Abstract Homer scaffolding proteins are important regulators of glutamatergic synapses that organize metabotropic glutamate receptor (mGluRs 1&5) signaling domains near the post-synaptic density (PSD) where they can induce short and long term synaptic inhibition. Neural activity and other stimuli induce a shift from the constitutively expressed, long Homer1b and 1c isoforms to the immediate early (IEG) Homer1a splice variant. Because Homer1a can interact with binding partners but cannot multimerize to promote scaffolding, it disrupts Homer scaffolds and disperses mGluR1 and 5 from the PSD. Thus, Homer1a acts as a natural dominant negative. Indeed, expression Homer1a can uncouple mGluR1 & 5 from postsynaptic processes such as short and long term depression while protecting mGluR coupling to non-synaptic effectors. By elevating Homer1a expression, cells profoundly change mGluR-effector coupling, acting as a molecular switch for mGluR signaling. In this way, regulation of Homer1 splicing is an important means of altering synaptic efficacy. To date however, much remains to be learned about the regulation and expression of Homer1a, including whether Homer1 mRNA splicing and translation occurs locally, near the post-synapse as is the case for many other synaptic proteins. Further, the mechanisms leading to Homer1a splicing are only poorly understood. These questions are relevant not only to the pathophysiological instance in disorders such as epilepsy, but also to the normal physiological situation in which Homer1a is more modestly up, and down regulated in response to more subtle changes in neural firing. To this end, we will generate a mouse using CRISPR in which the endogenous Homer1a protein will be C-terminally tagged with a green fluorescent protein (GFP) and the long isoforms with a red fluorophore (mScarlet-H) so that their expression levels, time course, and sub-cellular distribution can be analyzed following insult and pharmacological manipulation. These questions and others will benefit from the animal model we will generate from this proposal. To achieve this, we will pursue the following Specific Aims: Aim 1: To optimize and test the differentially tagged long and short Homer proteins in mouse embryonic stem cells. Aim 2: To generate a mouse in which the endogenous short and long Homer proteins are differentially tagged with green (GFP) and red (mScarlet-H) florescent proteins.

Project Narrative The proposed project will generate new tools to study alternative splicing and expression of Homer1a, a dominant negative post-synaptic scaffolding molecule with important functions in synaptic regulation. One splice variant, called Homer1a, exhibits regulated expression and disrupts Homer scaffolds as a dominant negative variant. We will generate a transgenic mouse in which Homer1a is tagged with a green fluorescent protein, and the long scaffolding forms of Homer encoded by the same gene will be tagged with a red fluorophore to allow for tracking of the time course, sub cellular distribution, and the ratio of the two isoforms under various conditions.