The acetylcholine receptor (AChR)-associated protein rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated rapsyn, we found that in denervated diaphragm rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more rapsyn suggesting that clustering of AChRs may require increased interaction with rapsyn. In similar experiments in cultured myotubes, rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsynAChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.

In acute experimental autoimmune encephalomyelitis (EAE), demyelination is induced by myelin‐specific CD4+ T lymphocytes and myelin‐specific antibodies. Recovery from the disease is initiated by cytokines which suppress T cell expansion and the production of myelin‐toxic molecules by macrophages. Th2/3 cell‐derived signals may also be involved in central nervous system (CNS) repair. Remyelination is thought to be initiated by the recruitment and differentiation of oligodendrocyte precursor cells (OPC) in demyelinated CNS lesions. Here, we report that unlike Th1 cytokines (TNF‐α, IFN‐γ), the Th2/3 cytokine TGF‐β induces primary microglia from C57BL/6 mice to secrete a chemotactic factor for primary OPC. We identified this factor to be the hepatocyte growth factor (HGF). Our studies show that TGF‐β‐1‐2‐3 as well as IFN‐β induce HGF secretion by microglia and that antibodies to the HGF receptor c‐Met abrogate OPC chemotaxis induced by TGF‐β2‐treated microglia. In addition we show spinal cord lesions in EAE induced in SJL/J mice to contain both OPC and HGF producing macrophages in the recovery phase, but not in the acute stage of disease. Taken these findings, TGF‐β may play a pivotal role in remyelination by inducing microglia to release HGF which is both a chemotactic and differentiation factor for OPC.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, a-dystrobrevin and utrophin, but failed to aggregate AChRs, a-and b-dystroglycan, syntrophin isoforms and rapsyn, an AChR±anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn ef®ciently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myo®bers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.

Central to synaptic function are protein scaffolds associated with neurotransmitter receptors. α7 neuronal nicotinic acetylcholine receptors (nAChRs) modulate network activity, neuronal survival and cognitive processes in the CNS, but protein scaffolds that interact with these receptors are unknown. Here we show that the PDZ-domain containing protein PICK1 binds to α7 nAChRs and plays a role in their clustering. PICK1 interacted with the α7 cytoplasmic loop in yeast in a PDZdependent way, and the interaction was confirmed in recombinant pull-down experiments and by co-precipitation of native proteins. Some α7 and PICK1 clusters were adjacent at the surface of SH-SY5Y cells and GABAergic interneurons in hippocampal cultures. Expression of PICK1 caused decreased α7 clustering on the surface of the interneurons in a PDZ-dependent way. These data show that PICK1 negatively regulates surface clustering of α7 nAChRs on hippocampal interneurons, which may be important in inhibitory functions of α7in the hippocampus.

Increased expression of the chondroitin proteoglycan NG2 is a prominent feature in central nervous system injury with unknown cellular source and biological relevance. Here, we describe the first detailed analysis of experimental autoimmune encephalomyelitis in NG2 knockout mice and NG2 knockout bone marrow chimeras. We show that both macrophages and oligodendrocyte progenitor cells express and secrete NG2 in response to transforming growth factor-β. A subpopulation of macrophages expresses NG2 within leucocyte infiltrates in the central nervous system, but only oligodendrocyte progenitor cells contribute to NG2 accumulation. Notably, NG2 plays no role in experimental autoimmune encephalomyelitis initiation, progression or recuperation. In concurrence, the immune response is unaltered in NG2-deficient mice as are the extent of central nervous system damage and degree of remyelination.