Homeoprotein transduction, critical periods and cortex plasticity – BrainEver

We have genetically down-regulated Otx2 expression in its main source and blocked its internalization by PV neurons using pharmacological tools. We are using biochemical approaches to identify Otx2 binding sites expressed by its target neurons and the transduction pathways involved. Since the Otx2 protein regulates transcription and translation we are in the process of identifying which genes are regulated at the transcription and translation
levels.

We have identified the choroid plexus as an Otx2 source and demonstrated that invalidating Otx2 gene in this structure decreases the amount of the protein in PV neurons and reopens plasticity in the adult.
We have identified the molecular basis of the recognition of the PV neurons by Otx2. Otx2 recognizes complex sugars expressed by PV neurons.
We have identified the domain of Otx2 involved in the latter recognition and used it as an antagonist of Otx2 capture by PV neurons, thus leading to plasticity reopening We have synthesized a «receptor sugar« by chemical means and shown that it binds Otx2 and blocks it internalization by PV neurons in vivo.

These results open the way to the development of pharmacological strategies to reopen cerebral cortex plasticity in the adult.

3 articles associated to this grant have already been published. The most important one (Spatazza et al. Choroid-Plexus Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity. Cell Reports. 2013. 3, 1815-1823.) demonstrates that Otx2 protein comes from the choroid plexus and that blocking its synthesis in this structure reopens plasticity in the visual cortex and allows one to cure adult amblyopic mice. Most importantly, the protein irrigates the entire cerebral cortex and may thus be a regulator of plasticity for several brain functions.

The main goal of this proposal is to study the role of Otx2, a homeoprotein (HP) transcription factor, in the regulation of the critical period for binocular vision in the mouse. We have recently published, in collaboration with the group of Takao Hensch, that the internalization of Otx2 by parvalbumin neurons (PV cells) in the mouse binocular visual cortex is necessary and sufficient to open the critical period (CP) for binocular vision at post-natal day 20 (P20) and to close it 20 days later (Sugiyama et al., Cell 134:508-520, 2008). The ins and outs of this observation will be developed below, but it is clear that many issues are raised by this first paper. A first question is the origin of the Otx2 imported by PV cells (thus not synthesized by them). Several possible routes will be investigated, including the visual pathway (from eye to thalamus to cortex), the pineal gland and the choroid plexus. A second question is the observed specificity of Otx2 internalization by PV cells. This specificity suggests the existence of binding sites at the PV cell surface and preliminary experiments indicate that complex sugars (GAGs) participate in this recognition. Part of the project will be devoted to the identification the GAGs involved in Otx2 recognition. Once internalized, Otx2 regulates PV cell maturation and physiology through the regulation of gene expression. This regulation can take place at the transcription level, but also possibly at the translation level. We shall identify these targets and analyze the function of some of them in CP regulation. In addition it is known that chromatin structure is modified during CP and we shall investigate if Otx2 plays a role also at this level. Most important is the observation made by several groups that in the adult, well after CP closure, plasticity can be reopened. Because Otx2 is internalized by PV cells throughout adulthood it is possible that interfering with this internalization or modulating the levels of expression of Otx2 in vivo that may allow plasticity reopening. Therefore, part of this project will be devoted to developing models for interfering with Otx2 transduction in the adult may lead to the development of novel strategies to regulate plasticity in the adult cortex. These studies will involve cell biological, biochemical, molecular and genetic approaches and should lead to a better understanding of visual cortex plasticity. However because Otx2 is found in most PV cells of the cortex, it is not impossible that the “visual cortex proof of concept” may have much larger consequences in term of understanding some aspects of brain plasticity during development and in the adult.