MCP-1 involvement in glial differentiation of neuroprogenitor cells through APP signaling.

Previously it has been reported that neural stem cells undergoing apoptotic stress have increased levels of amyloid precursor protein (APP) and increased APP expression results in glial differentiation. APP activity was also shown to be required for staurosporine-induced glial differentiation of neuroprogenitor cells. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed early during inflammation. The binding of MCP-1 to its chemokine receptor induces expression of novel transcription factor MCP-1-induced protein (MCPIP). MCPIP expression subsequently leads to cell death. Previous studies have shown that pro-apoptotic factors have the ability to induce neural differentiation. Therefore, we investigated if MCPIP expression leads to differentiation of NT2 neuroprogenitor cells. Results showed that MCPIP expression increased glial fibrillary acid protein (GFAP) expression and also caused distinct morphological changes, both indicative of glial differentiation. Similar results were observed with MCP-1 treatment. Interestingly, APP expression decreased in response to MCPIP. Instead, we found APP activity regulates expression of both MCP-1 and MCPIP. Furthermore, inhibition of either p38 MAPK or JAK signaling pathways significantly reduced APP's effect on MCP-1 and MCPIP. These data demonstrates the role APP has in glial differentiation of NT2 cells through MCP-1/MCPIP signaling. It is possible that increased APP expression after CNS injury could play a role in MCP-1 production, possibly promoting astrocyte activation at injured site.

MCP-1-induced migration of NT2 neuroprogenitor cells involving APP signaling.

Neuroprogenitor cells are an important resource because of their great potential to replace damaged cells in the brain caused by trauma and disease. Studies have shown that when neuroprogenitor cells are transplanted into the brain they migrate towards damaged areas, suggesting that these areas express factors that recruit migrating cells. Generally, after neuronal injury, there is a neuroinflammatory response that results in increased chemokine production. In this present study, we demonstrate that monocyte chemoattractant protein-1 (MCP-1) significantly induces the migration of NT2 neuroprogenitor cells. Activation of intracellular cyclic adenosine monophosphate or protein kinase C with forskolin and phorbol 12-myristate 13-acetate, respectively, was able to completely abolish the MCP-1-induced migration. Contrarily, neither extracellular signal-regulated kinase nor p38 mitogen-activated protein kinase was required for NT2 cells to respond to MCP-1. Previously, we showed that amyloid precursor protein (APP) activity increases MCP-1 expression in NT2 cells. We now demonstrate that NT2 cells expressing APP can induce migration of other neuroprogenitor cells. Utilizing a MCP-1 neutralizing antibody, we discovered that APP-induced migration was not caused solely by increased MCP-1 production. Interestingly, APP-increased expression of several C-C chemokines: MCP-1, regulated upon activation, normal T-cell expressed, and secreted (RANTES), and macrophage inflammatory protein alpha (MIP-1 alpha). This demonstrates the unique role APP has in regulating chemokine production, which directly affects cell migration. Taken together, these data provides greater detail of the chemotactic factors and intracellular signaling that direct neuroprogenitor cell migration, allowing for better understanding of cell migration during transplantation.

The phosphoprotein (P) and L binding sites reside in the N-terminus of the L subunit of the measles virus RNA polymerase.

Measles virus encodes an RNA-dependent RNA polymerase composed of the L and P proteins. Recent studies have shown that the L proteins of both Sendai virus and parainfluenza virus 3 form an L-L complex [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very Nterminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536.; Smallwood, S., Moyer, S.A., 2004. The L polymerase protein of parainfluenza virus 3 forms anoligomer and can interact with the heterologous Sendai virus L, P and C proteins. Virology 318, 439-450.; Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235-245.]. Using differentially tagged L proteins, we show here that measles L also forms an oligomer and the L-L binding site resides in the N-terminal 408 amino acids overlapping the P binding site in the same region of L. To identify amino acids important for binding P and L, site-directed mutagenesis of the L-408 protein was performed. Seven of twelve mutants in L-408 were unable to form a complex with measles P while the remainder did bind at least some P. In contrast, all of the mutants retained the ability to form the L-L complex, so different amino acids are involved in the L and P binding sites on L. Four of the 408 mutations defective in P binding were inserted into the full-length measles L protein and all retained L-L complex formation, but did not bind P. Full-length L mutants that did not bind P were also inactive in viral RNA synthesis, showing a direct correlation between P-L complex formation and activity.