Browsing by Subject "Neurofibromatosis type 1"

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    Dissecting the cellular and molecular mechanisms mediating neurofibromatosis type 1 related bone defects
    (2013-06) Rhodes, Steven David; Yang, Feng-Chun; Clapp, D. Wade; Robling, Alexander G.; Bidwell, Joseph P.
    Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.
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    Dissecting the role of the CRMP2–neurofibromin complex on pain behaviors
    (Wolters Kluwer, 2017-11) Moutal, Aubin; Wang, Yue; Yang, Xiaofang; Ji, Yingshi; Luo, Shizhen; Dorame, Angie; Bellampalli, Shreya S.; Chew, Lindsey A.; Cai, Song; Dustrude, Erik T.; Keener, James E.; Marty, Michael T.; Vanderah, Todd W.; Khanna, Rajesh; Psychiatry, School of Medicine
    Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.
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    Genetic disruption of the small GTPase RAC1 prevents plexiform neurofibroma formation in mice with neurofibromatosis type 1
    (Elsevier, 2020-07-17) Mund, Julie A.; Park, SuJung; Smith, Abbi E.; He, Yongzheng; Jiang, Li; Hawley, Eric; Roberson, Michelle J.; Mitchell, Dana K.; Abu-Sultanah, Mohannad; Yuan, Jin; Bessler, Waylan K.; Sandusky, George; Chen, Shi; Zhang, Chi; Rhodes, Steven D.; Clapp, D. Wade; Pediatrics, School of Medicine
    Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.
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    Guanfacine treatment improves ADHD phenotypes of impulsivity and hyperactivity in a neurofibromatosis type 1 mouse model
    (BMC, 2020-01-15) Lukkes, J. L.; Drozd, H. P.; Fitz, S. D.; Molosh, A. I.; Clapp, D. W.; Shekhar, A.; Psychiatry, School of Medicine
    BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a mutation in one copy of the neurofibromin gene (NF1+/-). Even though approximately 40-60% of children with NF1 meet the criteria for attention deficit hyperactivity disorder (ADHD), very few preclinical studies, if any, have investigated alterations in impulsivity and risk-taking behavior. Mice with deletion of a single NF1 gene (Nf1+/-) recapitulate many of the phenotypes of NF1 patients. METHODS: We compared wild-type (WT) and Nf1+/- mouse strains to investigate differences in impulsivity and hyperactivity using the delay discounting task (DDT), cliff avoidance reaction (CAR) test, and open field. We also investigated whether treatment with the clinically effective alpha-2A adrenergic receptor agonist, guanfacine (0.3 mg/kg, i.p.), would reverse deficits observed in behavioral inhibition. RESULTS: Nf1+/- mice chose a higher percentage of smaller rewards when both 10- and 20-s delays were administered compared to WT mice, suggesting Nf1+/- mice are more impulsive. When treated with guanfacine (0.3 mg/kg, i.p.), Nf1+/- mice exhibited decreased impulsive choice by waiting for the larger, delayed reward. Nf1+/- mice also exhibited deficits in behavioral inhibition compared to WT mice in the CAR test by repetitively entering the outer edge of the platform where they risk falling. Treatment with guanfacine ameliorated these deficits. In addition, Nf1+/- mice exhibited hyperactivity as increased distance was traveled compared to WT controls in the open field. This hyperactivity in Nf1+/- mice was reduced with guanfacine pre-treatment. CONCLUSIONS: Overall, our study confirms that Nf1+/- mice exhibit deficits in behavioral inhibition in multiple contexts, a key feature of ADHD, and can be used as a model system to identify alterations in neural circuitry associated with symptoms of ADHD in children with NF1.
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    Preclinical Evidence for the Use of Sunitinib Malate in the Treatment of Plexiform Neurofibromas
    (John Wiley & Sons, Inc., 2016-02) Ferguson, Michael J.; Rhodes, Steven D.; Jiang, Li; Li, Xiaohong; Yuan, Jin; Yang, Xianlin; Zhang, Shaobo; Vakili, Saeed T.; Territo, Paul; Hutchins, Gary; Yang, Feng-Chun; Ingram, David A.; Clapp, D. Wade; Chen, Shi; Department of Pediatrics, Indiana University School of Medicine
    Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1flox/− pNF murine model. Experimental Design Proliferation, β-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1+/− mast cells and fibroblasts, respectively. Krox20;Nf1flox/− mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [18F]DG-PET/CT imaging. Results Sunitinib suppressed multiple in vitro gain-in-functions of Nf1+/− mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1flox/− mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF. Conclusions Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1flox/− mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials.
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    THE ROLE OF PAK1 IN THE CELLULAR AND MOLECULAR COMPONENTS OF PLEXIFORM NEUROFIBROMAS
    (2008-10-10T18:12:42Z) McDaniel, Andrew S.; Clapp, D. Wade; Broxmeyer, Hal E.; Ingram, David A.; Srour, Edward F.
    Neurofibromatosis type I (NF1) is a common genetic disease that affects over 200,000 patients in North America, Europe, and Japan. Individuals with NF1 display a wide variety of pathologies; importantly, 15-40% of NF1 patients are affected by plexiform neurofibromas. Neurofibromas are complex tumors consisting of tumorgenic Schwann cells surrounded by endothelial cells, fibroblasts, and inflammatory mast cells. These peripheral nerve sheath tumors contribute significantly to the morbidity and mortality associated with NF1. Currently, no medical therapies exist for treating neurofibromas. Recent evidence indicates that the hematopoietic tumor microenvironment carries out a crucial function in the formation of plexiform neurofibromas. Neurofibromatosis is the result of mutations at the NF1 locus, which encodes the GTPase activating protein neurofibromin. Neurofibromin is a negative regulator of the proto-oncogene Ras. Ras hyperactivation is the molecular basis of NF1 associated phenotypes, and it has been demonstrated that restoration of Ras signaling to wild type levels can correct NF1 associated phenotypes in vitro and in vivo. In keeping with the long term goal of detecting potential molecular targets for medical therapies to treat human plexiform neurofibromas, we have identified the kinase Pak1 as a possible downstream intermediary of Ras signaling in NF1 deficient cells. Studies described here utilized murine genetic models to study the effects of genetic inactivation of Pak1 on molecular signaling and cellular functions related to neurofibromas. We demonstrate that inactivation of Pak1 leads to correction of SCF mediated gain-in-function phenotypes seen in Nf1 haploinsufficient mast cells, in vivo and in vitro. However, by using a conditional Nf1 knockout mouse that is a reliable model of plexiform neurofibroma formation, we shown that loss of Pak1 alone in the hematopoeitic compartement is not sufficient to prevent neurofibroma formation. Additionally, we describe a key role for Pak1 in regulating PDGF and TGF-β mediated fibroblast functions, in vitro and in vivo. These studies provide insight into the causes of debilitating tumors related to a common genetic disease, and this research could potentially lead to the development of medical therapies for these tumors, increasing the quality of life for tens of thousands of affected individuals each year.