Mark Marchionni

Neuregulin signaling through a PI3K/Akt/Bad pathway in Schwann cell survival.

beta-Neuregulin (betaNRG) is a potent Schwann cell survival factor that binds to and activates a heterodimeric ErbB2/ErbB3 receptor complex. We found that NRG receptor signaling rapidly activated phosphoinositide 3-kinase (PI3K) in serum-starved Schwann cells, while PI3K inhibitors markedly exacerbated apoptosis and completely blocked NRG-mediated rescue. NRG also rapidly signaled the phosphorylation of mitogen-activated protein kinase (MAPK) and the serine/threonine kinase Akt. The activation of Akt and MAPK in parallel pathways downstream from PI3K resulted in the phosphorylation of Bad at different serine residues. PI3K inhibitors that blocked NRG-mediated rescue also blocked the phosphorylation of Akt, MAPK, and Bad. However, selective inhibition of MEK-dependent Bad phosphorylation downstream from PI3K had no effect on NRG-mediated survival. Conversely, ectopic expression of wild-type Akt not only enhanced Bad phosphorylation but also enhanced autocrine- and NRG-mediated Schwann cell survival. Taken together, these results demonstrate that NRG receptor signaling through a PI3K/Akt/Bad pathway functions in Schwann cell survival.

Neuregulin Expression in PNS Neurons: Isoforms and Regulation by Target Interactions

Neuregulins have several important functions in the development of the peripheral nervous system, acting on both developing Schwann cells and muscle fibers. To determine whether these factors are also important for peripheral nerve regeneration, we have analyzed neuregulin expression in motor and sensory neurons by Northern blots and in situ hybridization. The results of this analysis show that the predominant neuregulin isoform expressed in these neurons is a novel transmembrane splice variant. After axotomy, there is a rapid decline in neuregulin expression in both motor and sensory neurons, but following reinnervation of target tissues, neuregulin expression returns to near normal levels. These results indicate that the normal expression of neuregulins in these neurons is maintained by the interactions with target tissues.

Control of Schwann Cell Survival and Proliferation: Autocrine Factors and Neuregulins

Postnatal rat Schwann cells secrete factors that prevent the programmed cell death (PCD) of low-density Schwann cells in serum-free culture. These autocrine survival signal(s) do not promote Schwann cell proliferation. Moreover, while NRG and bFGF, which promote proliferation, both rescue a subpopulation of neonatal Schwann cells from PCD, they do not rescue freshly isolated Schwann cells from older animals; other known protein factors tested also do not mimic the autocrine signal. These results suggest that Schwann cells switch their survival dependency around the time of birth from axonal signals such as NRG to autocrine signals. Such an arrangement would be advantageous for the regeneration of peripheral axons following injury. We also compared NRG-induced Schwann cell proliferation using autocrine signals or serum to promote survival. The autocrine signals increase the rate of NRG-stimulated proliferation of low-density Schwann cells in serum-free medium, whereas serum inhibits proliferation by inhibiting both the production of survival signals and the expression of erbB2 and erbB3 receptors; these inhibitions are all reversed by forskolin. In contrast, forskolin has no effect on proliferation when the cells are exposed to high levels of autocrine factors.

Schwann cell heparan sulfate proteoglycans play a critical role in glial growth factor/neuregulin signaling

Glial growth factors are proteins encoded by the neuregulin gene and are thought to signal via receptor tyrosine kinases. Many neuregulin gene products bind heparin, and we hypothesize that affinity for heparin may implicate cell surface heparan sulfate proteoglycans (HeSPGs) as co‐receptors for the soluble neuregulin gene product, recombinant human glial growth factor 2 (rhGGF2). Using primary rat Schwann cell cultures, we show that exogenous heparin and heparan sulfate block rhGGF2‐induced phosphorylation of putative neuregulin receptors, and block subsequent DNA synthesis; other glycosaminoglycans show no such effect. Inhibition of Schwann cell HeSPG biosynthesis by administration of β‐xyloside also blocks responsiveness to rhGGF2. In cell‐free binding assays, rhGGF2 binds heparin and heparan sulfate with high affinity, while suramin and suramin‐like molecules block this binding. These suramin‐like molecules reversibly block Schwann cell responsiveness to rhGGF2 with a rank order of potency identical to that in the cell‐free binding assay. Thus we demonstrate high affinity and specificity in the interaction of rhGGF2 with heparin‐like molecules, and show that three distinct perturbations of this interaction on Schwann cells (exogenous heparin/heparan sulfate treatment, inhibition of HeSPG biosynthesis, and treatment with suramin‐like molecules) result in a loss of responsiveness to rhGGF2. These results support a model in which HeSPGs are critical components that modulate extracellular rhGGF2 signaling interactions with appropriate receptor tyrosine kinases.