The cell of origin dictates the temporal course of neurofibromatosis-1 (Nf1) low-grade glioma formation.
ABSTRACT: Low-grade gliomas are one of the most common brain tumors in children, where they frequently form within the optic pathway (optic pathway gliomas; OPGs). Since many OPGs occur in the context of the Neurofibromatosis Type 1 (NF1) cancer predisposition syndrome, we have previously employed Nf1 genetically-engineered mouse (GEM) strains to study the pathogenesis of these low-grade glial neoplasms. In the light of the finding that human and mouse low-grade gliomas are composed of Olig2+ cells and that Olig2+ oligodendrocyte precursor cells (OPCs) give rise to murine high-grade gliomas, we sought to determine whether Olig2+ OPCs could be tumor-initiating cells for Nf1 optic glioma. Similar to the GFAP-Cre transgenic strain previously employed to generate Nf1 optic gliomas, Olig2+ cells also give rise to astrocytes in the murine optic nerve in vivo. However, in contrast to the GFAP-Cre strain where somatic Nf1 inactivation in embryonic neural progenitor/stem cells (Nf1flox/mut; GFAP-Cre mice) results in optic gliomas by 3 months of age in vivo, mice with Nf1 gene inactivation in Olig2+ OPCs (Nf1flox/mut; Olig2-Cre mice) do not form optic gliomas until 6 months of age. These distinct patterns of glioma latency do not reflect differences in the timing or brain location of somatic Nf1 loss. Instead, they most likely reflect the cell of origin, as somatic Nf1 loss in CD133+ neural progenitor/stem cells during late embryogenesis results in optic gliomas at 3 months of age. Collectively, these data demonstrate that the cell of origin dictates the time to tumorigenesis in murine optic glioma.
Project description:Low-grade glial neoplasms (astrocytomas) represent one of the most common brain tumors in the pediatric population. These tumors frequently form in the optic pathway (optic pathway gliomas, OPGs), especially in children with the neurofibromatosis type 1 (NF1)-inherited tumor predisposition syndrome. To model these tumors in mice, we have previously developed several Nf1 genetically-engineered mouse strains that form optic gliomas. However, there are three distinct macroglial cell populations in the optic nerve (astrocytes, NG2+ (nerve/glial antigen 2) cells and oligodendrocytes). The presence of NG2+ cells in the optic nerve raises the intriguing possibility that these cells could be the tumor-initiating cells, as has been suggested for adult glioma. In this report, we used a combination of complementary in vitro and novel genetically-engineered mouse strains in vivo to determine whether NG2+ cells could give rise to Nf1 optic glioma. First, we show that Nf1 inactivation results in a cell-autonomous increase in glial fibrillary acidic protein+ (GFAP+), but not in NG2+, cell proliferation in vitro. Second, similar to the GFAP-Cre transgenic strain that drives Nf1 optic gliomagenesis, NG2-expressing cells also give rise to all three macroglial lineages in vivo. Third, in contrast to the GFAP-Cre strain, Nf1 gene inactivation in NG2+ cells is not sufficient for optic gliomagenesis in vivo. Collectively, these data demonstrate that NG2+ cells are not the cell of origin for mouse optic glioma, and support a model in which gliomagenesis requires Nf1 loss in specific neuroglial progenitors during embryogenesis.
Project description:Children with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome are prone to the development of optic pathway gliomas resulting from biallelic inactivation of the NF1 gene. Recent studies have revealed the presence of other molecular alterations in a small portion of these NF1-associated brain tumors. The purpose of this study was to leverage Nf1 genetically engineered mouse strains to define the functional significance of these changes to optic glioma biology.Nf1+/- mice were intercrossed with Nf1(flox/flox) mice, which were then crossed with Nf1(flox/flox); GFAP-Cre mice, to generate Nf1(flox/mut); GFAP-Cre (FMC) mice. These mice were additionally mated with conditional KIAA1549:BRAF knock-in or Pten(flox/wt) mice to generate Nf1(flox/mut); f-BRAF; GFAP-Cre (FMBC) mice or Nf1(flox/mut); Pten(flox/wt); GFAP-Cre (FMPC) mice, respectively. The resulting optic gliomas were analyzed for changes in tumor volume, proliferation, and retinal ganglion cell loss.While KIAA1549:BRAF conferred no additional biological properties on Nf1 optic glioma, FMPC mice had larger optic gliomas with greater proliferative indices and microglial infiltration. In addition, all 3 Nf1 murine optic glioma strains exhibited reduced retinal ganglion cell survival and numbers; however, FMPC mice had greater retinal nerve fiber layer thinning near the optic head relative to FMC and FMBC mice.Collectively, these experiments demonstrate genetic cooperativity between Nf1 loss and Pten heterozygosity relevant to optic glioma biology and further underscore the value of employing genetically engineered mouse strains to define the contribution of discovered molecular alterations to brain tumor pathogenesis.
Project description:Neurofibromatosis type 1 (NF1) is one of the most common brain tumor predisposition syndromes, in which affected children are prone to the development of low-grade gliomas. While NF1-associated gliomas can be found in several brain regions, the majority arise in the optic nerves, chiasm, tracts, and radiations (optic pathway gliomas; OPGs). Owing to their location, 35-50% of affected children present with reduced visual acuity. Unfortunately, despite tumor stabilization following chemotherapy, vision does not improve in most children. For this reasons, more effective therapies are being sought that reflect a deeper understanding of the NF1 gene and the use of authenticated Nf1 genetically-engineered mouse strains. The implementation of these models for drug discovery and validation has galvanized molecularly-targeted clinical trials in children with NF1-OPG. Future research focused on defining the cellular and molecular factors that underlie optic glioma development and progression also has the potential to provide personalized risk assessment strategies for this pediatric population.
Project description:Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours<sup>1</sup>. Although the role of neurons in tumour progression has previously been demonstrated<sup>2</sup>, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood<sup>3,4</sup>, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)<sup>5</sup> to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.
Project description:Individuals with neurofibromatosis type 1 (NF1) are prone to develop optic pathway gliomas that can result in significant visual impairment. To explore the cellular basis for the reduced visual function resulting from optic glioma formation, we used a genetically engineered mouse model of Nf1 optic glioma (Nf1+/-(GFAP)CKO mice). We performed multimodal functional and structural analyses both before and after the appearance of macroscopic tumors. At 6 weeks of age, before obvious glioma formation, Nf1+/-(GFAP)CKO mice had decreased visual-evoked potential amplitudes and increased optic nerve axon calibers. By 3 months of age, Nf1+/-(GFAP)CKO mice exhibited pronounced optic nerve axonopathy and apoptosis of neurons in the retinal ganglion cell layer. Magnetic resonance diffusion tensor imaging showed a progressive increase in radial diffusivity between 6 weeks and 6 months of age in the optic nerve proximal to the tumor indicating ongoing deterioration of axons. These data suggest that optic glioma formation results in early axonal disorganization and damage, which culminates in retinal ganglion cell death. Collectively, this study shows that Nf1+/-(GFAP)CKO mice can provide a useful model for defining mechanisms of visual abnormalities in children with NF1 and lay the foundations for future interventional studies aimed at reducing visual loss.
Project description:Approximately 20% of patients with Neurofibromatosis type 1 (NF1) develop optic pathway gliomas (OPGs). Not all OPGs in NF1 necessarily become vision compromising and predicting which patients might develop visual decline is difficult at present time. Optical coherence tomography (OCT) has emerged as a useful tool able to directly assess the morphology and thickness of individual retinal layers. The ganglion cell layer (GCL) is composed of the retinal ganglion cells which receive information from photoreceptors via interneurons, while the retinal nerve fiber layer (RNFL) contains the retinal ganglion cell unmyelinated axons that merge to form the optic nerve. Lesions of the anterior visual pathway result in retrograde axonal degeneration from ganglion cell death and ultimately manifest as thinning of the RNFL and/or GCL. In this report we highlight a case of a 38 year-old woman with an NF1 associated left chiasmal and optic tract glioma who had normal visual fields and visual acuity. However, using OCT we demonstrate a homonymous pattern of GCL atrophy that corresponds with her left optic tract glioma. Given this homonymous pattern of atrophy in the GCL and the left optic tract lesion, one would expect a right homonymous hemianopia. To our knowledge this is the first reported case of a homonymous pattern of GCL-IPL atrophy in an adult with an NF1 related OPG involving the optic chiasm and optic tract, but without objective visual field or acuity deficits. This case is important because, mechanistically, it suggests that a necessary threshold of GCL atrophy may be needed before visual concerns can be detected and, secondly, it invites future studies to evaluate whether OCT may serve as a potential screening tool for those with NF1 related OPGs.
Project description:The purpose of this study was to investigate the progression of changes in retinal ganglion cells and optic nerve glia in neurofibromatosis-1 (NF1) genetically-engineered mice with optic glioma. Optic glioma tumors were generated in Nf1+/- mice lacking Nf1 expression in GFAP+ cells (astrocytes). Standard immunohistochemistry methods were employed to identify astrocytes (GFAP, S100beta), proliferating progenitor cells (sox2, nestin), microglia (Iba1), endothelial cells (CD31) and retinal ganglion cell (RGC) axons (Neurofilament 68k) in Nf1+/-, Nf1(GFAP)CKO (wild-type mice with Nf1 loss in glial cells), and Nf1+/-(GFAP)CKO (Nf1+/- mice with Nf1 loss in glial cells) mice. Ultrastructural changes in the optic chiasm and nerve were assessed by electron microscopy (EM). RGC were counted in whole retina preparations using high-resolution, mosaic confocal microscopy following their delineation by retrograde FluoroGold labeling. We found that only Nf1+/-(GFAP)CKO mice exhibited gross pre-chiasmatic optic nerve and chiasm enlargements containing aggregated GFAP+/nestin+ and S100beta+/sox2+ cells (neoplastic glia) as well as increased numbers of blood vessels and microglia. Optic gliomas in Nf1+/-(GFAP)CKO mice contained axon fiber irregularities and multilamellar bodies of degenerated myelin. EM and EM tomographic analyses showed increased glial disorganization, disoriented axonal projections, profiles of degenerating myelin and structural alterations at nodes of Ranvier. Lastly, we found reduced RGC numbers in Nf1+/-(GFAP)CKO mice, supporting a model in which the combination of optic nerve Nf1 heterozygosity and glial cell Nf1 loss results in disrupted axonal-glial relationships, subsequently culminating in the degeneration of optic nerve axons and loss of their parent RGC neurons.
Project description:Neurofibromatosis type 1 is the most commonly inherited human cancer predisposition syndrome. Neurofibromin (NF1) gene mutations lead to increased risk of neurofibromas, schwannomas, low grade, pilocytic optic pathway gliomas, as well as malignant peripheral nerve sheath tumors and glioblastomas. Despite the evidence for NF1 tumor suppressor function in glial cell tumors, the mechanisms underlying transformation remain poorly understood. In this report, we used morpholinos to knockdown the two nf1 orthologs in zebrafish and show that oligodendrocyte progenitor cell (OPC) numbers are increased in the developing spinal cord, whereas neurons are unaffected. The increased OPC numbers in nf1 morphants resulted from increased proliferation, as detected by increased BrdU labeling, whereas TUNEL staining for apoptotic cells was unaffected. This phenotype could be rescued by the forced expression of the GTPase-activating protein (GAP)-related domain of human NF1. In addition, the in vivo analysis of OPC migration following nf1 loss using time-lapse microscopy demonstrated that olig2-EGFP(+) OPCs exhibit enhanced cell migration within the developing spinal cord. OPCs pause intermittently as they migrate, and in nf1 knockdown animals, they covered greater distances due to a decrease in average pause duration, rather than an increase in velocity while in motion. Interestingly, nf1 knockdown also leads to an increase in ERK signaling, principally in the neurons of the spinal cord. Together, these results show that negative regulation of the Ras pathway through the GAP activity of NF1 limits OPC proliferation and motility during development, providing insight into the oncogenic mechanisms through which NF1 loss contributes to human glial tumors.
Project description:Optic pathway gliomas (OPGs) are present in 20% of children with neurofibromatosis 1 (NF1) but are less frequently observed in adults. Our goal was to determine the natural history of OPGs in children and adults with NF1.We analyzed the features of OPGs and other intracranial lesions on 1775 head MRI scans of 562 unselected adults and children with NF1 collected between 2003 and 2015. 52 (9.3%) of 562 patients in this study had an OPG diagnosed on their MRI. The median age at first scan with an OPG present was 12.7 years. Of the 52 OPG patients, the intraorbital optic nerves were affected in 29 patients (56%), the prechiasmatic optic nerves were affected in 32 patients (62%), the optic chiasm was affected in 17 patients (33%) and the optic radiations were affected in 19 patients (37%). 29 patients had two or more areas affected. One patient had a newly-appearing OPG, and 1 patient showed progression. The rate of progression over 5 years was 2.4% (95% CI: 0.4% to 16%). Four patients showed partial regression of their OPGs, but we observed no case of complete regression during this study. The rate of regression over 5 years was 8.9% (95% confidence intervals: 2.8% to 26%). We found the presence of UBOs and the presence of OPGs in individual patients to be highly associated (p?=?0.0061).OPGs are more common in older adults with NF1 than previously thought. The occurrences of unidentified bright objects (UBOs) and asymptomatic OPGs are associated with each other. This suggests the possibility that OPGs that remain asymptomatic may differ pathogenically from those that become symptomatic.
Project description:<h4>Objective</h4>To determine whether tumor size is associated with retinal nerve fiber layer (RNFL) thickness, a measure of axonal degeneration and an established biomarker of visual impairment in children with optic pathway gliomas (OPGs) secondary to neurofibromatosis type 1 (NF1).<h4>Methods</h4>Children with NF1-OPGs involving the optic nerve (extension into the chiasm and tracts permitted) who underwent both volumetric MRI analysis and optical coherence tomography (OCT) within 2 weeks of each other were included. Volumetric measurement of the entire anterior visual pathway (AVP; optic nerve, chiasm, and tract) was performed using high-resolution T1-weighted MRI. OCT measured the average RNFL thickness around the optic nerve. Linear regression models evaluated the relationship between RNFL thickness and AVP dimensions and volume.<h4>Results</h4>Thirty-eight participants contributed 55 study eyes. The mean age was 5.78 years. Twenty-two participants (58%) were female. RNFL thickness had a significant negative relationship to total AVP volume and total brain volume (p < 0.05, all comparisons). For every 1 mL increase in AVP volume, RNFL thickness declined by approximately 5 microns. A greater AVP volume of OPGs involving the optic nerve and chiasm, but not the tracts, was independently associated with a lower RNFL thickness (p < 0.05). All participants with an optic chiasm volume >1.3 mL demonstrated axonal damage (i.e., RNFL thickness <80 microns).<h4>Conclusions</h4>Greater OPG and AVP volume predicts axonal degeneration, a biomarker of vision loss, in children with NF1-OPGs. MRI volumetric measures may help stratify the risk of visual loss from NF1-OPGs.