CD40 is expressed and functional on neuronal cells

We show here that CD40 mRNA and protein are expressed by neuronal cells, and are increased in differentiated versus undifferentiated N2a and PC12 cells as measured by RT–PCR, western blotting and immunofluorescence staining. Additionally, immunohistochemistry reveals that neurons from adult mouse and human brain also express CD40 in situ. CD40 ligation results in a time‐dependent increase in p44/42 MAPK activation in neuronal cells. Furthermore, ligation of CD40 opposes JNK phosphorylation and activity induced by NGF‐β removal from differentiated PC12 cells or serum withdrawal from primary cultured neurons. Importantly, CD40 ligation also protects neuronal cells from NGF‐β or serum withdrawal‐induced injury and affects neuronal differentiation. Finally, adult mice deficient for the CD40 receptor demonstrate neuronal dysfunction as evidenced by decreased neurofilament isoforms, reduced Bcl‐xL:Bax ratio, neuronal morphological change, increased DNA fragmentation, and gross brain abnormality. These changes occur with age, and are clearly evident at 16 months. Taken together, these data demonstrate a role of CD40 in neuronal development, maintenance and protection in vitro and in vivo.

[1]  Kevin J. Tracey,et al.  Expression of TNF and TNF Receptors (p55 and p75) in the Rat Brain after Focal Cerebral Ischemia , 1997, Molecular medicine.

[2]  C. Jasmin,et al.  Chemokines and CD40 expression in human fibroblasts , 2000, European journal of immunology.

[3]  L. Greene,et al.  Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Woodgett,et al.  Cross‐linking CD40 on B cells preferentially induces stress‐activated protein kinases rather than mitogen‐activated protein kinases. , 1996, The EMBO journal.

[5]  C. Thompson,et al.  bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death , 1993, Cell.

[6]  W. Fanslow,et al.  Incorporation of an Isoleucine Zipper Motif Enhances the Biological Activity of Soluble CD40L (CD154)* , 1999, The Journal of Biological Chemistry.

[7]  J. Banchereau,et al.  Immune regulation by CD40-CD40-L interactions. , 1997, Frontiers in bioscience : a journal and virtual library.

[8]  F. Ruddle,et al.  Isolation of microtubule protein from cultured mouse neuroblastoma cells. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Mattson,et al.  Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. , 1999, Science.

[10]  G. Reichmann,et al.  Brain Dendritic Cells and Macrophages/Microglia in Central Nervous System Inflammation1 , 2001, The Journal of Immunology.

[11]  E. Hogan,et al.  Increased Calpain Expression Is Associated with Apoptosis in Rat Spinal Cord Injury: Calpain Inhibitor Provides Neuroprotection , 2000, Neurochemical Research.

[12]  N. Yoshida,et al.  The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. , 1994, Immunity.

[13]  J. Sutcliffe,et al.  Mature microglia resemble immature antigen‐presenting cells , 1998, Glia.

[14]  W. Hammerschmidt,et al.  Epstein–Barr virus‐mediated B‐cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor , 1998, The EMBO journal.

[15]  O. Linderkamp,et al.  Activation of the Ras signaling pathway by the CD40 receptor. , 1996, Journal of immunology.

[16]  P. Hodgkin,et al.  Integrating signals from IFN-gamma and IL-4 by B cells: positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a. , 1999, Journal of immunology.

[17]  V. Nguyen,et al.  Post‐transcriptional inhibition of CD40 gene expression in microglia by transforming growth factor‐β , 1998, European journal of immunology.

[18]  R. Flavell,et al.  Mice deficient for the CD40 ligand. , 1994, Immunity.

[19]  Q Cheng,et al.  NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  D. Lynch,et al.  CD40 ligand inhibits Fas/CD95‐mediated apoptosis of human blood‐derived dendritic cells , 1997, European journal of immunology.

[21]  A. Prochiantz,et al.  Developmental and regional regulation of rab3: A new brain specific “ras‐like” gene , 1989, Journal of neuroscience research.

[22]  J. Ham,et al.  Direct inhibition of c‐Jun N‐terminal kinase in sympathetic neurones prevents c‐jun promoter activation and NGF withdrawal‐induced death , 2001, Journal of neurochemistry.

[23]  L. Greene,et al.  Prevention of PC12 Cell Death by N-Acetylcysteine Requires Activation of the Ras Pathway , 1998, The Journal of Neuroscience.

[24]  M. Zhou,et al.  CD40 ligand upregulates expression of the IL-3 receptor and stimulates proliferation of B-lineage acute lymphoblastic leukemia cells in the presence of IL-3 , 2000, Leukemia.

[25]  A. Prochiantz,et al.  Developmental and regional expression of three new members of the ras‐gene family in the mouse brain , 1989, Journal of neuroscience research.

[26]  T. Kadowaki,et al.  Phosphatidylinositol 3-Kinase and NF-κB/Rel Are at the Divergence of CD40-Mediated Proliferation and Survival Pathways , 2000, The Journal of Immunology.

[27]  T. Molitor,et al.  Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. , 1992, Journal of immunology.

[28]  John Calvin Reed,et al.  Bcl-2 family proteins as ion-channels , 1998, Cell Death and Differentiation.

[29]  J. Banchereau,et al.  CD40 ligation of human keratinocytes inhibits their proliferation and induces their differentiation. , 1997, Journal of immunology.

[30]  P. Libby,et al.  Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[31]  H. Lassmann,et al.  Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. , 1993, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[32]  D. Cockayne,et al.  Genomic structure and chromosomal mapping of the murine CD40 gene. , 1992, Journal of immunology.

[33]  P. Cohen,et al.  Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases , 1991, Nature.

[34]  M. Iglesias,et al.  Extracellular‐Regulated Kinases and Phosphatidylinositol 3‐Kinase Are Involved in Brain‐Derived Neurotrophic Factor‐Mediated Survival and neuritogenesis of the Neuroblastoma Cell Line SH‐SY5Y , 1999, Journal of neurochemistry.

[35]  Sheila M. Thomas,et al.  Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases , 1992, Cell.

[36]  E. Engleman,et al.  Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro. , 1990, The Journal of clinical investigation.

[37]  J. Banchereau,et al.  CD40 Ligation on Human Cord Blood CD34+Hematopoietic Progenitors Induces Their Proliferation and Differentiation into Functional Dendritic Cells , 1997, The Journal of experimental medicine.

[38]  C. Kooten.  Immune regulation by CD40-CD40-l interactions - 2; Y2K update. , 2000 .

[39]  P. Rakic,et al.  JNK3 contributes to c‐Jun activation and apoptosis but not oxidative stress in nerve growth factor‐deprived sympathetic neurons , 2001, Journal of neurochemistry.

[40]  J Bajorath,et al.  Immune regulation by CD40 and its ligand GP39. , 1996, Annual review of immunology.

[41]  K. R. Ely,et al.  Differential Requirements for Tumor Necrosis Factor Receptor-associated Factor Family Proteins in CD40-mediated Induction of NF-κB and Jun N-terminal Kinase Activation* , 1999, The Journal of Biological Chemistry.

[42]  E. Clark,et al.  Differential increase of an alternatively polyadenylated mRNA species of murine CD40 upon B lymphocyte activation. , 1992, Journal of immunology.

[43]  N. Kamatani,et al.  Increased CD40 Expression on Muscle Cells of Polymyositis and Dermatomyositis: Role of CD40-CD40 Ligand Interaction in IL-6, IL-8, IL-15, and Monocyte Chemoattractant Protein-1 Production1 , 2000, The Journal of Immunology.

[44]  M. Mullan,et al.  CD45 Inhibits CD40L-induced Microglial Activation via Negative Regulation of the Src/p44/42 MAPK Pathway* , 2000, The Journal of Biological Chemistry.