The retromer complex component VPS35 prevents activation of the BACE1 and Aβ production and thus plays an essential role in limiting Alzheimer’s disease neuropathology.

Synapse formation requires proper interaction between pre- and postsynaptic cells. In anterograde signaling, neurons release factors to guide postsynaptic differentiation. However, less is known about how postsynaptic targets retrogradely regulate presynaptic differentiation or function. We found that muscle-specific conditional knockout of beta-catenin (Ctnnb1, also known as beta-cat) in mice caused both morphologic and functional defects in motoneuron terminals of neuromuscular junctions (NMJs). In the absence of muscle beta-catenin, acetylcholine receptor clusters were increased in size and distributed throughout a wider region. Primary nerve branches were mislocated, whereas secondary or intramuscular nerve branches were elongated and reduced in number. Both spontaneous and evoked neurotransmitter release was reduced at the mutant NMJs. Furthermore, short-term plasticity and calcium sensitivity of neurotransmitter release were compromised in beta-catenin-deficient muscle. In contrast, the NMJ was normal in morphology and function in motoneuron-specific beta-catenin-deficient mice. Taken together, these observations indicate a role for muscle beta-catenin in presynaptic differentiation and function, identifying a previously unknown retrograde signaling in the synapse formation and synaptic plasticity.

Netrins, a family of secreted molecules, play critical roles in axon guidance and cell migration during neuronal development 1,2. In addition to its role as a chemotropic molecule, netrin-1 also acts as a survival factor 3-7 . Both UNC5 (i.e. UNC5A, B, C or D) and DCC are transmembrane receptors for netrin-1 8 ,9. In the absence of netrin-1, DCC and UNC5 act as dependence receptors and trigger apoptosis 3,6, 10 . However, how netrin-1 suppresses the apoptotic activity of the receptors remains elusive. Here, we show that netrin-1 induces interaction of UNC5B with the brain specific GTPase PIKE-L. This interaction triggers activation of PI 3-kinase signaling, prevents UNC5B's proapoptotic activity and enhances neuronal survival. Moreover, this process tightly relies on Fyn as PIKE-L is tyrosine phosphorylated in response to netrin-1 and the netrin-1-mediated interaction of UNC5B with PIKE-L is inhibited in Fyn null mice. Thus, PIKE-L acts as a downstream survival effector for netrin-1 through UNC5B in the nervous system. PIKE-L is a brain specific GTPase, which binds and stimulates PI 3-kinase in a GTP-dependent manner 11, 12 . PIKE-L binds Homer, an adaptor protein for metabotropic glutamate receptor (mGluR). Activation of mGluRIs enhances formation of an mGluRI-Homer-PIKE-L complex, leading to activation of PI 3-kinase and prevention of neuronal apoptosis 13 . PIKE is also a substrate for caspases. PIKE can be phosphorylated on tyrosine residues by Fyn, leading to its resistance to caspase cleavage 14. To search for PIKE-L-binding proteins, we conducted yeast two-hybrid screening using GTPase domain as bait. Four out of twelve clones are both His and β-gal positive, one of which encodes the C-terminus of UNC5B ( Figure 1A). In HEK293 cells, transfected GFP-PIKE-L selectively binds to 569-946 fragment of UNC5B but not other fragments. Compared to the binding by the C-terminal motif (a.a. 569-946), truncation of death domain (a.a. 854-946) decreases UNC5B affinity to PIKE-L. Reciprocal immunoprecipitation reveals that the interaction occurs no matter PIKE-L or UNC5B is precipitated by its antibody ( Figure 1B, middle panels). Full-length UNC5B and its C-terminal fragment released after caspase cleavage 5 , 7 specifically interact with GTPase domain but not with other regions of PIKE-L, consistent with our yeast two-hybrid findings ( Figure 1B, right panels). We also 5To whom all correspondence should be addressed, Phone: 404-712-2814; Fax: 404-712-2979, kye@emory observed the robust interaction between endogenous PIKE-L and UNC5B in both the cortex and hippocampus of rat brain ( Figure 1C). Immunostaining of hippocampal and cortical primary neurons reveals that PIKE-L and UNC5B colocalize in the cell body and throughout all neuronal processes ( Figure 1D, left panel). The staining is specific as GST-PIKE-L (a.a. 268-384) antigen but not control GST abolishes PIKE-L staining in neurons ( Figure 1D, right panels).To examine whether netrin-1 modulates PIKE-L interaction with UNC5B, we cotransfected UNC5B into...

Focal adhesion kinase (FAK), a major cell adhesion-activated tyrosine kinase, has an important function in cell adhesion and migration. Here, we report a new signalling of FAK in regulating chromatin remodelling by its interaction with MBD2 (methyl CpG-binding protein 2), underlying FAK regulation of myogenin expression and muscle differentiation. FAK interacts with MBD2 in vitro, in myotubes, and in isolated muscle fibres. Such an interaction, increased in myotubes exposed to oxidative stress, enhances FAK nuclear localization. The nuclear FAK-MBD2 complexes alter heterochromatin reorganization and decrease MBD2 association with HDAC1 (histone deacetylase complex 1) and methyl CpG site in the myogenin promoter, thus, inducing myogenin expression. In line with this view are observations that blocking FAK nuclear localization by expressing dominant negative MBD2 or suppression of FAK expression by its miRNA in C2C12 cells attenuates myogenin induction and/or impairs muscle-terminal differentiation. Together, these results suggest an earlier unrecognized role of FAK in regulating chromatin remodelling that is important for myogenin expression and muscle-terminal differentiation, reveal a new mechanism of MBD2 regulation by FAK family tyrosine kinases, and provide a link between cell adhesion and chromatin remodelling.