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Ann Marie Craig, Ph.D.
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Professor of Psychiatry
Canada Research Chair in Neurobiology
Brain Research Centre
Koerner Pavillion Room F149
University of British Columbia
2211 Wesbrook Mall
Vancouver BC
Canada V6T 2B5
Tel Lab 604-827-3348
Tel Office 604-822-7283
FAX 604-822-7299
Email:
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CNS Synapse Assembly and Plasticity
We are interested in how nerve cells in the brain make connections and modify connections with experience. We study these questions of synapse development and synapse plasticity mainly from a cellular and molecular biology viewpoint. What are the molecular triggers that initiate central neuron synapse formation? How do glutamate and GABA receptors traffic to the synapse, and how are such processes regulated by activity? We use a combination of hippocampal neuron culture, molecular biology and genetics, live cell fluorescence imaging, and electrophysiology to answer these questions.
LAB PERSONNEL
Amanda Rooyakkers, Lab Manager:
Robert Cassidy, PhD, Research Associate
Frederick Dobie, Graduate Student
Yunhee Kang, PhD, Postdoctoral Fellow
Vivian Lam, Undergraduate Research Assistant
Michael Linhoff, Graduate Student
Jacqueline Rose, PhD, Postdoctoral Fellow
Kevin She, Graduate Student
Tabrez Siddiqui, PhD, Postdoctoral Fellow
Katherine Walzak, Graduate Student
Daisaku Yokomaku, PhD, Research Associate
Xiling Zhou, Cell Culture Expert
SOME ONGOING RESEARCH PROJECTS
Molecular Signals for GABA and Glutamate Synapse Development: Neurexins and Neuroligin
Ethan Graf, YunHee Kang, XueZhao Zhang, Frederick Dobie, Anushka Hauner, Shan-Xue Jin, Michael Linhoff, Huaiyang Wu
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Neurexin (blue) presented on non-neuronal cells induces local clustering of GABA receptor (red) on contacting hippocampal neuron dendrites. Unlike endogenous synaptic clusters, these induced clusters lack synapsin (green). Purified neurexins induce local clusters of GABA and glutamate receptors and postsynaptic scaffolding proteins via binding to dendritic neuroligins. (Graf et al., 2004)
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Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. β-Neurexins and neuroligins form a trans-synaptic link at synapses. We found that neurexin alone on a bead or on a fibroblast is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligin-1 localizes to glutamate postsynaptic sites, neuroligin-2 localizes to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins only to glutamate postsynaptic proteins. Thus the neurexin-neuroligin link is a core component contributing to both GABAergic and glutamatergic synaptogenesis.
Differences in neurexin and neuroligin isoform localization and binding affinities may contribute to appropriate differentiation and specificity of GABA versus glutamate synapses. Beta-neurexins containing the insert at splice site 4 selectively promote GABAergic over glutamatergic synapse development, and are expressed early in brain development. The longer α-neurexin variants are even more selective in promoting GABAergic and not glutamatergic postsynaptic differentiation, and may contribute directly to maintenance of GABA synapses. These conclusions from our neuron culture studies are supported by recently published in vivo mouse knockout analyses.
Mutations in neuroligins 3 and 4, and variations in neurexins, have been linked to autism and mental retardation in patient families. It is our hope that by studying the fundamental functions of these molecules we will contribute to understanding these disorders and developing effective therapies.
Novel Synapse-Promoting Molecules
Michael Linhoff, Katherine Walzak, Daisaku Yokomaku, Tabrez Siddiqui, Frederick Dobie, Robert Cassidy, Kevin She
The co-culture system of neurons with non-neuronal cells has been used by several labs to test the ability of candidate molecules to cluster presynaptic and postsynaptic proteins in contacting axons and dendrites. We used this system further to screen a cDNA expression library in pools to search for novel synaptogenic molecules. Michael Linhoff generated a cap-trapped full-length size-selected cDNA expression library and screened a total of
~100,000 clones in pools. After re-isolating neuroligin, a novel protein was identified that promotes presynaptic differentiation in contacting axons of cultured hippocampal neurons. Further studies support the idea that members of this small family of transmembrane proteins indeed function to promote synapse development. When expressed in COS or HEK cells and presented to axons, they trigger localized synaptic vesicle clustering. Recordings from such artificial synapses co-expressing glutamate receptors in the HEK cells indicates that these induced glutamate release sites are functional, much like bona fine presynaptic terminals. Recent data also indicate localization to glutamate postsynaptic sites. Current collaborative studies are underway to generate and characterize knockout mice to assess the in vivo function of this novel family of synaptogenic molecules.
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Voltage clamp recording from HEK cell co-expressing NMDA receptors and novel synapse-promoting protein and co-cultured with hippocampal neurons, indicating formation of functional glutamatergic terminals from the neurons onto the transfected HEK cell. (Linhoff and Craig, unpublished)
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Activity Regulation of Glutamate Synapses: NMDA Receptors and CaM Kinase II
Jacqueline Rose, Amanda Rooyakkers, Kevin She, Kimberly Harms, Kamal Sharma, Dan Fong, Shan-Xue Jin, collaboration with lab of Dr. Ana Luisa Carvalho
We are studying cellular models of learning and memory, of how neuronal activity regulates synapse stability, molecular composition, and functional efficacy. We found that long term local blockade of transmitter release at a subset of synapses onto a given postsynaptic neuron results in a local and selective reduction in GluR1 AMPA type glutamate receptor content. To follow more rapid dynamic changes, we are using live cell imaging of glutamate receptors and other signaling molecules including calcium-calmodulin activated kinase CaMKII tagged with variants of green fluorescent protein. Previous studies have shown that activation of NMDA receptors results in accumulation of CaMKII at synaptic sites, altering subsequent biochemical signaling. Using FRAP (fluorescence recovery after photobleaching), we found that the mobility of spine pools of CaMKII, but not NMDAR1 or PSD-95, is reduced following chemical long-term potentiation, a cellular model of memory. In current studies, we are characterizing a novel mode of trafficking of CaMKII within neurons.
A second major question we are currently addressing is the mechanism of synaptic accumulation and activity regulated trafficking of NMDA receptors. Engineered subunits of the NDMA receptor complex are being reintroduced into neuron cultures from knockout mice, allowing us to dissect molecular signals involved in NMDA receptor trafficking.
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GFP-CaMKII shows rapid and extensive mobility into bleached spines (red arrow) in control neurons, but becomes more clustered and less mobile in spines following induction of chemical long-term potentiation, a cellular model of memory. (Sharma et al., 2006)
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GABA Synapses: New Approaches for Live Cell Imaging and Proteomics
Robert Cassidy, YunHee Kang, Frederick Dobie, collaboration with lab of Dr. Rachel Wong
Compared with excitatory glutamatergic synapses, much less is known about the dynamics and molecular composition of inhibitory GABAergic synapses. To overcome limitations with antibody-based approaches and to allow for live imaging of synaptic GABA receptors, we generated transgenic mice expressing tagged GABA receptor subunits. The YFP (enhanced yellow fluorescent protein) tag was used to allow live cell imaging, and additional tags were added to allow for biochemical purification. Tagged subunits were expressed under control of the neuron-specific Thy1 promoter. In the resultant transgenic mouse lines, YFP is detected in puncta, colocalizing quite well with gephyrin immunoreactivity and opposite terminals labeled for GAD (glutamic acid decarboxylase). We are pursuing proteomics and imaging studies of GABAergic synapses using these transgenic mice. To visualize GABA synapses developing in neuron culture over many days, we also have set up a dedicated Nikon Perfect Focus System microscope with Okolabs environmental stage chamber.
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Confocal image of hippocampal CA1 region from a transgenic mouse expressing
tagged GABA receptors (green, YFP-GABA receptor; red, gephyrin immunostaining;
blue, DAPI). (Cassidy, Kang, Lewis, Wong, Craig, unpublished)
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SELECTED PUBLICATIONS
Kang Y, Zhang X, Dobie F, Wu H, Craig AM. 2008. Induction of GABAergic postsynaptic differentiation by α-neurexins. J. Biol. Chem. 283:2323-34.
Laezza F, Gerber BR, Lou JY, Kozel M, Hartman H, Craig AM, Ornitz DM, Nerbonne JM. 2007. The FGF14F145S mutation disrupts the interaction of FGF14 with voltage-gated Na+ channels and impairs neuronal excitability. J. Neurosci. 27:12033-44.
Dobie F, Craig AM. 2007. A fight for neurotransmission: SCRAPPER trashes RIM. Cell 130:775-7.
Liu Y, Wong TP, Aarts M, Rooyakkers A, Liu L, Lai TW, Wu DC, Lu J, Tymianski M, Craig AM, Wang YT. 2007. NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo. J. Neurosci. 27:2846-57.
Craig AM, Kang Y. 2007: Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17:43-52.
Laezza F, Wilding TJ, Sequeira S, Coussen F, Zhang XZ, Hill-Robinson R, Mulle C, Huettner JE, Craig AM. 2007. KRIP6: A novel BTB/kelch protein regulating function of kainate receptors. Mol. Cell. Neurosci. 34:539-50.
Schlief ML, West T, Craig AM, Holtzman DM, Gitlin JD. 2006: Role of the Menkes copper transporting ATPase in NMDA receptor-mediated neuronal toxicity. Proc. Natl. Acad. Sci. USA 103:14919-24.
Kang Y, Craig AM. 2006: Composition and assembly of GABAergic postsynaptic specializations. In: Molecular Mechanisms of Synaptogenesis (A Dityatev and A El-Husseini, eds.) Springer, New York, pp. 277-95.
Graf ER, Kang Y, Hauner A, Craig AM. 2006: Structure-Function and Splice Site Analysis of the Synaptogenic Activity of the Neurexin-1β LNS Domain. J. Neurosci. 26:4256-65.
Sharma K, Fong DK, Craig AM. 2006. Postsynaptic protein mobility in dendritic spines: Long-term regulation by synaptic NMDA receptor activation. Mol. Cell. Neurosci. 31:702-12.
Craig AM, Graf ER, Linhoff MW. 2006. How to build a central synapse: Clues from cell culture. Trends Neurosci. 29:8-20.
Lou J, Laezza F, Gerber BR, Xiao M, Yamada KA, Hartmann H, Craig AM, Nerbonne JM, Ornitz DM. 2005. Fibroblast growth factor 14 is an intracellular modulator of voltage-gated sodium channels. J. Physiol. 569:179-93.
Harms KJ, Tovar KR, Craig AM. 2005. Synapse-specific regulation of AMPA receptor subunit composition by activity. J. Neurosci. 25:6379-88.
Harms KJ, Craig AM. 2005. Synapse composition and organization following chronic activity blockade in cultured hippocampal neurons. J. Comp. Neurol. 490:72-84.
Waites CL, Craig AM, Garner CC. 2005. Mechanisms of vertebrate synaptogenesis. Annu. Rev. Neurosci. 28:251-74.
Graf E, Zhang X, Jin SX, Linhoff M, Craig AM. 2004. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119:1013-26.
Schlief ML, Craig AM, Gitlin JD. 2004. NMDA receptor activation mediates copper homeostasis in hippocampal neurons. J Neurosci. 25:239-246.
Levi S, Logan SM, Tovar KR, Craig AM. 2004. Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons. J. Neurosci. 24:207-17.
Wang X, Weiner JA, Levi S, Craig AM, Bradley A, Sanes JR. 2002. Gamma protocadherins are required for survival of spinal interneurons. Neuron 36:843-54.
Levi S, Grady RM, Henry MD, Campbell KP, Sanes JR, Craig AM. 2002. Dystroglycan is selectively associated with inhibitory GABAergic synapses but is dispensable for their differentiation. J. Neurosci. 22:4274-85.
Fong DK, Rao A, Crump FT, Craig AM 2002. Rapid synaptic remodeling by protein kinase C: reciprocal translocation of NMDA receptors and calcium/calmodulin-dependent kinase II. J. Neurosci. 22:2153-64.
Boudin H, Craig AM. 2001. Molecular determinants for PICK1 synaptic aggregation and mGluR7 receptor coclustering: role of the PDZ, coiled-coil, and acidic domains. J. Biol. Chem. 276: 30270-76.
Crump FT, Dillman KS, Craig AM. 2001. cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors. J. Neurosci. 21:5079-88.
Craig AM, Boudin H. 2001. Molecular heterogeneity of central synapses: afferent and target regulation. Nat. Neurosci. 4:569-78.
Boudin H, Doan A, Xia J, Shigemoto R, Huganir RL, Worley P, Craig AM. 2000. Presynaptic clustering of mGluR7 requires the PICK1 PDZ domain binding site. Neuron 28:485-97.
Rao A, Cha EM, Craig AM. 2000. Mismatched appositions of presynaptic and postsynaptic elements in isolated hippocampal neurons. J. Neurosci. 20:8344-53.
Allison DW, Chervin AS, Gelfand VI, Craig AM. 2000. Postsynpatic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: maintenance of core components independent of actin filaments and microtubules. J. Neurosci. 20:4545-54.
Stowell JN, Craig AM. 1999. Axon / dendrite targeting of metabotropic glutamate receptors by their cytoplasmic carboxy terminal domains. Neuron 22:525-36.
Serpinskaya AS, Feng G, Sanes JR, Craig AM. 1999. Synapse formation by hippocampal neurons from agrin-deficient mice. Dev. Biol. 205:65-78.
Craig AM. 1998. Activity and synaptic receptor targeting: the long view. Neuron 21:459-62.
Allison DW, Spector I, Gelfand VI, Craig AM. 1998. Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: Differential attachment of NMDA versus AMPA receptors. J. Neurosci. 18:2423-36.
Rao A, Kim E, Sheng M, Craig AM. 1998. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J. Neurosci. 18:1217-29.
Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM, Sheng M. 1997. Competitive binding of α-actinin and calmodulin to the NMDA receptor. Nature 385:439-42.
Rao A, Craig AM. 1997. Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 19:801-812.
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