Mouse Model Of Neurofibromatosis Type Research Articles

NF1 mutation-driven neuronal hyperexcitability sets a threshold for tumorigenesis and therapeutic targeting of murine optic glioma.

With the recognition that noncancerous cells function as critical regulators of brain tumor growth, we recently demonstrated that neurons drive low-grade glioma initiation and progression. Using mouse models of neurofibromatosis type 1 (NF1)-associated optic pathway glioma (OPG), we showed that Nf1 mutation induces neuronal hyperexcitability and midkine expression, which activates an immune axis to support tumor growth, such that high-dose lamotrigine treatment reduces Nf1-OPG proliferation. Herein, we execute a series of complementary experiments to address several key knowledge gaps relevant to future clinical translation. We leverage a collection of Nf1-mutant mice that spontaneously develop OPGs to alter both germline and retinal neuron-specific midkine expression. Nf1-mutant mice harboring several different NF1 patient-derived germline mutations were employed to evaluate neuronal excitability and midkine expression. Two distinct Nf1-OPG preclinical mouse models were used to assess lamotrigine effects on tumor progression and growth in vivo. We establish that neuronal midkine is both necessary and sufficient for Nf1-OPG growth, demonstrating an obligate relationship between germline Nf1 mutation, neuronal excitability, midkine production, and Nf1-OPG proliferation. We show anti-epileptic drug (lamotrigine) specificity in suppressing neuronal midkine production. Relevant to clinical translation, lamotrigine prevents Nf1-OPG progression and suppresses the growth of existing tumors for months following drug cessation. Importantly, lamotrigine abrogates tumor growth in two Nf1-OPG strains using pediatric epilepsy clinical dosing. Together, these findings establish midkine and neuronal hyperexcitability as targetable drivers of Nf1-OPG growth and support the use of lamotrigine as a potential chemoprevention or chemotherapy agent for children with NF1-OPG.

Neurofibromatosis type 1-dependent alterations in mouse microglia function are not cell-intrinsic

We previously discovered a sex-by-genotype defect in microglia function using a heterozygous germline knockout mouse model of Neurofibromatosis type 1 (Nf1 ± mice), in which only microglia from male Nf1 ± mice exhibited defects in purinergic signaling. Herein, we leveraged an unbiased proteomic approach to demonstrate that male, but not female, heterozygous Nf1 ± microglia exhibit differences in protein expression, which largely reflect pathways involved in cytoskeletal organization. In keeping with these predicted defects in cytoskeletal function, only male Nf1 ± microglia had reduced process arborization and surveillance capacity. To determine whether these microglial defects were cell autonomous or reflected adaptive responses to Nf1 heterozygosity in other cells in the brain, we generated conditional microglia Nf1-mutant knockout mice by intercrossing Nf1flox/flox with Cx3cr1-CreER mice (Nf1flox/wt; Cx3cr1-CreER mice, Nf1MG ± mice). Surprisingly, neither male nor female Nf1MG ± mouse microglia had impaired process arborization or surveillance capacity. In contrast, when Nf1 heterozygosity was generated in neurons, astrocytes and oligodendrocytes by intercrossing Nf1flox/flox with hGFAP-Cre mice (Nf1flox/wt; hGFAP-Cre mice, Nf1GFAP ± mice), the microglia defects found in Nf1 ± mice were recapitulated. Collectively, these data reveal that Nf1 ± sexually dimorphic microglia abnormalities are likely not cell-intrinsic properties, but rather reflect a response to Nf1 heterozygosity in other brain cells.

Cerebellum-dependent associative learning is not impaired in a mouse model of neurofibromatosis type 1

Individuals with Neurofibromatosis type 1 (NF1) experience a high degree of motor problems. The cerebellum plays a pivotal role in motor functioning and the NF1 gene is highly expressed in cerebellar Purkinje cells. However, it is not well understood to what extent NF1 affects cerebellar functioning and how this relates to NF1 motor functioning. Therefore, we subjected global Nf1+/− mice to a cerebellum-dependent associative learning task, called Pavlovian eyeblink conditioning. Additionally, we assessed general motor function and muscle strength in Nf1+/− mice. To our surprise, we found that Nf1+/− mice showed a moderately increased learning rate of conditioned eyeblink responses, as well as improved accuracy in the adaptive timing of the eyeblink responses. Locomotion, balance, general motor function, and muscle strength were not affected in Nf1+/− mice. Together, our results support the view that cerebellar function in Nf1+/− mice is unimpaired.

Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2

Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.

Treatment during a developmental window prevents NF1-associated optic pathway gliomas by targeting Erk-dependent migrating glial progenitors.

The mechanism of vulnerability to pediatric low-grade gliomas (pLGGs)-the most common brain tumor in children-during development remains largely unknown. Using mouse models of neurofibromatosis type 1 (NF1)-associated pLGGs in the optic pathway (NF1-OPG), we demonstrate that NF1-OPG arose from the vulnerability to the dependency of Mek-Erk/MAPK signaling during gliogenesis of one of the two developmentally transient precursor populations in the optic nerve, brain-derived migrating glial progenitors (GPs), but not local progenitors. Hyperactive Erk/MAPK signaling by Nf1 loss overproduced GPs by disrupting the balance between stem-cell maintenance and gliogenesis of hypothalamic ventricular zone radial glia (RG). Persistence of RG-like GPs initiated NF1-OPG, causing Bax-dependent apoptosis in retinal ganglion cells. Removal of three Mek1/Mek2 alleles or transient post-natal treatment with a low-dose MEK inhibitor normalized differentiation of Nf1-/- RG-like GPs, preventing NF1-OPG formation and neuronal degeneration. We provide the proof-of-concept evidence for preventing pLGGs before tumor-associated neurological damage enters an irreversible phase.

PAK1 inhibition reduces tumor size and extends the lifespan of mice in a genetically engineered mouse model of Neurofibromatosis Type 2 (NF2).

Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.

Identifying Bone Matrix Impairments in a Mouse Model of Neurofibromatosis Type 1 (NF1) by Clinically Translatable Techniques.

Three-to-four percent of children with neurofibromatosis type 1 (NF1) present with unilateral tibia bowing, fracture, and recalcitrant healing. Alkaline phosphatase (ALP) enzyme therapy prevented poor bone mineralization and poor mechanical properties in mouse models of NF1 skeletal dysplasia; but transition to clinical trials is hampered by the lack of a technique that (i) identifies NF1 patients at risk of tibia bowing and fracture making them eligible for trial enrollment and (ii) monitors treatment effects on matrix characteristics related to bone strength. Therefore, we assessed the ability of matrix-sensitive techniques to provide characteristics that differentiate between cortical bone from mice characterized by postnatal loss of Nf1 in Osx-creTet-Off ;Nf1flox/flox osteoprogenitors (cKO) and from wild-type (WT) mice. Following euthanasia at two time points of bone disease progression, femur and tibia were harvested from both genotypes (n ≥ 8/age/sex/genotype). A reduction in the mid-diaphysis ultimate force during three-point bending at 20 weeks confirmed deleterious changes in bone induced by Nf1 deficiency, regardless of sex. Pooling females and males, low bound water (BW), and low cortical volumetric bone mineral density (Ct.vBMD) were the most accurate outcomes in distinguishing cKO from WT femurs with accuracy improving with age. Ct.vBMD and the average unloading slope (Avg-US) from cyclic reference point indentation tests were the most sensitive in differentiating WT from cKO tibias. Mineral-to-matrix ratio and carbonate substitution from Raman spectroscopy were not good classifiers. However, when combined with Ct.vBMD and BW (femur), they helped predict bending strength. Nf1 deficiency in osteoprogenitors negatively affected bone microstructure and matrix quality with deficits in properties becoming more pronounced with duration of Nf1 deficiency. Clinically measurable without ionizing radiation, BW and Avg-US are sensitive to deleterious changes in bone matrix in a preclinical model of NF1 bone dysplasia and require further clinical investigation as potential indicators of an onset of bone weakness in children with NF1. © 2022 American Society for Bone and Mineral Research (ASBMR).

TMOD-06. MALIGNANT TRANSFORMATION OF PLEXIFORM NEUROFIBROMAS IN AN NF1 MUTANT MOUSE MODEL OF SPINAL IRRADIATION

Abstract PURPOSE Understanding the relationships between ionizing radiation (IR) and carcinogenesis, as well as other variables, including genetic background and sex, may minimize risks of transformation from benign to aggressive cancers while maintaining treatment efficacy. We aim to understand the relationship between IR, tumor microenvironments, and effect of sex on malignant transformation using a conditional knockout mouse model of Neurofibromatosis type 1 (NF1). METHODS Conditional knockout mouse models of plexiform neurofibromas (PeriCre+; Nf1fl/fl and PeriCre+; Nf1fl/-, n = 172) were treated with 0 Gy; 3 Gy x 5; 3 Gy x 10 of focal, fractionated spinal irradiation (SI) and aged until signs of illness. Histopathological analysis was performed on images from H&E-stained FFPE peripheral nerve tissues. RESULTS SI affected the survival of mice in a dose-dependent manner, with a significant decrease at 30Gy (median survival 254 and 218 days for 0 Gy and 30 Gy, p < 0.01, Log-rank test). Male mice had reduced overall survival at 15 and 30 Gy (median survival 368, 278 and 245 days for 0, 15 and 30Gy, p < 0.01, Log-rank test), but not in female mice, indicating sex-specific radiosensitivity. Differences in tumour microenvironments did not affect mouse survival after SI. Histopathological analysis is ongoing (43 out of 76 FFPE samples analyzed). Preliminary findings showed a total of 43 malignancies ranging from plexiform neurofibromas (PNs) to high-grade malignant peripheral nerve sheath tumours (MPNSTs). Increasing histologic aggressiveness was observed in higher SI doses, with high-grade MPNST (2.3%) exclusively found in mice irradiated at 30 Gy. CONCLUSION We present a novel mouse model to study malignant transformation in the context of NF1. Preliminary findings suggest increased malignant transformation at high doses of SI.

Disruption of Critical Period Plasticity in a Mouse Model of Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a common monogenic neurodevelopmental disorder associated with physical and cognitive problems. The cognitive issues are thought to arise from increased release of the neurotransmitter GABA. Modulating the signaling pathways causing increased GABA release in a mouse model of NF1 reverts deficits in hippocampal learning. However, clinical trials based on these approaches have so far been unsuccessful. We therefore used a combination of slice electrophysiology, in vivo two-photon calcium imaging, and optical imaging of intrinsic signal in a mouse model of NF1 to investigate whether cortical development is affected in NF1, possibly causing lifelong consequences that cannot be rescued by reducing inhibition later in life. We find that, in NF1 mice of both sexes, inhibition increases strongly during the development of the visual cortex and remains high. While this increase in cortical inhibition does not affect spontaneous cortical activity patterns during early cortical development, the critical period for ocular dominance plasticity is shortened in NF1 mice due to its early closure but unaltered onset. Notably, after environmental enrichment, differences in inhibitory innervation and ocular dominance plasticity between NF1 mice and WT littermates disappear. These results provide the first evidence for critical period dysregulation in NF1 and suggest that treatments aimed at normalizing levels of inhibition will need to start at early stages of development.SIGNIFICANCE STATEMENT Neurofibromatosis type 1 is associated with cognitive problems for which no treatment is currently available. This study shows that, in a mouse model of neurofibromatosis type 1, cortical inhibition is increased during development and critical period regulation is disturbed. Rearing the mice in an environment that stimulates cognitive function overcomes these deficits. These results uncover critical period dysregulation as a novel mechanism in the pathogenesis of neurofibromatosis type 1. This suggests that targeting the affected signaling pathways in neurofibromatosis type 1 for the treatment of cognitive disabilities may have to start at a much younger age than has so far been tested in clinical trials.

Increased epileptogenicity in a mouse model of neurofibromatosis type 1

RationaleNeurofibromatosis type 1 (NF1) is associated with higher rates of epilepsy compared to the general population. Some NF1 patients with epilepsy do not have intracranial lesions, suggesting the genetic mutation itself may contribute to higher rates of epilepsy in these patients. We have recently demonstrated increased seizure susceptibility in the Nf1+/− mouse, but it is unknown whether this model displays altered epileptogenicity, as has been reported in patients with NF1. The aim of this study was to determine whether the Nf1+/− mouse is more susceptible to electrical kindling-induced epileptogenesis. MethodsYoung male or female adult Nf1+/− or Nf1+/+ (wild-type; WT) mice were implanted with electrodes for neocortical or hippocampal kindling paradigms. Neocortical kindling was performed for 40 stimulation sessions followed by baseline EEG monitoring to detect possible SRSs. Hippocampal kindling was performed with a modified extended kindling paradigm, completed to a maximum of 80 sessions to try to induce spontaneous repetitive seizures (SRSs). Western blot assays were performed in naïve and kindled mice to compare levels of Akt and MAPK (ERK1/2), proteins downstream of the NF1 mutation. ResultsThe average initial neocortical after-discharge threshold (ADT) was significantly lower in the Nf1+/− group, which also required fewer stimulations to reach stage 5 seizure, had greater average seizure severity across all kindling sessions, had a greater number of convulsive seizures, and had a faster progression of after-discharge duration and Racine score during kindling. No WT mice exhibited SRS after neocortical kindling, versus 33% of Nf1+/− mice. The average initial hippocampal ADT was not significantly different between the WT and Nf1+/− groups, nor was there a difference in the number of stimulations required to reach the kindled state. The WT group had a significantly higher average seizure severity across all kindling sessions as compared with the Nf1+/− mice. The WT group also had faster progression of the Racine seizure score over the kindling sessions, mainly due to a faster increase in seizures severity early during the kindling process. However, SRSs were seen in 50% of Nf1+/− mice after modified extended kindling and in no WT mice. Western blots showed hippocampal kindling increased the ratio of phosphorylated/total Akt in both the WT and Nf1+/− mice, while neocortical kindling led to increased ratios of phosphorylated/total Akt and MAPK in Nf1+/− mice only. ConclusionsWe have demonstrated for the first time an increased rate of epileptogenesis in an animal model of NF1 with no known macroscopic/neoplastic brain lesions. This work provides evidence for the genetic mutation itself playing a role in seizures and epilepsy in patients with NF1, and supports the use of the Nf1+/− mouse model in future mechanistic studies.

Early administration of imatinib mesylate reduces plexiform neurofibroma tumor burden with durable results after drug discontinuation in a mouse model of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored. We evaluated the effect of imatinib dosed at 10-100mg/kg/day for 12weeks to one-month-old Nf1flox/flox ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden. Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control. Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.

Increased seizure susceptibility in a mouse model of neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder linked to higher rates of epilepsy as compared with the general population. Although some epilepsy cases in NF1 are related to intracranial lesions, epileptogenic lesions are not always identified. It is unknown whether the genetic mutation itself, which leads to lower levels of the tumor suppressor protein neurofibromin, alters seizure susceptibility. The purpose of this research was to determine whether Nf1+/− mice have altered seizure susceptibility to the chemical convulsants kainic acid and pilocarpine. Young adult Nf1+/− or WT control (Nf1+/+) mice were injected with either 20 mg/kg kainic acid or scopolamine 1 mg/kg and pilocarpine 300 mg/kg and assessed for various behavioral seizure parameters. Another subset of mice were implanted with intracranial electrodes and injected with 10 mg/kg kainic acid for electrographic seizure testing. Histological analyses were performed one week after kainic acid challenge to assess hippocampal damage. A higher proportion of Nf1+/− mice had behavioral seizures after kainic acid or pilocarpine challenge, with shorter seizure latency, longer seizure duration, and higher Racine scores compared to WT mice. Nf1+/− and WT mice with severe behavioral seizures demonstrated similar levels of hippocampal damage. EEG recordings confirmed decreased seizure latency and longer seizure duration in response to KA in the Nf1+/− group. These data demonstrate increased seizure susceptibility in a mouse model of NF1 and support the use of the Nf1+/− mouse for further investigations into the mechanistic link between NF1 and seizures.

Neurofibromin haploinsufficiency results in altered spermatogenesis in a mouse model of neurofibromatosis type 1.

The fertility of men with neurofibromatosis 1 (NF1) is reduced. Despite this observation, gonadal function has not been examined in patients with NF1. In order to assess the role of reduced neurofibromin in the testes, we examined testicular morphology and function in an Nf1+/- mouse model. We found that although Nf1+/- male mice are able to reproduce, they have significantly fewer pups per litter than Nf1+/+ control males. Reduced fertility in Nf1+/- male mice is associated with disorganization of the seminiferous epithelium, with exfoliation of germ cells and immature spermatids into the tubule lumen. Morphometric analysis shows that these alterations are associated with decreased Leydig cell numbers and increased spermatid cell numbers. We hypothesized that hyper-activation of Ras in Nf1+/- males affects ectoplasmic specialization, a Sertoli-spermatid adherens junction involved in spermiation. Consistent with this idea, we found increased expression of phosphorylated ERK, a downstream effector of Ras that has been shown to alter ectoplasmic specialization, in Nf1+/- males in comparison to control Nf1+/+ littermates. These data demonstrate that neurofibromin haploinsufficiency impairs spermatogenesis and fertility in a mouse model of NF1.

Insights into optic pathway glioma vision loss from mouse models of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 gene. The NF1-encoded protein (neurofibromin) is an inhibitor of the oncoprotein RAS and controls cell growth and survival. Individuals with NF1 are prone to developing low-grade tumors of the optic nerves, chiasm, tracts, and radiations, termed optic pathway gliomas (OPGs), which can cause vision loss. A paucity of surgical tumor specimens and of patient-derived xenografts for investigative studies has limited our understanding of human NF1-associated OPG (NF1-OPG). However, mice genetically engineered to harbor Nf1 gene mutations develop optic gliomas that share many features of their human counterparts. These genetically engineered mouse (GEM) strains have provided important insights into the cellular and molecular determinants that underlie mouse Nf1 optic glioma development, maintenance, and associated vision loss, with relevance by extension to human NF1-OPG disease. Herein, we review our current understanding of NF1-OPG pathobiology and describe the mechanisms responsible for tumor initiation, growth, and associated vision loss in Nf1 GEM models. We also discuss how Nf1 GEM and other preclinical models can be deployed to identify and evaluate molecularly targeted therapies for OPG, particularly as they pertain to future strategies aimed at preventing or improving tumor-associated vision loss in children with NF1.

Characterization of early communicative behavior in mouse models of neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder caused by mutation of the NF1 gene, in which 80% of affected children exhibit cognitive and behavioral issues. Based on emerging evidence that NF1 may be an autism predisposition gene, we examined autism spectrum disorder (ASD)-relevant early communicative behavior in Nf1 mouse models and observed alterations in both models. The changes in early communicative behavior in Nf1 mutant mice should motivate further studies into the causative factors and potential treatments for ASD arising in the context of NF1.

Whole tumor RNA-sequencing and deconvolution reveal a clinically-prognostic PTEN/PI3K-regulated glioma transcriptional signature.

The concept that solid tumors are maintained by a productive interplay between neoplastic and non-neoplastic elements has gained traction with the demonstration that stromal fibroblasts and immune system cells dictate cancer development and progression. While less studied, brain tumor (glioma) biology is likewise influenced by non-neoplastic immune system cells (macrophages and microglia) which interact with neoplastic glioma cells to create a unique physiological state (glioma ecosystem) distinct from that found in the normal tissue. To explore this neoplastic ground state, we leveraged several preclinical mouse models of neurofibromatosis type 1 (NF1) optic glioma, a low-grade astrocytoma whose formation and maintenance requires productive interactions between non-neoplastic and neoplastic cells, and employed whole tumor RNA-sequencing and mathematical deconvolution strategies to characterize this low-grade glioma ecosystem as an aggregate of cellular and acellular elements. Using this approach, we demonstrate that optic gliomas generated by altering the germline Nf1 gene mutation, the glioma cell of origin, or the presence of co-existing genetic alterations represent molecularly-distinct tumors. However, these optic glioma tumors share a 25-gene core signature, not found in normal optic nerve, that is normalized by microglia inhibition (minocycline), but not conventional (carboplatin) or molecularly-targeted (rapamycin) chemotherapy. Lastly, we identify a genetic signature conferred by Pten reduction and corrected by PI3K inhibition. This signature predicts progression-free survival in patients with either low-grade or high-grade glioma. Collectively, these findings support the concept that gliomas are composite ecological systems whose biology and response to therapy may be best defined by examining the tumor as a whole.

Social deficits in mouse models of Neurofibromatosis type 1 and Legius syndrome

Event Abstract Back to Event Social deficits in mouse models of Neurofibromatosis type 1 and Legius syndrome Sarah C. Borrie1*, Ellen Plasschaert1, Ype Elgersma2, Steven A. Kushner2, Eric Legius1 and Hilde Brems1 1 KU Leuven, Department of Human Genetics, Belgium 2 Erasmus Medical Center, Netherlands Neurofibromatosis type 1 (NF1) and Legius syndrome are neuro-cardio-facial-cutaneous (NCFC) disorders stemming from mutations in the RAS – MAPK pathway, in the NF1 and SPRED1 genes respectively. NF1 is a RAS-GAP protein, negatively regulating activation of RAS, while SPRED1 acts further downstream in the RAS-MAPK pathway, negatively regulating the activation of RAF1 by RAS. Common to these disorders are neurological problems including cognitive deficits and behavioral problems. Patients with NF1 exhibit deficits in memory and executive functioning, and have a high incidence of autism sprectrum disorder (ASD). Legius syndrome patients present a milder cognitive phenotype compared to NF1 patients, and ADHD and attentional problems are also reported. A mouse model of NF1, Nf1+/-, exhibits spatial memory and attentional impairments consistent with the human disorder, and the Spred1-/- mouse model for Legius syndrome also demonstrates spatial learning deficits. However, it is not known whether these models also recapitulate ASD-like symptoms seen in NF1. To further study this phenomenon, we studied social behaviors in Nf1+/- mice and Spred1-/- and Spred1+/- mice, to ask if social deficits are observed in these models, and whether any observed deficits can be rescued. Spred1-/- and Spred1+/- mice displayed abnormal social behavior in the automated tube test. Both genotypes won more encounters against wildtype littermates than expected by chance, indicative of disrupted social behavior. Other social behaviors were also impaired in Spred1-/- mice, including nesting, an intrinsic social behavior. Social deficits in Spred1-/- could be reversed in adult mice by inhibiting the RAS-MAPK pathway, suggesting that RAS – MAPK pathway overactivation underlies altered social behavior in this model. Studies in Nf1+/- mice show that similar social deficits are observed in the automated tube test. Other social behaviors are also being tested, and the role of RAS –MAPK signaling in mediating this phenotype in Nf1+/- is being investigated with pharmacological studies. Collectively these findings suggest that deficits in social behaviors that are core symptoms of NCFC syndromes can be reliably modelled in mouse models of NF1 and Legius syndrome, and that disruptions in RAS-MAPK signaling are important for these behavior alterations. Keywords: RAS-MAPK, NF1, Legius syndrome, ASD, Social Behaviour Conference: 12th National Congress of the Belgian Society for Neuroscience, Gent, Belgium, 22 May - 22 May, 2017. Presentation Type: Poster Presentation Topic: Disorders of the Nervous System Citation: Borrie SC, Plasschaert E, Elgersma Y, Kushner SA, Legius E and Brems H (2019). Social deficits in mouse models of Neurofibromatosis type 1 and Legius syndrome. Front. Neurosci. Conference Abstract: 12th National Congress of the Belgian Society for Neuroscience. doi: 10.3389/conf.fnins.2017.94.00027 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 24 Apr 2017; Published Online: 25 Jan 2019. * Correspondence: Dr. Sarah C Borrie, KU Leuven, Department of Human Genetics, Leuven, Belgium, sarah.borrie@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sarah C Borrie Ellen Plasschaert Ype Elgersma Steven A Kushner Eric Legius Hilde Brems Google Sarah C Borrie Ellen Plasschaert Ype Elgersma Steven A Kushner Eric Legius Hilde Brems Google Scholar Sarah C Borrie Ellen Plasschaert Ype Elgersma Steven A Kushner Eric Legius Hilde Brems PubMed Sarah C Borrie Ellen Plasschaert Ype Elgersma Steven A Kushner Eric Legius Hilde Brems Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Activity of Selumetinib in Neurofibromatosis Type 1–Related Plexiform Neurofibromas

BackgroundEffective medical therapies are lacking for the treatment of neurofibromatosis type 1–related plexiform neurofibromas, which are characterized by elevated RAS–mitogen-activated protein kinase (MAPK) signaling.MethodsWe conducted a phase 1 trial of selumetinib (AZD6244 or ARRY-142886), an oral selective inhibitor of MAPK kinase (MEK) 1 and 2, in children who had neurofibromatosis type 1 and inoperable plexiform neurofibromas to determine the maximum tolerated dose and to evaluate plasma pharmacokinetics. Selumetinib was administered twice daily at a dose of 20 to 30 mg per square meter of body-surface area on a continuous dosing schedule (in 28-day cycles). We also tested selumetinib using a mouse model of neurofibromatosis type 1–related neurofibroma. Response to treatment (i.e., an increase or decrease from baseline in the volume of plexiform neurofibromas) was monitored by using volumetric magnetic resonance imaging analysis to measure the change in size of the plexiform neurofibroma.ResultsA total of 24 children (median age, 10.9 years; range, 3.0 to 18.5) with a median tumor volume of 1205 ml (range, 29 to 8744) received selumetinib. Patients were able to receive selumetinib on a long-term basis; the median number of cycles was 30 (range, 6 to 56). The maximum tolerated dose was 25 mg per square meter (approximately 60% of the recommended adult dose). The most common toxic effects associated with selumetinib included acneiform rash, gastrointestinal effects, and asymptomatic creatine kinase elevation. The results of pharmacokinetic evaluations of selumetinib among the children in this trial were similar to those published for adults. Treatment with selumetinib resulted in confirmed partial responses (tumor volume decreases from baseline of ≥20%) in 17 of the 24 children (71%) and decreases from baseline in neurofibroma volume in 12 of 18 mice (67%). Disease progression (tumor volume increase from baseline of ≥20%) has not been observed to date. Anecdotal evidence of decreases in tumor-related pain, disfigurement, and functional impairment was observed.ConclusionsOur early-phase data suggested that children with neurofibromatosis type 1 and inoperable plexiform neurofibromas benefited from long-term dose-adjusted treatment with selumetinib without having excess toxic effects. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01362803.)

A randomized placebo-controlled lovastatin trial for neurobehavioral function in neurofibromatosis I.

ObjectiveLovastatin has been shown to reverse learning deficits in a mouse model of Neurofibromatosis Type 1 (NF1), a common monogenic disorder caused by a mutation in the Ras‐MAPK pathway and associated with learning disabilities. We conducted a randomized double‐blind placebo‐controlled trial to assess lovastatin's effects on cognition and behavior in patients with NF1.MethodForty‐four NF1 patients (mean age 25.7+/−11.6 years; 64% female) were randomly assigned to 14 weeks of lovastatin (N = 23; maximum dose of 80 mg/day for adult participants and 40 mg/day for children) or placebo (N = 21). Based on findings in the mouse model, primary outcome measures were nonverbal learning and working memory. Secondary outcome measures included verbal memory, attention, and self/parent‐reported behavioral problems, as well as tolerability of medication. Participants also underwent neuroimaging assessments at baseline and 14 weeks, to determine whether neural biomarkers were associated with treatment response. Linear mixed models assessed for differential treatment effects on outcome measures.ResultsTwelve participants dropped from the study prior to completion (8 placebo, 4 lovastatin), resulting in 32 completers (15 placebo, 17 lovastatin). Lovastatin was well‐tolerated, with no serious adverse events. Differential improvement favoring lovastatin treatment was observed for one primary (working memory; effect size f 2 = 0.70, P < 0.01) and two secondary outcome measures (verbal memory, f 2 = 0.19, P = 0.02, and adult self‐reported internalizing problems, f 2 = 0.26, P = 0.03). Exploratory moderator analyses revealed that higher baseline neural activity in frontal regions was associated with larger treatment effects.InterpretationThese preliminary results suggest beneficial effects of lovastatin on some learning and memory functions, as well as internalizing symptoms in patients with NF1.

RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth

Solid cancers develop within a supportive microenvironment that promotes tumor formation and growth through the elaboration of mitogens and chemokines. Within these tumors, monocytes (macrophages and microglia) represent rich sources of these stromal factors. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1) low-grade brain tumor (optic glioma), we have previously demonstrated that microglia are essential for glioma formation and maintenance. To identify potential tumor-associated microglial factors that support glioma growth (gliomagens), we initiated a comprehensive large-scale discovery effort using optimized RNA-sequencing methods focused specifically on glioma-associated microglia. Candidate microglial gliomagens were prioritized to identify potential secreted or membrane-bound proteins, which were next validated by quantitative real-time polymerase chain reaction as well as by RNA fluorescence in situ hybridization following minocycline-mediated microglial inactivation in vivo. Using these selection criteria, chemokine (C-C motif) ligand 5 (Ccl5) was identified as a chemokine highly expressed in genetically engineered Nf1 mouse optic gliomas relative to nonneoplastic optic nerves. As a candidate gliomagen, recombinant Ccl5 increased Nf1-deficient optic nerve astrocyte growth in vitro. Importantly, consistent with its critical role in maintaining tumor growth, treatment with Ccl5 neutralizing antibodies reduced Nf1 mouse optic glioma growth and improved retinal dysfunction in vivo. Collectively, these findings establish Ccl5 as an important microglial growth factor for low-grade glioma maintenance relevant to the development of future stroma-targeted brain tumor therapies.