The nonclassical MHC class I Qa-1 expressed in layer 6 neurons regulates activity-dependent plasticity via microglial CD94/NKG2 in the cortex.

During developmental critical periods, circuits are sculpted by a process of activity-dependent competition. The molecular machinery involved in regulating the complex process of responding to different levels of activity is now beginning to be identified. Here, we show that the nonclassical major histocompatibility class I (MHCI) molecule Qa-1 is expressed in the healthy brain in layer 6 corticothalamic neurons. In the visual cortex, Qa-1 expression begins during the critical period for ocular dominance (OD) plasticity and is regulated by neuronal activity, suggesting a role in regulating activity-dependent competition. Indeed, in mice lacking Qa-1, OD plasticity is perturbed. Moreover, signaling through CD94/NKG2, a known cognate Qa-1 heterodimeric receptor in the immune system, is implicated: selectively targeting this interaction phenocopies the plasticity perturbation observed in Qa-1 knockouts. In the cortex, CD94/NKG2 is expressed by microglial cells, which undergo activity-dependent changes in their morphology in a Qa-1­dependent manner. Our study thus reveals a neuron­microglial interaction dependent upon a nonclassical MHCI molecule expressed in L6 neurons, which regulates plasticity in the visual cortex. These results also point to an unexpected function for the Qa-1/HLA-E (ligand) and CD94/NKG2 (receptor) interaction in the nervous system, in addition to that described in the immune system.

Enhanced potency of the metalloprotease inhibitor TAPI-2 by multivalent display.

Metalloproteases regulate a vast array of critical cellular processes such as proliferation, migration, repair, and invasion/metastasis. In so doing, metalloproteases have been shown to play key roles in the pathogenesis of multiple disorders including arteriosclerosis, arthritis, cancer metastasis, and ischemic brain injury. Therefore, much work has focused on developing metalloprotease inhibitors to provide a potential therapeutic benefit against the progression of these and other diseases. In order to produce a more potent inhibitor of metalloproteases, we synthesized multivalent displays of a metalloprotease inhibitor derived from the ring-opening metathesis polymerization (ROMP). Specifically, multivalent ligands of a broad-spectrum metalloprotease inhibitor, TAPI-2, were generated upon conjugation of the amine-bearing inhibitor with the ROMP-derived N-hydroxysuccinimide ester polymer. By monitoring the metalloprotease dependent cleavage of the transmembrane protein Semaphorin4D (Sema4D), we demonstrated an enhancement of inhibition by multivalent TAPI-2 compared to monovalent TAPI-2. To further optimize the potency of the multivalent inhibitor, we systematically varied the polymer length and inhibitor ligand density (mole fraction, χ). We observed that while ligand density plays a modest role in the potency of inhibition caused by the multivalent TAPI-2 display, the length of the polymer produces a much greater effect on inhibitor potency, with the shortest polymer achieving the greatest level of inhibition. These findings validate the use of multivalent display to enhance the potency of metalloprotease inhibitors and further, suggest this may be a useful approach to enhance potency of other small molecule towards their targets.

Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus.

While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.