- Insights into Neurofibromatosis-Associated Pain
- Chipping Away at the Origin of Malignant Peripheral Nerve Sheath Tumors
- Identifying Novel Functions of Neurofibromin
Neurofibromatosis type 1 (NF1) is characterized by the formation of painful benign and malignant tumors in the peripheral nervous system. While patients with NF1 experience varying levels of pain, some report an increased pain response and some develop chronic pain syndromes. The cause of NF1 has been linked to a decrease in neurofibromin due to loss of its expression by mutation of the NF1 gene. One of the functions of neurofibromin is to reduce the level of activated Ras, an important signaling molecule, in the cells. Decreased neurofibromin leads to an abnormally high level of active Ras in NF1 neurons. Nerve growth factor (NGF), which signals by increasing active Ras, has been found to be an important mediator of the enhanced pain sensation that occurs in inflamed sensory neurons. Dr. Cynthia Hingtgen, with support from a Department of Defense Neurofibromatosis Research Program Fiscal Year 2002 New Investigator Award, is investigating whether the enhanced painful sensations experienced by people with NF1 could result from altered control of Ras activity due to decreased neurofibromin levels. Through a comparison of sensory neurons from normal mice and NF1 mice, Dr. Hingtgen found that NF1 neurons have enhanced excitability, firing with much less stimulation and firing more frequently from a single stimulation. Treatment of the normal neurons with NGF prior to stimulation resulted in responses similar to the NF1 neurons, indicating that they share a common mechanism of action, most likely through Ras activation. Dr. Hingtgen also noted that the NF1 neurons released significantly more neuropeptides than normal neurons did following stimulation, further exacerbating the pain response and inflammation around the responding sensory neurons and leading to the potential establishment of chronic pain. Dr. Hingtgen hopes that further exploration of this mechanism will yield specific targets for therapeutic intervention to alleviate neurofibromatosis-associated pain.
Hingtgen C, Roy S, and Clapp D. 2006. Stimulus-evoked release of neuropeptides is enhanced in sensory neurons from mice with a heterozygous mutation of the Nf1 gene. Neuroscience 137:637-645.
Wang Y, Nicol G, Clapp D, and Hingtgen C. 2005. Sensory neurons from Nf1 haploinsufficient mice exhibit increased excitability. Journal of Neurophysiology 94:3670-3676.
Approximately 30% of individuals with neurofibromatosis type 1 (NF1) develop benign tumors known as plexiform neurofibromas, which can undergo transformation to malignant peripheral nerve sheath tumors (MPNSTs) in a subset of patients. MPNSTs, which are hypothesized to arise from neoplastic Schwann cells, are generally resistant to conventional treatment regimens and are a major cause of mortality of NF1. The transformation of Schwann cells to malignant tumors is a process that remains vastly unexplored. However, recent research findings from Dr. Nancy Ratner at the Cincinnati Children's Hospital Medical Center and her colleague, Dr. Shyra Miller, provide the first glimpses of the molecular underpinnings of malignant transformation. Their innovative research, supported by a Fiscal Year 2000 (FY00) Idea Award and an FY03 Investigator-Initiated Research Award, examined the genetic differences between NF1-associated malignant tumor cells and their normal human Schwann cell counterparts. Specifically, Dr. Ratner and Dr. Miller compared the genetic profiles of primary Schwann cells to malignant cell lines using state-of-the-art oligonucleotide microarray chips. Although the rates of proliferation and gene expression profiles in the malignant cell lines were somewhat variable, the team was able to identify and validate a 159-gene signature that distinguished MPNST cells from normal Schwann cells. Many of these genes have been implicated in other cancers, suggesting that therapeutics developed for other types of tumors may be useful for the treatment of MPNSTs. Importantly, TWIST1, which is involved in tumor migration, invasion, and drug resistance, was overexpressed in all malignant cell lines tested and was found to promote migration. This research has great potential to identify new diagnostic biomarkers and drug targets to combat malignancies associated with NF1.
Miller SJ, Rangwala F, Williams J, Ackerman P, Kong S, Jegga AG, Kaiser S, Aronow BJ, Frahm S, Kluwe L, Mautner V, Upadhyaya M, Muir D, Wallace M, Hagen J, Quelle DE, Watson MA, Perry A, Gutmann DH, and Ratner N. 2006. Large-scale molecular comparison of human schwann cells to malignant peripheral nerve sheath tumor cell lines and tissues. Cancer Research 66:2584-2591.
Neurofibromatosis type 1 (NF1) is a genetic disease characterized by the development of nerve tumors, bone lesions, learning disabilities, and other potentially devastating clinical complications. The disorder is associated with mutations in the NF1 tumor suppressor gene, which encodes a protein called neurofibromin. The only clearly defined function of neurofibromin is to downregulate the activity of Ras, a protein involved in tumor formation, through a region known as the GTPase-activating protein-related domain (GRD). Although some individuals with NF1 carry inactivating mutations in the neurofibromin GRD, many do not, suggesting that other regions of the protein may play a critical role in normal cells.
Dr. Klaus Scheffzek of the European Molecular Biology Laboratory is applying his expertise in biophysics and X-ray crystallography to elucidate the functions of neurofibromin. With the support of a fiscal year 1999 (FY99) New Investigator Award, Dr. Scheffzek's group is using crystallography to characterize the structures of individual segments of neurofibromin and compare the structures to those of other proteins to obtain clues to the potential functions of each segment. This approach led to the identification and crystallization of a novel bipartite module of neurofibromin, NF1-Sec-PH, that is adjacent to the GRD. Importantly, mutations in the NF1-Sec-PH domain have been identified in NF1 patients, indicating that this region may be critical for the maintenance of normal cellular and molecular function.
NF1-Sec-PH is composed of two distinct segments, a lipid-binding Sec14 homologous segment (NF1-Sec) and a pleckstrin homology (PH)-like domain (NF1-PH) that has not been detected previously by other approaches. Sec14 homology domains are found in many signaling proteins and have been implicated in the regulation of protein localization within cells, whereas PH-like domains in other proteins bind phosphatidylinositol in lipid membranes and interact with multiple signaling molecules. A protrusion of NF1-PH appears to stabilize the closed conformation of the NF1-Sec lipid-binding cavity, indicating that NF1-PH may control the interaction of the neighboring NF1-Sec segment with its unknown natural ligands. Moreover, Dr. Scheffzek determined that NF1-Sec-PH binds phospholipids, suggesting that this region may contribute to recruitment of neurofibromin to Ras at the cell membrane. Identification of proteins that interact with NF1-Sec-PH may provide further insight into neurofibromin activity in normal cellular processes.
Collaborating with Dr. Andre Bernards (Boston), Dr. Scheffzek's group is also working to isolate and crystallize full-length neurofibromin with funding from an FY04 NFRP Concept Award, since determination of the structure of the entire protein is necessary for comprehensive understanding of its functions. These efforts may ultimately lead to improved understanding of the mechanisms underlying NF1 pathogenesis and facilitate the development of targeted therapies for affected individuals.
D'Angelo I, Welti S, Bonneau S, and Scheffzek K. 2005. A novel bipartite phospholipid-binding module in the neurofibromatosis type 1 protein. EMBO Reports 7:174-179.
Bonneau F, D'Angelo I, Welti S, Stier G, Ylanne J, and Scheffzek K. 2004. Expression, purification and preliminary crystallographic characterization of a novel segment from the neurofibromatosis type 1 protein. Acta Crystallographica Section D 60:2364-2367.