Stimulus-linked RNA and protein synthesis is required for establishment of long-term neuroplasticity. To identify molecular mechanisms underlying long-term neuroplasticity, we have used differential cDNA techniques to clone a novel immediate-early gene (IEG) that is rapidly induced in neurons of the hippocampus and cortex by physiological synaptic activity. Analysis of the deduced amino acid sequence indicates homology to members of the pentraxin family of secreted lectins that include C-reactive protein and serum amyloid P component. Regions of homology include an 8 amino acid "pentraxin signature" sequence and a characteristic pentraxin calcium-binding domain. We have termed this gene and the encoded protein Narp (from neuronal activity-regulated pentraxin). Biochemical analyses confirm the presence of a functional signal sequence, and Narp is secreted by transfected COS-1 cells in culture. Additionally, Narp binds to agar matrix in a calcium-dependent manner consistent with the lectin properties of the pentraxin family. When cocultured with Narp-secreting COS-1 cells, neurons of cortical explants exhibit enhanced growth of neuronal dendritic processes. Neurite outgrowth-promoting activity is also observed using partially purified Narp and can be specifically immunodepleted, demonstrating that Narp is the active principle. Narp is fully active at a concentration of approximately 40 ng/ml, indicating a potency similar to known peptide growth factors. Because Narp is rapidly regulated by neuronal activity, its lectin and growth-promoting activities are likely to play role in the modification of cellular properties that underlie long-term plasticity.

Podocytes are morphologically complex cells, the junctions of which form critical elements of the final filtration barrier. Disruption of their foot processes and slit diaphragms occur early in the development of many glomerular diseases. Here, we biochemically purified fractions enriched with slit diaphragm proteins and performed a proteomic analysis to identify new components of this important structure. Several known slit diaphragm proteins were found, such as podocin and nephrin, confirming the validity of the purification scheme. However, proteins on the apical membrane such as podocalyxin were neither enriched nor identified in our analysis. The chloride intracellular channel protein 5 (CLIC5), predominantly expressed in podocytes, was enriched in these fractions and localized in the foot process apical and basal membranes. CLIC5 colocalized and associated with the ezrin/radixin/moesin complex and with podocalyxin in podocytes in vivo. It is important to note that CLIC5−/− mice were found to have significantly decreased foot process length, widespread foot process abnormalities, and developed proteinuria. The ezrin/radixin/moesin complex and podocalyxin were significantly decreased in podocytes from CLIC5−/− mice. Thus, our study identifies CLIC5 as a new component that is enriched in and necessary for foot process integrity and podocyte function in vivo.

Glomerulosclerosis correlates with a reduction in podocyte number that occurs through mechanisms that include apoptosis. Whether glial cell line-derived neurotrophic factor (GDNF), a growth factor that is critical for neural and renal development, is a survival factor for injured podocytes was investigated. Ret, the GDNF receptor tyrosine kinase, was upregulated in podocytes in the passive Heymann nephritis and puromycin aminonucleoside (PA) nephrosis rat models of podocyte injury. In addition, Ret mRNA and protein were upregulated in mouse podocytes in vitro after injury that was induced by sublytic C5b-9 and PA. GDNF, which also was induced during podocyte injury, inhibited significantly the apoptosis of podocytes that was induced by ultraviolet C irradiation. Knockdown of Ret expression by small interference RNA in podocytes exacerbated apoptosis that was induced by both ultraviolet C and PA. Ret knockdown, upon injury, decreased AKT phosphorylation, suggesting that the phosphoinositol-3 kinase/AKT pathway mediated the survival effect of GDNF on podocytes. Consistent with this hypothesis, the selective phosphoinositol-3 kinase inhibitor LY294002 blocked the survival-promoting effects of GDNF. In conclusion, GDNF is a novel podocyte survival factor. Furthermore, Ret is highly upregulated during podocyte injury in vitro and in vivo, suggesting that Ret activation is a critical adaptive response for podocyte remodeling and repair.

Glial cell line-derived neurotrophic factor (GDNF) is a neuronal growth factor critical for the development and maintenance of central and peripheral neurons. GDNF is expressed in targets of innervation and provides support to several populations of large, projection neurons. To determine whether GDNF promotes retrograde survival over long axonal distances to cell bodies, we used a compartmentalized culture system. GDNF supported only modest and transient survival of postnatal sympathetic neurons when applied to their distal axons, in contrast to dorsal root ganglion (DRG) sensory neurons in which GDNF promoted survival equally well from either distal axons or cell bodies. Ret, the receptor tyrosine kinase for GDNF, underwent rapid proteasomal degradation in the axons of sympathetic neurons. Interestingly, the level of activated Ret in DRG neurons was sustained in the axons and also appeared in the cell bodies, suggesting that Ret was not degraded in sensory axons and was retrogradely transported. Pharmacologic inhibition of proteasomes only in the distal axons of sympathetic neurons caused an accumulation of activated Ret in both the axons and cell bodies during GDNF stimulation. Furthermore, exposure of the distal axons of sympathetic neurons to both GDNF and proteasome inhibitors, but neither one alone, promoted robust survival, identical to GDNF applied directly to the cell bodies. This differential responsiveness of sympathetic and sensory neurons to target-derived GDNF was attributable to the differential expression and degradation of the Ret9 and Ret51 isoforms. Therefore, the local degradation of Ret in axons dictates whether GDNF family ligands act as retrograde survival factors.