Exploring the genetic basis of neuronal connectivity
Research in the Paradis laboratory seeks to bridge the gap between molecular and systems neuroscience by defining the genes that instruct neurons to establish and modify their synaptic connections and dendritic morphology. Neurons convey information by making synaptic connections onto other neurons, and receive information via synaptic inputs onto their dendritic arbor. To understand how appropriate information flow occurs within neuronal networks, it is first necessary to define the genes and signaling pathways that instruct specific connections between neurons. To this end, research in the Paradis lab is roughly organized around three main questions:
1. Molecular mechanisms of inhibitory synapse formation
Synapses are specialized sites of cell-cell contact that mediate communication between neurons in the nervous system. There are two main types of synaptic connections in the mammalian brain: excitatory glutamatergic synapses and inhibitory GABAergic synapses. The balance between the excitatory and inhibitory inputs a neuron receives regulates the overall activity of neuronal networks. Intensive investigation by a large number of labs over the past thirty years has led to the identification of molecules that are localized to and function at glutamatergic synapses in processes such as presynaptic neurotransmitter release. In addition, information derived in large part from live imaging of glutamatergic synapses while they are forming suggests that synapse formation is a progression of discrete processes (McAllister, 2007; Ziv and Garner, 2001). 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. In contrast, far less is known about inhibitory, GABAergic synapse formation.
We discovered a previously unknown role for the class 4 Semaphorin Sema4D as an important regulator of GABAergic synapse formation while having no apparent effect on glutamatergic synapse formation (Fig.1) (Kuzirian et al., 2013; Paradis et al., 2007; Raissi et al., 2013). Semaphorins are a large family of secreted and transmembane glycoproteins that were first described as repulsive axon guidance molecules in the developing nervous system over twenty years ago (Zhou et al., 2008). Subsequently, Semaphorin function has been implicated in a variety of developmental processes both within and outside of the nervous system including: retinal lamination, neuronal migration, synapse formation, dendrite morphogenesis, lymphocyte specification and signaling, cell migration, and vascular and heart morphogenesis (Kruger et al., 2005; Matsuoka et al., 2011; Tran et al., 2007; Yazdani and Terman, 2006). In fact, prior to our discovery that Sema4D is required for GABAergic synapse formation, there was little appreciation of a synaptogenic function for Semaphorins (notable exceptions being Godenschwege, Murphey (Godenschwege et al., 2002) in Drosophila and Sahay, Kolodkin (Sahay et al., 2005) in rodents). Since our first report in 2007, there is a growing appreciation in the field that multiple Semaphorins and their receptors are critical mediators of synaptogenesis (Ding et al., 2012; Kuzirian et al., 2013; O'Connor et al., 2009; Raissi et al., 2013; Tran et al., 2009). Interestingly, aside from our work, these other studies exclusively pertain to glutamatergic synapse formation.
We demonstrated that addition of a soluble domain of Sema4D is sufficient to initiate GABAergic synapse formation within 30 minutes and further, that these newly formed synapses appear functional within 2 hours of Sema4D addition (Fig.1) (Kuzirian et al., 2013). This effect is entirely dependent on the presence of PlexinB1 (Kuzirian et al., 2013), a high-affinity receptor for Sema4D (Tamagnone et al., 1999). Our discovery represents the first description of a ligand-receptor pair that bi-directionally regulates GABAergic synapse formation on a rapid time-scale. As very few studies have addressed the nature of events involved in constructing a GABAergic synapse, we sought to gain insight into the dynamics of GABAergic synapse assembly in response to Sema4D by time-lapse imaging of GABAergic synapses while they are forming in real time. Time-lapse imaging of GFP-Gephyrin puncta revealed that Sema4D drives the addition of new postsynaptic assemblies of Gephyrin scaffolding proteins by splitting pre-existing assemblies of these proteins within 10 minutes of Sema4D treatment (Kuzirian et al., 2013).
Our results lead us to hypothesize that GABAergic synaptic components are poised and ready to quickly respond to reception of a pro-synaptogenic signal. Previous live imaging studies of GABAergic synapse formation (Dobie and Craig, 2011) described a gradual accumulation of synaptic components over the course of a few hours. In fact, splitting of existing GFP-Gephyrin puncta had been observed during ongoing GABAergic synapse development; however images were only acquired every 30 minutes and are thus not comparable to our study (Dobie and Craig, 2011) . Our data suggest that addition of Sema4D triggers the action of the native GABAergic synapse formation machinery, consistent with loss of function studies that demonstrate a requirement for Sema4D for proper development of GABAergic synapses (Paradis et al., 2007) . We are currently working to ascertain if the rapidity of this effect is unique to Sema4D signaling, or if other GABAergic synaptogenic factors (Fazzari et al., 2010; Terauchi et al., 2010) have a previously unappreciated capacity for driving GABAergic synapse formation on a similar time scale. This is a crucial question because the answer promises to yield important insight into common mechanisms of GABAergic synapse assembly. Further, the ability of GABAergic synapses to rapidly form in response to a specific signal implies a novel mechanism by which the neuronal circuits can quickly alter their excitatory/inhibitory balance, perhaps in response to a pathological stimulus such as a seizure.
Given the rapid nature of the effect, we wondered if the action of Sema4D could be used to drive inhibition in the context of runaway neuronal excitation, such as that observed during seizure activity in mammalian brains. To address this question, we used organotypic hippocampal slice culture as an in vitro model of epileptiform activity to demonstrate that acute Sema4D treatment rapidly (within 2 hours) and dramatically alters the hyperexcitability found in these slices in a manner consistent with a Sema4D-mediated increase in network inhibition (Fig. 2). This finding suggests a possible use of Sema4D, as well as other molecules that instruct formation of GABAergic synapses, as a disease-modifying treatment for epilepsy and other seizure disorders. In fact, we have filed a patent application (Provisional US Patent Application No. 61/756,809) based on this idea. Our idea is simple, untested, and has high impact potential: on command, we instruct neurons to assemble more inhibitory synapses in the brain, thus permanently rewiring circuits and ameliorating the symptoms of disorders such as epilepsy. We have begun an exploration of this hypothesis by establishing a number of models of seizure disorders. We will test the hypothesis that Sema4D treatment prior to or during a seizure protocol will delay acquisition of epileptic activity.
2. Activity-regulated gene expression and circuit function
Surprisingly little is known about the molecular mechanisms by which changes in neuronal activity are translated into changes in neuronal morphology and synaptic connections. The relative ease with which the visual experience of animals can be altered in the laboratory renders the visual system an excellent choice with which to study this problem. For example, as our in vitro studies demonstrated that the activity-dependent GTPase Rem2 negatively regulates dendritic complexity in cultured cortical neurons (Ghiretti et al., 2014), we chose to use the Xenopus laevis tadpole visual system to examine Rem2 function in vivo. The X. laevis visual system is a rich experimental paradigm with which to probe the function of activity-dependent genes such as Rem2, as dendritic complexity of optic tectum neurons in tadpoles exposed to a visual stimulus is enhanced in an activity-dependent manner (Fig. 3) (Sin et al., 2002). This paradigm also allows us to distinguish between the function of a gene that is constitutively expressed versus the function of a gene that is expressed specifically in response to increased neuronal activity, because we can co-manipulate gene expression and sensory experience. Using this approach, we identified Rem2 as a critical regulator of activity-dependent dendritic complexity, as dialing Rem2 expression up or down, in the context of increased sensory experience, decreases or increases dendritic complexity, respectively (Fig. 4) (Ghiretti et al., 2013; Ghiretti et al., 2014) .
When we began our work, a number of studies had demonstrated that increased neuronal activity promotes the growth of the dendritic arbor (Redmond et al., 2002; Sin et al., 2002) . Accordingly, a handful of molecules that are themselves activity-regulated and function to promote dendritic arborization were identified (e.g. NeuroD, CREST, BDNF, CPG15, and certain CaMK family members) (Leslie and Nedivi, 2011; Wayman et al., 2008) , with there being little appreciation that neuronal activity also turns on signal transduction pathways that function to restrict dendritic complexity.
In contrast, only a few studies have explored the potential for growth-restricting effects of neuronal depolarization. For example, expression of a constitutively active form of the Rho GTPase blocks the light-induced increase in dendritic complexity observed in the X. laevis tectal system (Sin et al., 2002). Further, expression of constitutively active CaMKII in X. laevis tectal neurons restricts dendritic growth (Wu and Cline, 1998) . Lastly, knockout of the NR2B subunit of the NMDA receptor in the CNS increases dendritic arborization in certain neuronal subtypes (Espinosa et al., 2009) . These results imply the existence of signaling pathways, such as the one defined by Rem2 in our lab, that are directly responsive to sensory experience yet function to inhibit dendritic complexity. Thus, our studies of Rem2 provide molecular evidence that integration of physiologically relevant, activity-dependent positive and negative signals instructs dendritic complexity in response to sensory experience.
Rem2 is a member of the Rad/Rem/Rem2/Gem/Kir (RGK) protein family, a Ras-related subfamily of small GTPases, and is highly expressed in the CNS. Previous to our work, the primary function of Rem2 and RGK family members was thought to be inhibition of voltage gated calcium channel function and regulation of actin cytoskeleton rearrangements (Correll et al., 2008) . However, these conclusions were based almost exclusively on overexpression studies of RGK family members in heterologous cell types. Thus, despite the fact that Rem2 was known to be well-expressed in the nervous system, before our work a comprehensive analysis of Rem2 function in neurons had never been conducted. We established verifiable functions of Rem2 using loss of function approaches in hippocampus and cortex and X. laevis optic tectum: Rem2 is a positive regulator of synapse formation and a negative regulator of dendritic complexity (Ghiretti et al., 2013; Ghiretti et al., 2014; Ghiretti and Paradis, 2011; Moore et al., 2013) . In addition, our loss of function studies failed to uncover a definitive role for Rem2 in mediating calcium channel function (Moore et al., 2013), leading us to hypothesize that this may not be a relevant function of endogenous Rem2.
Finally, we demonstrated that Rem2 is regulated by neuronal activity via multiple, distinct mechanisms (Fig. 5). First, calcium entry into neurons in response to neuronal depolarization rapidly increases Rem2 mRNA expression (Ghiretti et al., 2014). Second, calcium activation of CaMK signaling pathways regulates Rem2 function (Ghiretti et al., 2013) . We hypothesize that these different layers of activity-dependent regulation of Rem2 function provide the neuron with the ability to execute changes in synapses and dendrite morphology on discreet time scales.
Our molecular insight into the activity-dependence of Rem2 function presents an unparalleled opportunity to test hypotheses regarding experience-dependence structural and functional plasticity in an intact nervous system. One example of an experimental approach that we are taking to address this question is to investigate the role of Rem2 in experience-dependent plasticity (i.e. ocular dominance plasticity) in visual cortex. These experiments are performed in close collaboration with the lab of Dr. Steve Van Hooser at Brandeis.
3. The role of TDP-43 in neurodegenerative disease
The RNA-binding protein TDP-43 plays a central role in both familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 regulates the splicing, mRNA transport, and stability of several thousand mRNAs, many of which encode proteins that play important roles in neuronal arbor elaboration and synaptic function. However, the specific misregulated TDP-43 targets and the resulting effects on neuronal morphology and function that lead to neurodegeneration remain unclear. To gain insight into ALS and FTD disease mechanisms,we use primary cortical neuronal cultures as a model to study TDP-43 dependent phenotypic changes that occur prior to neurodegeneration. Cortical neurons are relevant to both ALS and FTD since the motor cortex is affected in ALS and the frontal and temporal cortical regions of the brain atrophy in FTD. The overall goal of this line of research is to elucidate cellular and molecular mechanisms by which dysfunction of the ALS gene TDP-43 contributes to disease pathogenesis and in so doing, create new avenues of investigation for the development of therapeutics. These experiments are performed in close collaboration with the lab of Dr. Avital Rodal at Brandeis.