundee, UK
Centro de Bíologia Molecular Severo Ochoa – CSIC, Universidad Autómona de Madrid, Madrid, Spain
Interdisciplinary Neuroscience Institute, Bordeaux, France
University of Lyon 1, Lyon, France
Célia Barreto Carvalho
University of Azores, Ponta Delgada, Portugal
Physics Department, University of Coimbra, Coimbra, Portugal
Life and Health Sciences Research Institute, University of Minho, Braga, Portugal
Sandra de Macedo-Ribeiro
IBMC, University of Porto, Porto, Portugal
- "la Caixa" Foundation
- European Commission
Ribeiro L.F., Catarino T., Carvalho M., Cortes L., Santos SD, Opazo P., Ribeiro L.R. Oliveiros B., Choquet D., Esteban J.A., Peça J., Carvalho A.L. (2020) Ligand-independent activity of the ghrelin receptor modulates AMPA receptors trafficking and supports memory formation. Science Signaling (accepted for publication). BioRxive doi: 10.1101/2020.02.05.934463
Silva M.M., Rodrigues B., Fernandes J., Santos S.D., Carreto L., Santos M.A.S., Pinheiro P., Carvalho A.L. (2019) MicroRNA-186-5p controls GluA2 surface expression and synaptic scaling in hippocampal neurons. PNAS 116(12), 5727-5736. DOI: 10.1073/pnas.1900338116
Fernandes D., Santos S.D., Coutinho, E., Whitt J.L., Rondão T., Leite M.I., Buckley C., Lee H.K., Carvalho A.L. (2019) Disrupted AMPA receptor function upon genetic or antibody-mediated loss of autism-associated CASPR2. Cerebral Cortex 29(12):4919-4931. DOI: 10.1093/cercor/bhz032
Lima Caldeira, G., Peca, J., & Carvalho, A. L. (2019). New insights on synaptic dysfunction in neuropsychiatric disorders. Curr Opin Neurobiol, 57, 62-70. doi: 10.1016/j.conb.2019.01.004
Edfawy, M., Guedes, J. R., Pereira, M. I., Laranjo, M., Carvalho, M. J., Gao, X., . . . Peca, J. (2019). Abnormal mGluR-mediated synaptic plasticity and autism-like behaviours in Gprasp2 mutant mice. Nat Commun, 10(1), 1431. doi: 10.1038/s41467-019-09382-9
Kellermayer B., Ferreira J.S., Dupuis J., Levet F., Grillo-Bosch D., Bard L., Linares-Loyez J., Bouchet D., Choquet D., Rusakov D.A., Bon P., Sibarita J.B., Cognet L., Sainlos M., Carvalho A.L., Groc L. (2018) Differential Nanoscale Topography and Functional Role of GluN2-NMDA Receptor Subtypes at Glutamatergic Synapses. Neuron 100, 106-119 e107. DOI: 10.1016/j.neuron.2018.09.012
Agís‑Balboa RC, Pinheiro PS, Rebola N, Kerimoglu C, Benito E, Gertig M, Bahari-Javan S, Jain G, Burkhardt S, Delalle I, Jatzko A, Dettenhofer M, Zunszain PA, Schmitt A, Falkai P, Pape JC, Binder EB, Mulle C, Fischer A & Sananbenesi F (2017) Formin 2 links neuropsychiatric phenotypes at young age to an increased risk for dementia. EMBO J 36, 2815‑2828.
Matos, C. A., Nobrega, C., Louros, S. R., Almeida, B., Ferreiro, E., Valero, J., Pereira de Almeida, L., Macedo-Ribeiro, S., and Carvalho, A. L. (2016) Ataxin-3 phosphorylation decreases neuronal defects in spinocerebellar ataxia type 3 models. J Cell Biol 212, 465-480. DOI: 10.1083/jcb.201506025; commentary in J Cell Biol 212, 355.
Vieira, M. M., Schmidt, J., Ferreira, J. S., She, K., Oku, S., Mele, M., . . . Carvalho, A. L. (2016). Multiple domains in the C-terminus of NMDA receptor GluN2B subunit contribute to neuronal death following in vitro ischemia. Neurobiol Dis, 89, 223-234. doi: 10.1016/j.nbd.2015.11.007
Ferreira, J. S., Schmidt, J., Rio, P., Aguas, R., Rooyakkers, A., Li, K. W., Smit, A. B., Craig, A. M., and Carvalho, A. L. (2015) GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome. J Neurosci 35, 8462-8479. DOI: 10.1523/JNEUROSCI.3567-14.2015; commentary in BrainFacts.org, Lighting Up the Synapses. (http://www.brainfacts.org/brain-anatomy-and-function/cells-and-circuits/2015/image-of-the-week-lighting-up-the-synapses); cover image.
GROUP LEADER AND PRINCIPAL INVESTIGATOR
Ana Luísa Carvalho (PhD)
Ana Luísa Carvalho is an Associate Professor at the Department of Life Sciences, University of Coimbra, and group leader of the Synapse Biology group at the Center for Neuroscience and Cell Biology, University of Coimbra. She did her PhD at the University of Coimbra, in collaboration with the Johns Hopkins University, USA, and did a sabbatical year at the University of British Columbia, Canada. She is interested in the cellular and molecular mechanisms of synaptic function and plasticity and in elucidating how changes in synapses and neuronal circuits contribute to psychiatric disorders.
Paulo Pinheiro (PhD)