The function of PNNs has remained largely mysterious since their first description by Camillo Golgi over a century ago (Celio and Blumcke, 1994
; Hartig et al., 1999
). We have found that the internalization of Otx2 by a sub-population of cells in the neocortex (PV-cells) is facilitated by the recognition of one or several sugar sequences in the PNN, probably enriched in chondroitin-6-sulfate moieties (CS-D and/or CS-E). This recognition requires a short Otx2 domain with GAG-binding molecular characteristics, which can be antagonized in vivo
to reduce endogenous Otx2 internalization, down-regulate PV expression and WFA binding sites to reactivate plasticity in the binocular visual cortex (). We thus propose that PNNs provide Otx2 binding sites facilitating internalization. This unexpected PNN function does not rule out other functions related (or not) to cortical plasticity.
Homeodomain and homeoprotein internalization is not believed to be receptor-dependent and the third helix of the homeodomain (Penetratin), which is necessary and sufficient for internalization, does not bind a chiral receptor (Joliot and Prochiantz, 2004
). However, negative charges are important for homeoprotein internalization and the homeodomain alone shows a higher affinity for cells expressing specific sugars at their surfaces. In particular, Antennapedia is preferentially captured in vitro
by neurons expressing α2,8-polysialic acid (PSA) (Joliot et al., 1991b
). This absence of specificity in vitro
does not mean that extracellular homeoproteins do not recognize specific cells in vivo
and our present data strongly suggest that GAG sequences may participate in this “sugar code” for homeoprotein recognition.
In fact, most studies indicate that the efficiency of internalization of these transduction proteins and peptides is increased by their interaction with complex sugars (Ziegler, 2008
; Ziegler and Seelig, 2008
). Here, we suggest that this recognition is specific, reminiscent of the mode of action of several morphogens and growth factors (eg. FGF 1/2) that must bind a proteoglycan before they activate a transducing receptor (Jastrebova et al., 2006
). In some cases, the diffusion of a morphogen requires it to be attached to complex sugars until it actually binds a receptor (Bulow and Hobert, 2006
; Gallet et al., 2008
In our homeoprotein infusion experiments, Otx2 internalization by PV-cells results from an exchange of the protein between its carrier (PSA) and a binding site (the acceptor) at the cell surface. We demonstrate that an Otx2 domain of 15 amino acids participates in this recognition and that the replacement of an RK-doublet by an AA-doublet abolishes specificity. The importance of this short sequence is further supported by differences of in vitro affinities for specific sugars () and by their distinct physiological effects in vivo. Indeed, when infused into the cortex, the three peptides localize in a similar non-specific manner around the site of infusion. However, in contrast to RK-peptide, the AA and Scb peptides do not significantly change Otx2 accumulation inside PV-cells and have no effect on the expression of PV or that of PNNs. We also noticed that the degree of reduced Otx2 accumulation by the RK peptide could vary between experiments (from 40 to 80%) but was always higher than the reduction by ChABC (~20%). Reactivation of plasticity was observed regardless of this variation.
We therefore propose that the Otx2 RK domain enables binding to an “acceptor site” containing a chondroitin-6-sulfate. As GAG sulfation patterns determine most of their binding properties, the avidity of Otx2 for 6-sulfated CS is of particular interest. Indeed, in the rat posterior cortex, the amount of 6-sulfated GAG has been found to drop dramatically between P21 and adulthood (where they represent 3% of the GAG) (Carulli et al., 2011
). Moreover, very recent evidence suggests a developmental increase in the 4-sulfation/6-sulfation (4S/6S) ratio of CSPGs may be required for the accumulation of Otx2 (Miyata et al, 2012
). Thus, the recognition of Otx2 by PV-cells might, at least in part, stem from the progressive refinement of the PNN sulfation profile.
Although we do not exclude the possibility that a glycoprotein could transduce a specific signal across the membrane, our present understanding of homeoprotein action (Brunet et al., 2005
; Brunet et al., 2007
; Wizenmann et al., 2009
) favors the idea of a non-cell autonomous regulation of translation and transcription by Otx2 following its internalization. The actual targets at both levels are presently under investigation and, once identified, will be analyzed at a functional level. Notably, both gain- and loss-of-function (Sugiyama et al., 2008
) as well as this study indicate that Otx2 internalization enhances expression of several markers of PV-cell maturation, including PNN formation itself.
From a physiological point of view, the ongoing positive feedback of nascent PNNs attracting Otx2 for their own continued maintenance throughout life may serve to prevent plasticity in adulthood (). Importantly, PV-cells receive the most potent and direct input from thalamus (Erisir and Dreusicke, 2005
; Cruikshank et al., 2007
), and have themselves been shown to display a robust bidirectional plasticity that becomes limited with age (Yazaki-Sugiyama et al., 2009
). Our results provide a mechanistic explanation for the earlier success in reopening visual plasticity by diffuse treatment with chondroitinases in the adult rat (Pizzorusso et al., 2002
). Resetting V1b to a juvenile state by function-blocking Otx2 antibody (Sugiyama et al., 2008
) or the RK-peptide here offer a more precise therapeutic tool.
Otx2 may thus serve as a “master regulator” of visual cortex plasticity during development and throughout adulthood. How might CP induction and closure be linked? Our initial report showed that Otx2 not only opens a CP, but also leads to its premature closure (Sugiyama et al., 2008
). This suggests that a threshold level of Otx2 initiates plasticity, which is eventually exceeded to limit plasticity in adulthood. A similar two-threshold model is proposed to explain the direct effects of GABA maturation on CP timecourse (Feldman, 2000
; Spolidoro et al., 2009
). PV-cell development establishes the crucial excitatory-inhibitory balance conducive to plasticity (Hensch, 2005
), but then these cells acquire PNNs which terminate further rewiring by sustained Otx2 internalization (). Loss of Otx2 in adulthood then leads to transient loss of PNNs and a reactivation of plasticity until both Otx2 and PNN levels recover ().
Plasticity thus reflects a threshold amount of Otx2 within PV cells with therapeutic relevance for recovery from amblyopia (). It will be important to evaluate the Otx2 content of PV-cells in other paradigms (e.g. environmental conditions, fluoxetine) reported to support adult visual plasticity (Morishita and Hensch, 2008
; Spolidoro et al., 2009
; Bavelier et al., 2010
). As appropriate delivery methods are devised, RK-related agents may not only restore vision to human amblyopes but also potentially rescue other defects of CP brain development. Therapeutic strategies adjusting intercellular transfer of Otx2 into PV-circuits may, for instance, be adapted more generally to rescue neurodevelopmental disorders of similar cellular etiology, such as autism spectrum disorders, fear extinction or schizophrenia (Gogolla et al., 2009a
; Gogolla et al., 2009b
; Lewis et al., 2005