Netrin-1 mediates neuronal survival through PIKE-L interaction with the dependence receptor UNC5B

Abstract

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...

Netrin-1 exerts oncogenic activities through enhancing Yes-associated protein stability

“…In the presence of netrin-1, the multimerized receptors recruit different adapter proteins and activate PI3K/Akt, ERK1/2, and small GTPase RhoA/Rac-1/Cdc-42 to affect cell survival, motility, invasion, and morphogenesis through cytoskeletal rearrangements (22)(23)(24). It is reported that PP1A acts downstream of RhoA in LPA-induced YAP dephosphoryaltion (25) and PI3K and ERK1/2 signaling is involved in the regulation of PP1A activity in cancer cells (26,27).…”

PIKEis essential for oligodendroglia development and CNS myelination

“…The essential functions of PIKE in neuronal survival, development, synaptic plasticity, and memory formation have been clearly demonstrated in our previous reports (8)(9)(10)(11)(12)16). Accumulating evidence suggests that oligodendroglia also plays critical roles in governing neuronal functions, for example, via myelination-dependent saltatory conductance and proper signal transmission in the brain, which is essential for cognitive and motor behaviors (27).…”

“…As such, ablation or inactivation of PIKE-L might block the extracellular factors from using this pathway to initiate OL maturation and myelination. We have shown previously that PIKE-L is a downstream effecter of netrin 1 to protect neurons from apoptosis through substantiating the activity of PI3K/Akt (9). Given that netrin 1 also regulates OPC migration and differentiation (34), PIKE-L may be an executor of netrin 1 to promote OL differentiation.…”

“…It participates in numerous cellular events to regulate neuronal activity and survival. Our previous studies show that PIKE-L interacts with both netrin receptor (UNC5H) and metabotropic glutamate receptor I (mGlu 1 ) to prevent apoptotic cell death (8,9). In addition, PIKE-L controls cell-surface trafficking of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid receptor and the formation of long-term potentiation in the postsynaptic neurons (10).…”

Repair and regeneration properties of Ginkgo biloba after ischemic brain injury

“…It is known that attractive and repulsive guidance cues dictate post-stroke axonal sprouting as well as migration of neuroblasts towards the site of the injury (Carmichael, 2008). Netrin-1 overexpression promotes neuronal migration and aids cell survival (Tang et al, 2008). Thus, EGb 761-mediated over-expression of netrin-1 signifies its ability to promote a suitable environment for the migration of neuroblasts.…”