Role of the transcription factor NeuroD2 in synaptic integration in the postnatal brain – SynD2

Neurogenesis persists lifelong in two regions of the mammalian brain: the hippocampal dentate gyrus and the olfactory bulb. In the latter region different types of neurons are continuously generated, from neural stem cells located a long distance away, in the periventricuar zone (PVZ). New bulbar neurons are interesting candidates for future cell replacement therapies in brain damage and disease. Furthermore, bulbar neurogenesis is a convenient model to study endogenous mechanisms that may be used to circumvent problems with current transplantation strategies. While the molecular mechanisms governing precursor cell proliferation, migration and neuronal differentiation start being elucidated, the ones that regulate synaptic integration into the preexisting circuitry remain largely unknown. In the present project, we will study the mechanisms of synaptic integration in the postnatal brain, using a molecular entry point: the transcription factor NeuroD2. This gene was isolated from a genetic screen in the host lab and our preliminary data indicate a major function in synaptogenesis. First, a genetic screen showed that NeuroD2 transcript is enriched in neurons that are synaptically integrating into the bulb. Second, electroporating NeuroD2 shRNAs or grafting NeuroD2 null neural stem cells in the PVZ induced bulbar neurons with less synapses. Third and finally, OB size was decreased while global OB apoptosis was increased in NeuroD2 null mice, a sign of reduced neuronal integration. All these data indicate a function of NeuroD2 in postnatal synaptogenesis.
Our project is divided into three parts:

1. Determining the function of NeuroD2 in bulbar synaptogenesis in living postnatal mice
We will determine if NeuroD2 regulates formation, elimination and/or stabilization of synapses. In order to dissociate between these events, we will analyze dynamic changes in spines of new OB neurons after down-regulation or over-expression of NeuroD2 using two-photon imaging in anesthetized mice. Second, we will use patch clamp electrophysiological recordings on OB slices to determine the functional impact of NeuroD2 at the synapse. And third, we will measure calcium dynamics in single spines of anaesthetized mice to confirm electrophysiological recordings.

2. Does NeuroD2 mediate activity-dependent synaptogenesis in the postnatal OB?
Sensory experience influences neuronal network formation by selecting activated synapses during brain development. This activity-dependent synapse selection also exists in the adult OB where olfactory enrichment and deprivation are thought to increase and decrease synaptogenesis, respectively. We will study whether NeuroD2 is functionally involved in this phenomenon. Therefore, we will determine whether blocking NeuroD2 expression alters activity-dependent synaptic changes. Furthermore, we will establish by which way neuronal activity modulates NeuroD2 by measuring both its phosphorylation status and its transcription before and after induction of neural activity.

3. Determining if NeuroD2 regulates synaptogenesis in excitatory neocortical neurons and measure autism-related behavior in NeuroD2 null mice
Since NeuroD2 is expressed in neocortical excitatory neurons from development to adulthood, we will test whether NeuroD2 regulates synapse formation / stability also in those neurons. If yes we will perform a battery of autism-related behavioral tests on NeuroD2 null mice because autism is correlated to synapse alterations in the neocortex and because linkage analyses indicate that NeuroD2 is located at a linkage peak for autism patients.