Research
Molecular Mechanisms of Synapse Development
The complex circuitry of the mammalian brain enables the execution of fundamental cognitive processes such as learning, speech, and memory. A given neuron, through its synaptic connections, either excites or inhibits other neurons in the circuit and the correct balance of excitation and inhibition must be established and maintained for proper circuit function. It is now widely believed that aberrant changes in the balance of excitation and inhibition can have pathological consequences for brain function as demonstrated by the manifestation of devastating neurological impairments, including epilepsy, mental retardation, and autism spectrum disorders. Further, these disorders are distressingly common as autism affects roughly 0.5% of children while epilepsy affects 1% of people worldwide.
To appreciate how synapse dysfunction contributes to these widespread neurological impairments, it is important to first understand how synapses are formed, maintained, and function in the non-pathological state. Information derived in part from live imaging of synapses while they are forming suggests that synapse formation is a progression of discrete processes. These include contact between an axon and a dendrite, recruitment of the appropriate components to the presynaptic active zone and the postsynaptic specialization, and activity-dependent synaptic refinement, i.e. a subset of synapses are eliminated while others are stabilized and maintained.
However, while biochemical and candidate gene approaches have led to the identification of a large number of molecules that function at synapses, the process of synapse formation itself remains poorly understood. Some of the outstanding questions in the field include: which proteins are required for excitatory and inhibitory synapse formation, and what is their mechanism of action? How does a neuron maintain the correct balance of excitatory and inhibitory synapses in order to function appropriately within a neural circuit? The goal of our research is to define the molecular program that underlies neural circuit formation and function by elucidating the mechanisms of excitatory and inhibitory synapse development.
To begin to address these questions, we developed a novel, forward genetic RNA interference (RNAi)-based screen in cultured hippocampal neurons that has identified new molecules required for synapse formation (Paradis et al., Neuron53: 217). In brief, we transfected cultured hippocampal neurons with short, interfering RNAs (siRNAs) targeting candidate synaptogenic molecules. We then assayed synapse density onto these neurons by immunostaining to quantify the density of glutamatergic, excitatory synapses (Figures 1, 2). In our initial screen of 160 genes, we identified four genes that are required in the postsynaptic neuron for the proper development of excitatory synapses: the activity-regulated GTPase Rem2, the adhesion molecules cadherin-11 and cadherin-13, and the transmembrane protein Semaphorin 4B (Sema4B).
Figure 1. Schematic of assay used to quantify synapse density. Because we are scoring the number of synapses formed onto the transfected neuron and not by the transfected neuron we are assaying the function of the candidate genes in the postsynaptic neuron. Note that approximately 10% of neurons in our cultures are transfected.
Figure 2. Assay for synapse density. Neurons are fixed and stained for markers of excitatory synapses after 14 days in vitro and then imaged on a confocal microscope using a 60X objective. The GFP signal marks transfected neurons, PSD-95 staining marks the postsynaptic density of excitatory synapses, Synapsin is a presynaptic marker common to all synapses. Synapse density is the number of co-localized PSD-95 and Synapsin puncta on a transfected neuron (white arrowhead in merged image) divided by the total area of the transfected cell.
Next, we evaluated the relevance of these genes for inhibitory synapse density using immunostaining and quantifying the density of GABAergic, inhibitory synapses. Remarkably, these four molecules are also required for inhibitory synapse formation, suggesting that common mechanisms mediate excitatory and inhibitory synapse formation. Quite unexpectedly, we also discovered that RNAi-mediated knockdown of another class 4 Semaphorin, Sema4D, in the postsynaptic neuron led to a decrease in the density of inhibitory synapses while having no apparent effect on excitatory synapse density. Thus, our experiments identify Sema4D as one of only a few molecules that preferentially regulate inhibitory synapse formation, which has important implications for the mechanisms underlying inhibitory synapse formation.
1) The role of Class 4 Semaphorin Signaling in Synapse Formation (Funding provided by the Richard and Susan Smith Family Foundation and by the March of Dimes):
The Semaphorin family of proteins is conserved from flies to humans and twenty mammalian homologs have been described. The defining characteristic of the family is the Sema domain, a conserved, cysteine-rich region of about 500 amino acids at the N-terminal of the protein (Yazdani and Terman, Genome Biol 7: 211). Semaphorins exist as transmembrane, GPI-linked, or secreted proteins. Semaphorin family members have been implicated in a variety of biological processes including immune system function, angiogenesis, cell migration, and cardiovascular development. At present, the Semaphorin family is perhaps best known by the function of the secreted, class 3 Semaphorin family members that act as repulsive molecules to guide axons to their targets in the developing nervous system. In contrast to the secreted class 3 Semaphorins, relatively little is known about the function of the transmembrane class 4 Semaphorins in the nervous system.
Our discovery that Sema4B and Sema4D regulate excitatory and/or inhibitory synapse formation suggests a previously underappreciated role for Semaphorin family members as important regulators at the synapse. To elucidate the role of class 4 Semaphorin signaling in synapse formation, we will define the intracellular signaling mechanisms that underlie the function of Sema4B and Sema4D in this process. These experiments will provide us with a unique opportunity to uncover the similarities and differences between the signal transduction pathways that mediate excitatory and inhibitory synapse formation.
Also, as Sema4B and Sema4D are transmembrane proteins, the possibility of both "forward" (Sema4 engagement by its receptor initiates a signal transduction cascade within the cell expressing the receptor) and "reverse" (Sema4 engagement by its receptor initiates a signal transduction cascade within the cell expressing Sema4) signaling exists. Thus, we will identify the pertinent receptors for Sema4B or Sema4D in the hippocampus and ask if these receptors are playing a role in synapse formation.
2) Cadherin family members in synapse development:
The idea that Cadherin family members could be involved in synapse formation is based on a number of observations. For example, there are over 100 family members with restricted expression patterns in the nervous system. And, functional studies from the Takeichi lab using a function blocking dominant negative N-Cadherin construct have implicated Cadherin family members in synapse formation and dendritic spine morphogenesis (Togashi et al., Neuron 35: 77). Despite these intriguing studies, much remains to be learned about how Cadherins regulate synapse development. A most pressing issue is determing which Cadherin family member regulates synapse formation in a particular neuronal subtype. None of the functional studies performed thus far have addressed this issue, and we believe that our RNAi screen technology has the potential the answer this question.
Of the 22 Cadherin family members that we assayed in our screen thus far, we have demonstrated a role for two Cadherin family members in synapse formation: Cadherin-11 and Cadherin-13. Interestingly, we found that over-expression of Cadherin-11, but not Cadherin-13, increases synapse number. This result is the first example of a Cadherin family member that is both necessary and sufficient for synapse formation and suggests for the first time that individual Cadherin family members are working through distinct mechanisms to mediate synaptic development. Current experiments aim to address at which step in synapse formation these Cadherins function and to understand the Cadherin-dependent signaling pathways that regulate synapse formation. To accomplish these goals, we are employing live, time-lapse imaging studies of synapses while they are forming in the presence or absence of Cadherins and a heterologous cell/neuron co-culture assay.
3) Gene discovery efforts (Funding provided by the National Institute on Drug Abuse):
It has been demonstrated that addiction to stimulants such as cocaine causes changes in neuronal structure and synapse density (Robinson and Kolb, 2004). Thus, a current hypothesis to explain the persistent features of drug addiction, including drug cravings and relapse, posits that changes in synaptic structure and neuronal connectivity underlie these features of the disease (Chao and Nestler, 2004; Robinson and Kolb, 2004). To understand how aberrant synapse number or function contributes to the symptoms of drug addiction, it is important to first understand how synapses are formed, maintained, and function in the non-pathological state. Protein kinases are excellent candidates to be mediators of synapse formation. Thus far, a number of kinases have been implicated in regulating synapse formation or function, lending support to the hypothesis that protein kinases have critical functions at the synapse. In addition, it has been demonstrated that protein kinases are activated in response to administration of cocaine, and it is hypothesized that this activation leads to changes in neuronal structure and synapses in response to drug exposure (Russo et al., 2008) (Norrholm et al., 2003). To test this hypothesis, further investigation into the role of protein kinases in synapse formation is warranted. To this end, we propose to take a forward genetic, RNA-interference (RNAi)-based approach to decrease expression of protein kinases in cultured mammalian neurons and assay the effect on synapse formation.