Specific transfer of Otx2 homeoprotein into GABAergic interneurons expressing parvalbumin (PV) is necessary and sufficient to open, then close, a critical period (CP) of plasticity in the developing mouse visual cortex. The accumulation of endogenous Otx2 in PV-cells suggests the presence of specific Otx2 binding sites. Here, we find that perineuronal nets (PNNs) on the surface of PV-cells permit the specific, constitutive capture of Otx2. We identify a 15 amino-acid domain containing an arginine-lysine doublet (RK-peptide) within Otx2, bearing prototypic traits of a glycosaminoglycan (GAG) binding sequence that mediates Otx2 binding to PNNs and specifically Chondroitin sulfate D and E with high affinity. Accordingly, PNN hydrolysis by Chondroitinase ABC reduces the amount of endogenous Otx2 in PV-cells. Direct infusion of RK-peptide similarly disrupts endogenous Otx2 localization to PV-cells, reduces PV and PNN expression and reopens plasticity in adult mice. The closure of one eye during this transient window reduces cortical acuity and is specific to the RK motif, as an AA variant or scrambled peptide fail to reactivate plasticity. Conversely, this transient reopening of plasticity in the adult restores binocular vision in amblyopic mice. Thus, one function of PNNs is to facilitate the persistent internalization of Otx2 by PV-cells to maintain CP closure. The pharmacological use of the Otx2 GAG-binding domain offers a novel, potent therapeutic tool with which to restore cortical plasticity in the mature brain.

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of ␥-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser 940) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser 940 is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser 940 increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser 940 increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition. Cation-chloride cotransporters (CCC)3 regulate Cl Ϫ homeostasis in cells and the generation of transmembrane chloride gradients (1). Adult mammalian neurons maintain low intracellular Cl Ϫ concentrations, which arise principally from the activity of the potassium chloride cotransporter-2 (KCC2). The maintenance of such low levels of intracellular Cl Ϫ ions is responsible for hyperpolarizing Cl Ϫ currents upon activation of GABA A and Gly receptors, which are responsible for fast synaptic inhibition in the adult central nervous system (2-5). Molecular studies have demonstrated that KCC2 is a member of a CCC superfamily and that these transporters are composed of 12-transmembrane domains with N-and C-terminal cytoplasmic domains (2, 6, 7). KCC2 is expressed exclusively in neurons throughout the adult brain. Developmentally KCC2 is first detected around 10 days in vitro in cultured rat neurons, which is coincident with the emergence of hyperpolarizing GABA A receptor-mediated Cl Ϫ currents (4, 8). Gene knock-out of KCC2 has revealed that ablating the expression of this protein results in early postnatal death. Neurons derived from these animals exhibit compromised GABA A receptor-mediated synaptic inhibition (9).Under pathological conditions such as epilepsy or ischemic brain injury, deficits in the expression of KCC2 are evident together with decreased efficacy of GABAergic inhibition and with the emergence of depola...

KCC2 is a neuron-specific K+-Cl− cotransporter that maintains a low intracellular Cl− concentration essential for hyperpolarizing inhibition mediated by GABAA receptors. Deficits in KCC2 activity occur in disease states associated with pathophysiological glutamate release. However, the mechanisms by which elevated glutamate levels alter KCC2 function are unknown. The phosphorylation of KCC2 residue S940 is known to regulate its surface activity. Here we demonstrated in dissociated rat neurons that NMDA receptor activity and Ca2+ influx caused the dephosphorylation of S940 leading to a loss of KCC2 function that lasted greater than 20 minutes. PP1 mediated the dephosphorylation events of S940 that coincided with a deficit in hyperpolarizing GABAergic inhibition due to the loss of KCC2 activity. Blocking dephosphorylation of S940 reduced the glutamate-induced downregulation of KCC2 and significantly improved the maintenance of hyperpolarizing GABAergic inhibition. Reducing the downregulation of KCC2 thus has therapeutic potential in the treatment of neurological disorders.

Summary Brain plasticity is often restricted to critical periods in early life. Here, we show that a key regulator of this process in visual cortex, Otx2 homeoprotein, is synthesized and secreted globally from the choroid plexus. Consequently, Otx2 is maintained in selected GABA cells unexpectedly throughout the mature forebrain. Genetic disruption of choroid-expressedOtx2 impacts these distant circuits and in primary visual cortex reopens binocular plasticity to restore vision in amblyopic mice. The potential to regulate adult cortical plasticity through the choroid plexus underscores the importance of this structure in brain physiology and offers novel therapeutic approaches to recovery from neurodevelopmental disorders more broadly.