Netrins are a family of secreted molecules that are important for axonal outgrowth and guidance in the developing nervous system. However, the signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. Here we report that the netrin receptor DCC (deleted in colorectal cancer) interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK was expressed in developing brains and was localized with DCC in cultured neurons. Netrin-1 induced FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolished netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation.

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.

We previously demonstrated the anti-inflammatory effects and renal tissue protection in response to adenosine A(2A)-receptor (A(2A)R) activation in acute renal injury. We sought to extend these studies and determine the efficacy of A(2A)R agonists in a chronic model of renal injury. We hypothesized that A(2A) agonists mediate renal tissue protection in diabetic nephropathy by reducing glomerular inflammation. Diabetes was induced with single intravenous injection of streptozotocin in Sprague-Dawley rats (50 mg/kg). Increases in urinary albumin excretion (UAE) and plasma creatinine at week 6 in the diabetes group (26- and 6-fold over control, respectively) were markedly reduced by continuous subcutaneous administration of ATL146e (10 ng x kg(-1) x min(-1)), a selective A(2A) agonist. The increase in UAE in the diabetes group was associated with a significant reduction in the expression of slit diaphragm-associated molecules compared with control (nephrin; P < 0.05 and podocin; P < 0.005) that was reversed by ATL146e treatment. Diabetes led to an increase in urinary excretion of monocyte chemoattractant protein-1 (705% of control), TNF-alpha (1,586% of control), IFN-gamma (298% of control), kidney fibronectin mRNA (457% of control), and glomerular infiltration of macrophages (764% of control), effects significantly reduced by ATL146e treatment. Mesangial expansion and basement membrane thickness were reduced with ATL146e. To further confirm the selectivity of ATL146e, we used wild-type (WT) or A(2A)knockout (A(2A)-KO) mice. Four weeks after diabetes, UAE increased significantly in both WT and A(2A)-KO diabetic mice (3.0- and 3.3-fold over control). A(2A) agonist treatment blocked the increase in UAE in WT diabetic mice (P < 0.001), whereas it had no effect on the A(2A)-KO diabetic mice. These results demonstrate that chronic A(2A)R activation in diabetic rats 1) ameliorates histological and functional changes in kidneys induced by diabetes and 2) causes reduced inflammation associated with diabetic nephropathy.

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...

Alzheimer’s disease (AD), one of the most dreaded neurodegenerative disorders, is characterized by cortical and cerebrovascular Aβ (amyloid β peptide) deposits, neurofibrillary tangles, chronic inflammation, and neuronal loss. Increased bone fracture rates and reduced bone density are commonly observed in patients with AD, suggesting a common denominator(s) between both disorders. However, very few studies are available that have addressed this issue. Here, we present evidence for a function of amyloid precursor protein (APP) and Aβ in regulating osteoclast (OC) differentiation in vitro and in vivo. Tg2576 mice, which expresses Swedish mutation of APP (APPswe) under the control of prion promoter (1,2), exhibit biphasic effects on OC activation, with an increase of OC in younger mice (< 4 month old), but a decrease in older Tg2576 mice (> 4 month old). The increase of OC in young Tg2576 mice appears to be mediated by Aβ oligomers and RAGE (receptor for advanced glycation end products) expression in BMMs. However, the decrease of OC formation and activity in older Tg2576 mice may be due to the increase of sRAGE in aged Tg2576 mice, an inhibitor of RANKL induced osteoclastogenesis. These results suggest an unexpected function of APPswe/Aβ, reveal a mechanism underlying altered bone remodeling in AD patients, and implicate APP/Aβ and RAGE as common denominators for both AD and osteoporosis.