Doctor of Philosophy in Neuroscience (PhD)
The proper functioning of the brain and central nervous system (CNS) requires the precise formation of synapses between neurons. The two main neurotransmitter systems for fast synaptic communication in the CNS are excitatory glutamate and inhibitory gamma-aminobutyric acid. A growing body of evidence has begun to uncover several shared and divergent rules for the establishment of each of these two types of synapses.At the molecular level, a number of key proteins have been shown to be involved in the initial formation and subsequent development of synaptic connection, including cell adhesion molecules (CAMs). Among the CAMs, neurexins and neuroligins are important synaptogenic proteins that act trans-synaptically to organize synapses: binding of axonal beta-neurexins by neuroligins is sufficient to cause development of a presynaptic specialization at that site, while binding of dendritic neuroligin-1 or neuroligin-2 by beta-neurexins is sufficient to cause development of postsynaptic excitatory or inhibitory specializations, respectively. In Chapter 2, we explore the role of alpha-neurexins in synapse organization. We find alpha-neurexins are able to specifically induce the formation of inhibitory synapses, presumably through clustering of postsynaptic neuroligin-2. Moreover, we find that the expression of various splice variants of alpha- and beta-neurexins is regulated both during development and by activity, suggesting a physiological role for alternative splicing in the modulation of synapse assembly.At the cellular level, it is now clear from live imaging studies that synapses and their formation are highly dynamic processes. A number of studies have established the temporal recruitment of pre- and postsynaptic components to nascent synapses and how synapse formation can influence neuron growth. However, these studies have focused on excitatory synapses. In Chapter 3, we explore the cellular mechanisms of inhibitory synapse formation and modulation. We find that entire synapses are highly mobile and can undergo dynamic structural modulation. New synapses are formed by gradual accumulation of components from diffuse cytoplasmic pools, with a significant contribution of presynaptic vesicles from previously recycling sites. These results provide new insights into the mechanisms of inhibitory synapse formation and how it is both similar and different from excitatory synapse formation.
Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system and is typically studied in the visual system in vivo where individual synapses are difficult to visualize. Here, we developed a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. GluN1 -/- (KO) mouse hippocampal neurons were cultured alone or in defined ratios with wild type (WT) neurons. Synapse development was assessed by immunofluorescence for PSD-95 apposed to VGlut1. Synapse density was specifically enhanced only onto minority WT neurons co-cultured with majority KO neighbour neurons and this increased synapse density was dependent on activity through NMDA receptors. This enhanced synaptic density onto NMDA receptor-competent neurons in minority co-culture represents a cell culture paradigm for studying synaptic competition. Trafficking of NMDA receptors to the cell surface is critical for proper brain function. Recent evidence suggest that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. We show that glutamate binding is required for trafficking of NMDA receptors to the cell surface by expressing a panel of GluN2B ligand binding mutants in heterologous cells and primary rodent neurons and found that glutamate efficacy correlates with surface expression. Such a correlation was found even with inhibition of endocytosis indicating differences in forward trafficking. These results indicate that ligand binding is critical for receptor trafficking to the cell surface. NMDA receptors mediate many forms of synaptic plasticity. GluN2B is proposed to bind and recruit CaMKII to synapses to mediate multiple forms of synaptic plasticity. We find that accumulation of CFP-CaMKIIα at synapses is induced in wild-type but not in KO neurons by bath stimulation of NMDA receptors or by a chemical long-term potentiation protocol. Stimulated synaptic accumulation of CFP-CaMKIIα was rescued in KO neurons by YFP-GluN2B or chimeric GluN2A/2B tail but not by GluN2A, chimeric GluN2B/2A tail, or GluN2B with point mutations in the CaMKII binding site. Thus, activity-regulated synaptic aggregation of CaMKII is dependent on the cytoplasmic CaMKII binding site of GluN2B and not on differential permeation properties between GluN2B and GluN2A.
The brain consists of billions of neurons. During development, these neuronsmust migrate to their proper position and form connections with neighboring neuronsto form networks. The specificity and maturation of these connections, or synapses,are critical for proper brain function, including learning, memory and cognition. Manycell adhesion molecules (CAMs) are involved in the formation and maturation ofsynapses, including the well-characterized neuroligin-neurexin pair. In this study, twonew synapse modifying proteins, calsyntenin and MDGA, are characterized using invitro assays and primary hippocampal neuron cultures. Calsyntenin-3 was identifiedin an un-biased screen to search for new synaptogenic proteins. It is a post-synaptictransmembrane protein that induces the formation of excitatory and inhibitorypresynaptic specializations in contacting axons via extracellular cadherin and LNSdomains. Overexpression of calsyntenin-3 in neurons increases presynaptic proteinclustering. Interestingly, calsyntenin-3 binds to α-neurexins with high affinity,suggesting presynaptic induction is mediated through trans-synaptic signaling withneurexins. MDGAs are a family of synaptic GPI-linked proteins that bind neuroligin-2with high affinity. MDGA1 blocks the presynaptic induction activity of neuroligin-2,through blocking binding to neurexins, via extracellular immunoglobulin domains.Overexpression of MDGA1 in neurons specifically decreases inhibitory synapses,while knockdown increases inhibitory synapses. Interestingly, like other synapticproteins including neurexin and neuroligin, MDGAs have recently been linked toautism spectrum disorders and schizophrenia. Thus, the characterization of thesynapse-promoting calsyntenin-3 and the synapse-reducing MDGA1 shed new light on the mechanisms by which synaptogenesis is regulated. Investigating the complexinterplay between molecular players during synaptogenesis is critical not only forunderstanding normal brain development, but also for providing insight intoneurodevelopmental disorders.
The leucine rich repeat transmembrane neuronal (LRRTM) proteins are a family of four synaptogenic cell adhesion molecules that instruct excitatory presynaptic differentiation and mediate postsynaptic differentiation. LRRTM1 and LRRTM2 are most potent at inducing presynaptic differentiation and have been shown to interact with neurexins at glutamatergic synapses. LRRTM4 has been recently identified as a major component of native AMPA-type glutamate receptor complexes, and is expressed at very high levels in dentate gyrus granule cells. Similar to neurexins, neuroligins, and several other synapse organizing proteins, LRRTMs are linked to psychiatric disorders such as autism spectrum disorders. LRRTM4 is also linked to risk of attempted suicide in females based on a recent genome-wide association study of over 2500 patients with bipolar disorder. Our project on LRRTM1 and LRRTM2 involved determining the role of these proteins in synapse development and function using LRRTM1 and 2 double knockout mice. Our results indicated that LRRTM1 and 2 are essential for normal excitatory synapse development and function in CA1 region but not the dentate gyrus. Our project on LRRTM4 assessed the role of this protein in synapse development. Using targeted deletion in mice, our results revealed that LRRTM4 is essential for normal excitatory synapse development and function in dentate gyrus granule cells but not in CA1 hippocampal pyramidal neurons. In addition, it was shown that LRRTM4 differentiates from LRRTM1 and 2 in terms of binding partners as it binds heparan sulfate proteoglycans (HSPGs). Experiments indicated that HSPGs are essential to mediate the synaptogenic activity of LRRTM4. Overall, our results from LRRTM1 and 2 and LRRTM4 projects indicate that members of the LRRTM family function in a cell-type specific manner through different presynaptic molecular pathways. This emphasizes the complexity of synapse-organizing protein networks and the importance of studying region specific roles of these synaptic proteins.