Oligodendrocyte RasG12V Expressed in its Endogenous Locus Disrupts Myelin Structure Through Increased MAPK, Nitric Oxide, and Notch Signaling

Costello syndrome (CS) is a gain of function Rasopathy caused by heterozygous activating mutations in the HRAS gene. Patients show brain dysfunction that can include abnormal brain white matter. Transgenic activation of HRas in the entire mouse oligodendrocyte lineage resulted in myelin defects and behavioral abnormalities, suggesting roles for disrupted myelin in CS brain dysfunction. Here we studied a mouse model in which the endogenous HRas gene is conditionally replaced by mutant HRasG12V in mature oligodendrocytes, to separate effects in mature myelinating cells from developmental events. Increased myelin thickness due to decompaction was detectable within one month of HRasG12V expression in the corpus callosum of adult mice. Increases in active ERK and Nitric Oxide (NO) were present in HRas mutants and inhibition of NO synthase (NOS) or MEK each partially rescued myelin decompaction. In addition, genetic or pharmacologic inhibition of Notch signaling improved myelin compaction. Complete rescue of myelin structure required dual drug treatments combining MAPK, NO or Notch inhibition; with MEK + NOS blockade producing the most robust effect. We suggest that individual or concomitant blockade of these pathways in Costello syndrome patients may improve aspects of brain function

Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of neurofibromatosis type 1.

Background: Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes affected individuals to formation of benign neurofibromas, peripheral nerve tumors that can be associated with significant morbidity. Loss of the NF1 Ras-GAP protein causes increased Ras-GTP, and we previously found that inhibiting MEK signaling downstream of Ras can shrink established neurofibromas in a genetically engineered murine model. Procedures: We studied effects of MEK inhibition using 1.5 mg/kg/day PD-0325901 prior to neurofibroma onset in the Nf1 flox/flox;Dhh-Cre mouse model. We also treated mice with established tumors at 0.5 and 1.5 mg/kg/day dosees of PD-0325901. We monitored tumor volumes using MRI and volumetric measurements, and measured pharmacokinetic and pharmacodynamic endpoints. Results: Early administration significantly delayed neurofibroma development as compared to vehicle controls. When treatment was discontinued neurofibromas grew, but no rebound effect was observed and neurofibromas remained significantly smaller than controls. Low dose treatment of mice with PD-0325901 resulted in neurofibroma shrinkage equivalent to that observed at higher doses. Tumor cell proliferation decreased, although less than at higher doses with drug. Tumor blood vessels per area correlated with tumor shrinkage. Conclusions: Neurofibroma development was not prevented by MEK inhibition, beginning at 1 month of age, but tumor size was controlled by early treatment. Moreover, treatment with PD-0325901 at very low doses may shrink neurofibromas while minimizing toxicity. These studies highlight how genetically engineered mouse models can guide clinical trial design

Oligodendrocyte Nf1 Controls Aberrant Notch Activation and Regulates Myelin Structure and Behavior

The RASopathy neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic disorders. In NF1 patients, neurological issues may result from damaged myelin, and mice with a neurofibromin gene (Nf1) mutation show white matter (WM) defects including myelin decompaction. Using mouse genetics, we find that altered Nf1 gene-dose in mature oligodendrocytes results in progressive myelin defects and behavioral abnormalities mediated by aberrant Notch activation. Blocking Notch, upstream mitogen-activated protein kinase (MAPK), or nitric oxide signaling rescues myelin defects in hemizygous Nf1 mutants, and pharmacological gamma secretase inhibition rescues aberrant behavior with no effects in wild-type (WT) mice. Concomitant pathway inhibition rescues myelin abnormalities in homozygous mutants. Notch activation is also observed in Nf1+/− mouse brains, and cells containing active Notch are increased in NF1 patient WM. We thus identify Notch as an Nf1 effector regulating myelin structure and behavior in a RASopathy and suggest that inhibition of Notch signaling may be a therapeutic strategy for NF1

Genetically engineered minipigs model the major clinical features of human neurofibromatosis type 1.

Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies

Substance P-immunoreactivity in the developing human retinogeniculate pathway

Substance P has been immunohistochemically localized in the human optic nerves and lateral geniculate nuclei during the prenatal period from 13-14 to 37 weeks of gestation. Substance P-immunoreactive fibres were present in the optic nerves and lateral geniculate nuclei in all these ages thereby providing direct evidence of this undecapeptide being associated with the retinogeniculate pathway. At 16-17 weeks, greater numbers of fibres were observed than in the later ages. It is likely that the reduction in number of optic nerve fibres seen quantitatively during prenatal life may partly be due to the loss of substance P fibres

How Loss of Neurofibromin in Oligodendrocytes Affects the Brain

Neurofibromatosis type 1 patients are predisposed to central nervous system (CNS) phenotypes including enlarged brains, delayed acquisition of motor skills, brain tumors, and cognitive deficits. Imaging and pathologic analysis suggest that changes in white matter myelination may underlie both the enlargement of white matter tracts that contributes to megancephaly, and/or hyper-intense signals visualized on MRI. To study the role(s) of Nf1 and HRasin oligodendrocytes, we examined the optic nerve and corpus callosum,myelinated fiber tracts.We studiedNf1heterozygous mice,tamoxifen-induced Nf1 loss in mature oligodendrocytes (Plp-CreERT), and a new transgenic model in which the CNPase promoter drives expression of HRasG12V. Activated HRas and loss of Nf1 within oligo-lineage cells (PLPCre; Nf1fl+; &PLPCre; Nf1fl/fl) resulted in optic nerve enlargement. The corpus callosum of CNP-HRasG12V mice was also enlarged. Electron microscopy analysis revealed 3 phenotypes within the enlarged optic nerves. 1)When Nf1 was lost or HRas was activated within oligodendrocytes, the myelin was decompacted due to splitting at the intraperiod lines. The transgenic Nf1+/- mice, in which Nf1 loss is not restricted to oligo-lineage cells, displayed lesser myelin decompaction, and these mice did not have significantly enlarged optic nerves. 2) Enlarged axons accompanied the decompacted myelin within all models. 3) All Nf1 and Ras mouse models also showed an expansion of the perivascular astrocyticendfeet surrounding the vasculature. These phenotypes were also found within the corpus callosum. Thus, myelin and vascular phenotypesare not limited to a single myelinated fiber tract. These studies reveal a cell autonomous role for the Nf1/Ras pathway in the regulation of myelin compaction, and a non-cell autonomous role in the regulation of astrocyticendfeet surrounding brain capillaries

How Loss of Neurofibromin in Oligodendrocytes Affects the Brain

Neurofibromatosis type 1 patients are predisposed to central nervous system (CNS) phenotypes including enlarged brains, delayed acquisition of motor skills, brain tumors, and cognitive deficits. Imaging and pathologic analysis suggest that changes in white matter myelination may underlie both the enlargement of white matter tracts that contributes to megancephaly, and/or hyper-intense signals visualized on MRI. To study the role(s) of Nf1 and HRasin oligodendrocytes, we examined the optic nerve and corpus callosum,myelinated fiber tracts.We studiedNf1heterozygous mice,tamoxifen-induced Nf1 loss in mature oligodendrocytes (Plp-CreERT), and a new transgenic model in which the CNPase promoter drives expression of HRasG12V. Activated HRas and loss of Nf1 within oligo-lineage cells (PLPCre; Nf1fl+; &PLPCre; Nf1fl/fl) resulted in optic nerve enlargement. The corpus callosum of CNP-HRasG12V mice was also enlarged. Electron microscopy analysis revealed 3 phenotypes within the enlarged optic nerves. 1)When Nf1 was lost or HRas was activated within oligodendrocytes, the myelin was decompacted due to splitting at the intraperiod lines. The transgenic Nf1+/- mice, in which Nf1 loss is not restricted to oligo-lineage cells, displayed lesser myelin decompaction, and these mice did not have significantly enlarged optic nerves. 2) Enlarged axons accompanied the decompacted myelin within all models. 3) All Nf1 and Ras mouse models also showed an expansion of the perivascular astrocyticendfeet surrounding the vasculature. These phenotypes were also found within the corpus callosum. Thus, myelin and vascular phenotypesare not limited to a single myelinated fiber tract. These studies reveal a cell autonomous role for the Nf1/Ras pathway in the regulation of myelin compaction, and a non-cell autonomous role in the regulation of astrocyticendfeet surrounding brain capillaries

Perinatal or Adult \u3cem\u3eNf1\u3c/em\u3e Inactivation using Tamoxifen-inducible PlpCre Each Cause Neurofibroma Formation

OBJECTIVES Neurofibromas are tumors initiated by biallelic mutation of the NF1 tumor suppressor gene in the Schwann cell lineage. One idea within the field suggests that Nf1loss must occur within progenitor cells present within a critical window during Schwann cell development in order for neurofibromas to form. To test this hypothesis and to examine whethermyelinating Schwann cells can serve as aneurofibroma cell of origin, Nf1 loss was induced at perinatal or adult timepoints using a tamoxifen-inducible Plp-CreERT driver. RESULTS Perinatal loss of Nf1 resulted in small neurofibromas late in life, while adult loss caused large neurofibromas and morbidity beginning 4 months after onset of Nf1loss. PLP-CreERT recombination (EGFP+ cells) occurred in: satellite cells, S100β+ myelinating Schwann cells, and p75+ cells. Plp-CreERTnerves and neurofibromas contained cells with Remak bundle disruption; however, no recombination within GFAP+ non-myelinating Schwann cells was identified. Extramedullarylympho-hematopoietic expansion that contained EGFP+/Sca-1+ stromal cells amongst EGFP-negative lympho-hematopoietic cells was also observed. CONCLUSIONS/SIGNIFICANCE Neurofibroma formation is not restricted to loss of Nf1 in embryonic life, but can be triggered by Nf1 loss throughout life.Although all neurofibroma models and human samples have Remak bundle disruption (leading to the assumption that Nf1 loss within the non-myelinating Schwann cell may be vital for tumor formation), there was no EGFP+ recombination within GFAP+ non-myelinating Schwann cells – eliminating the GFAP+ non-myelinating Schwann cell as the cell of origin for neurofibroma formation

Oral 1821-3 - Ras Signaling and No in Oligodendrocytes Modulate Permeability of the Blood–Brain Barrier

Background: Ras is a small G-protein switch, activated when GTP-bound; in the GTP-bound state Ras signals to downstream effector proteins. GTPase inactivating proteins (GAPs) including neurofibromain (Nf1) accelerate hydrolysis of Ras-GTP to inactive Ras-GDP. We showed that activation of Ras or loss of Nf1 in brain oligodendrocytes correlates with myelin decompaction, down-regulation of claudins, and down-regulation and mis-localization of connexins (Mayes et al., Cell Reports, 2013). Non-cell autonomous defects in perivascular astrocytes and blood–brain barrier (BBB) are also observed. The blood–brain barrier becomes leaky, implicating a soluble mediator. NO and nitric oxide synthases (NOS1-3) are up-regulated in mutant white matter. Treating mice with the NOS inhibitor L-NAME or the antioxidant NAC corrected cellular phenotypes. Consistent with recent data implicating white matter changes in learning, CNP-HRasG12V mice displayed locomotor hyperactivity which could be rescued by antioxidant treatment. Results: Using flow cytometry to define reactive species in cells in sorted brain non-neuronal cells we have now identified alterations in reactive species (NO, SO and/or peroxynitrites) in oligodendrocyte precursors, oligodendrocytes, astrocytes, pericytes and endothelial cells when Ras-GTP is elevated in oligodendrocytes. We also found that many changes are absent in Nf1+/− mice, when cells in the brain in addition to oligodendrocytes are hemizygous for Nf1 loss. Mating mice in which oligodendrocyte only have activated Ras with Nf1+/− mice confirmed that effects on reactive species are mediated by hemizygosity in Nf1+/− astrocytes, pericytes and/or endothelial cells. FACs analysis showed that NO is significantly diminished in oligodendrocytes in these mutants, and shuttled to surrounding cells of the vasculature. Conclusion: We conclude that signaling between oligodendrocytes and cells of white matter blood vessels contributes to homeostasis of the blood–brain barrier. NR is supported by the NIH, the DOD Program in Neurofibromatosis and the Children\u27s Tumor Foundation