NF1 mutation drives neuronal activity-dependent initiation of optic glioma.

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 tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, 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 childhood3,4, 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)5 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.

A common single nucleotide variant in the cytokine receptor-like factor-3 (CRLF3) gene causes neuronal deficits in human and mouse cells.

Single nucleotide variants in the general population are common genomic alterations, where the majority are presumed to be silent polymorphisms without known clinical significance. Using human induced pluripotent stem cell (hiPSC) cerebral organoid modeling of the 1.4 megabase Neurofibromatosis type 1 (NF1) deletion syndrome, we previously discovered that the cytokine receptor-like factor-3 (CRLF3) gene, which is co-deleted with the NF1 gene, functions as a major regulator of neuronal maturation. Moreover, children with NF1 and the CRLF3L389P variant have greater autism burden, suggesting that this gene might be important for neurologic function. To explore the functional consequences of this variant, we generated CRLF3L389P-mutant hiPSC lines and Crlf3L389P-mutant genetically engineered mice. While this variant does not impair protein expression, brain structure, or mouse behavior, CRLF3L389P-mutant human cerebral organoids and mouse brains exhibit impaired neuronal maturation and dendrite formation. In addition, Crlf3L389P-mutant mouse neurons have reduced dendrite lengths and branching, without any axonal deficits. Moreover, Crlf3L389P-mutant mouse hippocampal neurons have decreased firing rates and synaptic current amplitudes relative to wild type controls. Taken together, these findings establish the CRLF3L389P variant as functionally deleterious and suggest that it may be a neurodevelopmental disease modifier.

Neurofibromatosis type 1 (Nf1)-mutant mice exhibit increased sleep fragmentation.

Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder in which affected children and adults are at a higher risk of sleep disorders. In an effort to identify potential sleep disturbances in a small animal model, we used a previously reported Nf1 conditional knockout (Nf1CKO ) mouse strain. In contrast to Nf1 mutant flies, the distribution of vigilance states was intact in Nf1CKO mice. However, Nf1CKO mice exhibited increased non-REM sleep (NREM)-to-wake and wake-to-NREM transitions. This sleep disruption was accompanied by decreased bout durations during awake and NREM sleep states under both light and dark conditions. Moreover, Nf1CKO mice have higher percentage delta power during awake and NREM sleep states under all light conditions. Taken together, Nf1CKO mice phenocopy some of the sleep disturbances observed in NF1 patients and provide a tractable platform to explore the molecular mechanisms governing sleep abnormalities in NF1.

Contextual signaling in cancer.

The formation and maintenance of an organism are highly dependent on the orderly control of cell growth, differentiation, death, and migration. These processes are tightly regulated by signaling cascades in which a limited number of molecules dictate these cellular events. While these signaling pathways are highly conserved across species and cell types, the functional outcomes that result from their engagement are specified by the context in which they are activated. Using the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome as an illustrative platform, we discuss how NF1/RAS signaling can create functional diversity at multiple levels (molecular, cellular, tissue, and genetic/genomic). As such, the ability of related molecules (e.g., K-RAS, H-RAS) to activate distinct effectors, as well as cell type- and tissue-specific differences in molecular composition and effector engagement, generate numerous unique functional effects. These variations, coupled with a multitude of extracellular cues and genomic/genetic changes that each modify the innate signaling properties of the cell, enable precise control of cellular physiology in both health and disease. Understanding these contextual influences is important when trying to dissect the underlying pathogenic mechanisms of cancer relevant to molecularly-targeted therapeutics.

NF1 germline mutation differentially dictates optic glioma formation and growth in neurofibromatosis-1.

Neurofibromatosis type 1 (NF1) is a common neurogenetic condition characterized by significant clinical heterogeneity. A major barrier to developing precision medicine approaches for NF1 is an incomplete understanding of the factors that underlie its inherent variability. To determine the impact of the germline NF1 gene mutation on the optic gliomas frequently encountered in children with NF1, we developed genetically engineered mice harboring two representative NF1-patient-derived Nf1 gene mutations (c.2542G>C;p.G848R and c.2041C>T;p.R681X). We found that each germline Nf1 gene mutation resulted in different levels of neurofibromin expression. Importantly, only R681X(CKO) but not G848R(CKO), mice develop optic gliomas with increased optic nerve volumes, glial fibrillary acid protein immunoreactivity, proliferation and retinal ganglion cell death, similar to Nf1 conditional knockout mice harboring a neomycin insertion (neo) as the germline Nf1 gene mutation. These differences in optic glioma phenotypes reflect both cell-autonomous and stromal effects of the germline Nf1 gene mutation. In this regard, primary astrocytes harboring the R681X germline Nf1 gene mutation exhibit increased basal astrocyte proliferation (BrdU incorporation) indistinguishable from neo(CKO) astrocytes, whereas astrocytes with the G848R mutation have lower levels of proliferation. Evidence for paracrine effects from the tumor microenvironment were revealed when R681X(CKO) mice were compared with conventional neo(CKO) mice. Relative to neo(CKO) mice, the optic gliomas from R681X(CKO) mice had more microglia infiltration and JNK(Thr183/Tyr185) activation, microglia-produced Ccl5, and glial AKT(Thr308) activation. Collectively, these studies establish that the germline Nf1 gene mutation is a major determinant of optic glioma development and growth through by both tumor cell-intrinsic and stromal effects.

Elucidating the impact of neurofibromatosis-1 germline mutations on neurofibromin function and dopamine-based learning.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurologic condition characterized by significant clinical heterogeneity, ranging from malignant cancers to cognitive deficits. Recent studies have begun to reveal rare genotype-phenotype correlations, suggesting that the specific germline NF1 gene mutation may be one factor underlying disease heterogeneity. The purpose of this study was to define the impact of the germline NF1 gene mutation on brain neurofibromin function relevant to learning. Herein, we employ human NF1-patient primary skin fibroblasts, induced pluripotent stem cells and derivative neural progenitor cells (NPCs) to demonstrate that NF1 germline mutations have dramatic effects on neurofibromin expression. Moreover, while all NF1-patient NPCs exhibit increased RAS activation and reduced cyclic AMP generation, there was a neurofibromin dose-dependent reduction in dopamine (DA) levels. Additionally, we leveraged two complementary Nf1 genetically-engineered mouse strains in which hippocampal-based learning and memory is DA-dependent to establish that neuronal DA levels and signaling as well as mouse spatial learning are controlled in an Nf1 gene dose-dependent manner. Collectively, this is the first demonstration that different germline NF1 gene mutations differentially dictate neurofibromin function in the brain.

Neuronal NF1/RAS regulation of cyclic AMP requires atypical PKC activation.

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder in which affected individuals are prone to learning, attention and behavioral problems. Previous studies in mice and flies have yielded conflicting results regarding the specific effector pathways responsible for NF1 protein (neurofibromin) regulation of neuronal function, with both cyclic AMP (cAMP)- and RAS-dependent mechanisms described. Herein, we leverage a combination of induced pluripotent stem cell-derived NF1 patient neural progenitor cells and Nf1 genetically engineered mice to establish, for the first time, that neurofibromin regulation of cAMP requires RAS activation in human and mouse neurons. However, instead of involving RAS-mediated MEK/AKT signaling, RAS regulation of cAMP homeostasis operates through the activation of atypical protein kinase C zeta, leading to GRK2-driven Gαs inactivation. These findings reveal a novel mechanism by which RAS can regulate cAMP levels in the mammalian brain.

DHX34 and NBAS form part of an autoregulatory NMD circuit that regulates endogenous RNA targets in human cells, zebrafish and Caenorhabditis elegans.

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species. Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.

Stem cell modeling of nervous system tumors.

Nervous system tumors, particularly brain tumors, represent the most common tumors in children and one of the most lethal tumors in adults. Despite decades of research, there are few effective therapies for these cancers. Although human nervous system tumor cells and genetically engineered mouse models have served as excellent platforms for drug discovery and preclinical testing, they have limitations with respect to accurately recapitulating important aspects of the pathobiology of spontaneously arising human tumors. For this reason, attention has turned to the deployment of human stem cell engineering involving human embryonic or induced pluripotent stem cells, in which genetic alterations associated with nervous system cancers can be introduced. These stem cells can be used to create self-assembling three-dimensional cerebral organoids that preserve key features of the developing human brain. Moreover, stem cell-engineered lines are amenable to xenotransplantation into mice as a platform to investigate the tumor cell of origin, discover cancer evolutionary trajectories and identify therapeutic vulnerabilities. In this article, we review the current state of human stem cell models of nervous system tumors, discuss their advantages and disadvantages, and provide consensus recommendations for future research.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Nf1 mutation disrupts activity-dependent oligodendroglial plasticity and motor learning in mice.

Neurogenetic disorders, such as neurofibromatosis type 1 (NF1), can cause cognitive and motor impairments, traditionally attributed to intrinsic neuronal defects such as disruption of synaptic function. Activity-regulated oligodendroglial plasticity also contributes to cognitive and motor functions by tuning neural circuit dynamics. However, the relevance of oligodendroglial plasticity to neurological dysfunction in NF1 is unclear. Here we explore the contribution of oligodendrocyte progenitor cells (OPCs) to pathological features of the NF1 syndrome in mice. Both male and female littermates (4-24 weeks of age) were used equally in this study. We demonstrate that mice with global or OPC-specific Nf1 heterozygosity exhibit defects in activity-dependent oligodendrogenesis and harbor focal OPC hyperdensities with disrupted homeostatic OPC territorial boundaries. These OPC hyperdensities develop in a cell-intrinsic Nf1 mutation-specific manner due to differential PI3K/AKT activation. OPC-specific Nf1 loss impairs oligodendroglial differentiation and abrogates the normal oligodendroglial response to neuronal activity, leading to impaired motor learning performance. Collectively, these findings show that Nf1 mutation delays oligodendroglial development and disrupts activity-dependent OPC function essential for normal motor learning in mice.

Next Directions in the Neuroscience of Cancers Arising outside the CNS.

SUMMARY: The field of cancer neuroscience has begun to define the contributions of nerves to cancer initiation and progression; here, we highlight the future directions of basic and translational cancer neuroscience for malignancies arising outside of the central nervous system.

Dhx34 and Nbas function in the NMD pathway and are required for embryonic development in zebrafish.

The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved surveillance mechanism that is present in all eukaryotes. It prevents the synthesis of truncated proteins by selectively degrading mRNAs harbouring premature termination codons (PTCs). The core NMD effectors were originally identified in genetic screens in Saccharomyces cerevisae and in the nematode Caenorhabditis elegans, and subsequently by homology searches in other metazoans. A genome-wide RNAi screen in C. elegans resulted in the identification of two novel NMD genes that are essential for proper embryonic development. Their human orthologues, DHX34 and NAG/NBAS, are required for NMD in human cells. Here, we find that the zebrafish genome encodes orthologues of DHX34 and NAG/NBAS. We show that the morpholino-induced depletion of zebrafish Dhx34 and Nbas proteins results in severe developmental defects and reduced embryonic viability. We also found that Dhx34 and Nbas are required for degradation of PTC-containing mRNAs in zebrafish embryos. The phenotypes observed in both Dhx34 and Nbas morphants are similar to defects in Upf1, Smg-5- or Smg-6- depleted embryos, suggesting that these factors affect the same pathway and confirming that zebrafish embryogenesis requires an active NMD pathway.

Neurons as stromal drivers of nervous system cancer formation and progression.

Similar to their pivotal roles in nervous system development, neurons have emerged as critical regulators of cancer initiation, maintenance, and progression. Focusing on nervous system tumors, we describe the normal relationships between neurons and other cell types relevant to normal nerve function, and discuss how disruptions of these interactions promote tumor evolution, focusing on electrical (gap junctions) and chemical (synaptic) coupling, as well as the establishment of new paracrine relationships. We also review how neuron-tumor communication contributes to some of the complications of cancer, including neuropathy, chemobrain, seizures, and pain. Finally, we consider the implications of cancer neuroscience in establishing risk for tumor penetrance and in the design of future anti-tumoral treatments.

Neurofibromin 1 mutations impair the function of human induced pluripotent stem cell-derived microglia.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.

RAS and beyond: the many faces of the neurofibromatosis type 1 protein.

Neurofibromatosis type 1 is a rare neurogenetic syndrome, characterized by pigmentary abnormalities, learning and social deficits, and a predisposition for benign and malignant tumor formation caused by germline mutations in the NF1 gene. With the cloning of the NF1 gene and the recognition that the encoded protein, neurofibromin, largely functions as a negative regulator of RAS activity, attention has mainly focused on RAS and canonical RAS effector pathway signaling relevant to disease pathogenesis and treatment. However, as neurofibromin is a large cytoplasmic protein the RAS regulatory domain of which occupies only 10% of its entire coding sequence, both canonical and non-canonical RAS pathway modulation, as well as the existence of potential non-RAS functions, are becoming apparent. In this Special article, we discuss our current understanding of neurofibromin function.

Generation of human induced pluripotent stem cell-derived cerebral organoids for cellular and molecular characterization.

Human induced pluripotent stem cell (hiPSC)-derived cerebral organoids (COs) can serve as an in vitro model for studying normal and pathologic human brain development. Here, we optimized existing protocols to streamline the generation of forebrain COs from hiPSCs. We employ these COs to define the impact of disease-causing mutations on cell fate, differentiation, maturation, and morphology relevant to neurodevelopmental disorders. Although limited to forebrain CO identity, this schema requires minimal external interference and is amenable to low-throughput biochemical assays. For complete details on the use and execution of this profile, please refer to Anastasaki et al. (2020) and Wegscheid et al. (2021).

Human induced pluripotent stem cell engineering establishes a humanized mouse platform for pediatric low-grade glioma modeling.

A major obstacle to identifying improved treatments for pediatric low-grade brain tumors (gliomas) is the inability to reproducibly generate human xenografts. To surmount this barrier, we leveraged human induced pluripotent stem cell (hiPSC) engineering to generate low-grade gliomas (LGGs) harboring the two most common pediatric pilocytic astrocytoma-associated molecular alterations, NF1 loss and KIAA1549:BRAF fusion. Herein, we identified that hiPSC-derived neuroglial progenitor populations (neural progenitors, glial restricted progenitors and oligodendrocyte progenitors), but not terminally differentiated astrocytes, give rise to tumors retaining LGG histologic features for at least 6 months in vivo. Additionally, we demonstrated that hiPSC-LGG xenograft formation requires the absence of CD4 T cell-mediated induction of astrocytic Cxcl10 expression. Genetic Cxcl10 ablation is both necessary and sufficient for human LGG xenograft development, which additionally enables the successful long-term growth of patient-derived pediatric LGGs in vivo. Lastly, MEK inhibitor (PD0325901) treatment increased hiPSC-LGG cell apoptosis and reduced proliferation both in vitro and in vivo. Collectively, this study establishes a tractable experimental humanized platform to elucidate the pathogenesis of and potential therapeutic opportunities for childhood brain tumors.

Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1.

Neuronal activity is emerging as a driver of central and peripheral nervous system cancers. Here, we examined neuronal physiology in mouse models of the tumor predisposition syndrome Neurofibromatosis-1 (NF1), with different propensities to develop nervous system cancers. We show that central and peripheral nervous system neurons from mice with tumor-causing Nf1 gene mutations exhibit hyperexcitability and increased secretion of activity-dependent tumor-promoting paracrine factors. We discovered a neurofibroma mitogen (COL1A2) produced by peripheral neurons in an activity-regulated manner, which increases NF1-deficient Schwann cell proliferation, establishing that neurofibromas are regulated by neuronal activity. In contrast, mice with the Arg1809Cys Nf1 mutation, found in NF1 patients lacking neurofibromas or optic gliomas, do not exhibit neuronal hyperexcitability or develop these NF1-associated tumors. The hyperexcitability of tumor-prone Nf1-mutant neurons results from reduced NF1-regulated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel function, such that neuronal excitability, activity-regulated paracrine factor production, and tumor progression are attenuated by HCN channel activation. Collectively, these findings reveal that NF1 mutations act at the level of neurons to modify tumor predisposition by increasing neuronal excitability and activity-regulated paracrine factor production.

Kinase-activating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activity during zebrafish development and are sensitive to small molecule inhibitors.

The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug.