Neural regulation of sleep and metabolic homeostasis are critical in many aspects of human health. Despite extensive epidemiological evidence linking sleep dysregulation with obesity, diabetes, and metabolic syndrome, little is known about the neural and molecular basis for the integration of sleep and metabolic function. The RAS GTPase-activating gene Neurofibromin (Nf1) has been implicated in the regulation of sleep and metabolic rate, raising the possibility that it serves to integrate these processes, but the effects on sleep consolidation and physiology remain poorly understood. A key hallmark of sleep depth in mammals and flies is a reduction in metabolic rate during sleep. Here, we examine multiple measures of sleep quality to determine the effects of Nf1 on sleep-dependent changes in arousal threshold and metabolic rate. Flies lacking Nf1 fail to suppress metabolic rate during sleep, raising the possibility that loss of Nf1 prevents flies from integrating sleep and metabolic state. Sleep of Nf1 mutant flies is fragmented with a reduced arousal threshold in Nf1 mutants, suggesting Nf1 flies fail to enter deep sleep. The effects of Nf1 on sleep can be localized to a subset of neurons expressing the GABAA receptor Rdl. Sleep loss has been associated with changes in gut homeostasis in flies and mammals. Selective knockdown of Nf1 in Rdl-expressing neurons within the nervous system increases gut permeability and reactive oxygen species (ROS) in the gut, raising the possibility that loss of sleep quality contributes to gut dysregulation. Together, these findings suggest Nf1 acts in GABA-sensitive neurons to modulate sleep depth in Drosophila.

Mutations of the neurofibromin 1 gene cause neurofibromatosis type 1, a disease in which learning and behavioral abnormalities are common. The disease is completely penetrant but shows variable phenotypic expression in patients. The repertoire of regulatory interactions utilized by neurons to control neurofibromin 1 expression is poorly understood. Here, we examined the contribution of microRNAs into this regulatory network. Using reporter assays, we provided evidence that miR-128 and to a lesser extent miR-137 and miR-103 reduced neurofibromin 1 reporter levels through specific binding to Nf1 3'-UTR. Mutations in all three predicted binding sites eliminated the reporter response. MiR-128 and miR-137, unlike miR-103 that showed a more ubiquitous expression, were predominantly expressed in brain with a distribution that resembled neurofibromin 1 expression in different tissues as well as during the course of neuronal development. In the nervous system, all three microRNAs showed highest expression in neurons and least in Schwann cells and astrocytes. Overexpression of miR-128 alone or with miR-103 and miR-137 significantly reduced endogenous neurofibromin 1 protein levels, while antisense inhibition of these microRNAs enhanced translation of endogenous neurofibromin 1 and reporter in primary cultures of hippocampal neurons. These findings revealed a significant additional mechanism by which neurofibromin 1 is regulated in neurons and implicated new candidates for the treatment of multifarious neurofibromatosis type 1 cognitive symptoms.

Neurofibromin 1 (NF1) is a tumor suppressor that has been previously reported to regulate RAS‑MAPK signaling. The present study investigated the possible relationship between NF1 expression and anti‑EGFR antibody (cetuximab) sensitivity in colorectal cancer cell lines. In addition, primary or metastatic colorectal cancer samples from patients treated with cetuximab were assessed for the association of cetuximab sensitivity. The quantities of the NF1 transcript, NF1‑related pathway enrichment and NF1 mutation profile were measured and investigated using RNA sequencing and targeted DNA sequencing. Based on growth inhibition and colony formation assay results, cell lines were designated to be cetuximab‑sensitive (NCI‑H508 and Caco2) or cetuximab‑resistant (KM12C and SM480). Western blotting revealed NF1 was highly expressed in cetuximab‑sensitive cell lines whilst there was little expression in their cetuximab‑resistant counterparts. Knocking down NF1 expression using small interfering RNA in the cetuximab‑sensitive cell lines enhanced the phosphorylation of MEK and ERK according to western blotting. NF1 knockdown also reduced apoptosis, as observed by the decreased number of apoptotic bodies by DAPI nuclear staining and reduced cleavage of caspase and poly‑(ADP ribose) polymerase. NF1 overexpression by transfection with GTPase‑activating protein‑related domain subunit rendered the cetuximab‑resistant cell lines, KM12C and SW480, more susceptible to cetuximab‑induced apoptosis. RNA sequencing of 111 RAS and BRAFV600 wild‑type tumor samples collected from cetuximab‑treated patients with metastatic colorectal cancer revealed that the pre‑treatment NF1 expression levels were not associated with the cetuximab response. However, tumor samples obtained after cetuximab treatment displayed slightly lower NF1 transcript levels compared with those in the pre‑treatment samples, suggesting that exposure to the anti‑EGFR antibody may be associated with reduced NF1 expression levels. Next‑generation sequencing revealed that the frequency of inactivating mutations in NF1 were rare (1.8%) in patients with colorectal cancer and were not associated with the protein expression levels of NF1 except for in a small number of cases (0.5%), where the biallelic inactivation of NF1 was observed. To conclude, the present study showed that modification of NF1 expression can affect sensitivity to cetuximab in colorectal cancer cell lines, though a limitation exists in terms of its potential application as a biomarker for RAS and BRAFV600 wild‑type tumors.

The protein giant neurofibromin (320kDa) is the protein product of the NF1 tumor suppressor gene, alterations of which are responsible for the pathogenesis of neurofibromatosis type 1 (NF1). Neurofibromin is a Ras-specific GTPase activating protein (RasGAP) that, 15 years after the cloning of the gene, remains the only clearly defined function of the protein. In a structural proteomics approach, we aimed at defining functions beyond RasGAP activity based on the discovery of structural modules. Given the poor outcome of domain prediction tools, we have undertaken a fragment solubility survey covering the full protein sequence, with the aim of defining new domain boundaries or fragments that could be investigated by biochemical methods including structural analysis. More than 200 constructs have been expressed and tested for solubility in small scale assays. Boundaries were chosen based upon secondary structure predictions, sequence conservation among neurofibromin orthologues and chemical properties of amino acids. Using this strategy we recently discovered a novel bipartite module in neurofibromin. We have expanded our study to include ESPRIT, a library-based construct screen, to perform fragment testing at a finer level with respect to the choice of terminal residues. Our study confirms earlier notions about the challenges neurofibromin presents to the biochemist and points to strategies whereby the success rate may be enhanced in the future.

Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40-50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines. In vitro knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation in vivo. By integrating in silico prediction algorithms with in vitro experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression via down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.

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.

In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/-, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.

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.

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

Neurofibromin, a protein encoded by the NF1 gene, is mutated in neurofibromatosis 1, one of the most common genetic diseases. Oral manifestations are common and a high prevalence of hyposalivation was recently described in individuals with neurofibromatosis 1. Although neurofibromin is ubiquitously expressed, its expression levels vary depending on the tissue type and developmental stage of the organism. The role of neurofibromin in the development, morphology, and physiology of salivary glands is unknown and a detailed expression of neurofibromin in human normal salivary glands has never been investigated.

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

Various behavioral and cognitive states exhibit circadian variations in animals across phyla including Drosophila melanogaster, in which only ~0.1% of the brain's neurons contain circadian clocks. Clock neurons transmit the timing information to a plethora of non-clock neurons via poorly understood mechanisms. Here, we address the molecular underpinning of this phenomenon by profiling circadian gene expression in non-clock neurons that constitute the mushroom body, the center of associative learning and sleep regulation. We show that circadian clocks drive rhythmic expression of hundreds of genes in mushroom body neurons, including the Neurofibromin 1 (Nf1) tumor suppressor gene and Pka-C1. Circadian clocks also drive calcium rhythms in mushroom body neurons via NF1-cAMP/PKA-C1 signaling, eliciting higher mushroom body activity during the day than at night, thereby promoting daytime wakefulness. These findings reveal the pervasive, non-cell-autonomous circadian regulation of gene expression in the brain and its role in sleep.

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/- mice. Nf1+/- mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/- mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/- mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/- mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/- mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

Neurofibromatosis type 1 is a chronic multisystemic genetic disorder that results from loss of function in the neurofibromin protein. Neurofibromin may regulate metabolism, though the underlying mechanisms remain largely unknown. Here we show that neurofibromin regulates metabolic homeostasis in Drosophila via a discrete neuronal circuit. Loss of neurofibromin increases metabolic rate via a Ras GAP-related domain-dependent mechanism, increases feeding homeostatically, and alters lipid stores and turnover kinetics. The increase in metabolic rate is independent of locomotor activity, and maps to a sparse subset of neurons. Stimulating these neurons increases metabolic rate, linking their dynamic activity state to metabolism over short time scales. Our results indicate that neurofibromin regulates metabolic rate via neuronal mechanisms, suggest that cellular and systemic metabolic alterations may represent a pathophysiological mechanism in neurofibromatosis type 1, and provide a platform for investigating the cellular role of neurofibromin in metabolic homeostasis.

Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

An understudied symptom of the genetic disorder Neurofibromatosis type 1 (NF1) is chronic idiopathic pain. We used targeted editing of Nf1 in rats to provide direct evidence of a causal relationship between neurofibromin, the protein product of the Nf1 gene, and pain responses. Our study data identified a protein-interaction network with collapsin response meditator protein 2 (CRMP2) as a node and neurofibromin, syntaxin 1A, and the N-type voltage-gated calcium (CaV2.2) channel as interaction edges. Neurofibromin uncouples CRMP2 from syntaxin 1A. Upon loss/mutation of neurofibromin, as seen in patients with NF1, the CRMP2/Neurofibromin interaction is uncoupled, which frees CRMP2 to interact with both syntaxin 1A and CaV2.2, culminating in increased release of the pro-nociceptive neurotransmitter calcitonin gene-related peptide (CGRP). Our work also identified the CRMP2-derived peptide CNRP1, which uncoupled CRMP2's interactions with neurofibromin, syntaxin 1A, as well as CaV2.2. Here, we tested if CRISPR/Cas9-mediated editing of the Nf1 gene, which leads to functional remodeling of peripheral nociceptors through effects on the tetrodotoxin-sensitive (TTX-S) Na+ voltage-gated sodium channel (NaV1.7) and CaV2.2, could be affected using CNRP1, a peptide designed to target the CRMP2-neurofibromin interface. The data presented here shows that disrupting the CRMP2-neurofibromin interface is sufficient to reverse the dysregulations of voltage-gated ion channels and neurotransmitter release elicited by Nf1 gene editing. As a consequence of these effects, the CNRP1 peptide reversed hyperalgesia to thermal stimulation of the hindpaw observed in Nf1-edited rats. Our findings support future pharmacological targeting of the CRMP2/neurofibromin interface for NF1-related pain relief.

Neurofibromatosis type 1 (NF1), a genetic disorder linked to inactivating mutations or a homozygous deletion of the Nf1 gene, is characterized by tumorigenesis, cognitive dysfunction, seizures, migraine, and pain. Omic studies on human NF1 tissues identified an increase in the expression of collapsin response mediator protein 2 (CRMP2), a cytosolic protein reported to regulate the trafficking and activity of presynaptic N-type voltage-gated calcium (Cav2.2) channels. Because neurofibromin, the protein product of the Nf1 gene, binds to and inhibits CRMP2, the neurofibromin-CRMP2 signaling cascade will likely affect Ca channel activity and regulate nociceptive neurotransmission and in vivo responses to noxious stimulation. Here, we investigated the function of neurofibromin-CRMP2 interaction on Cav2.2. Mapping of >275 peptides between neurofibromin and CRMP2 identified a 15-amino acid CRMP2-derived peptide that, when fused to the tat transduction domain of HIV-1, inhibited Ca influx in dorsal root ganglion neurons. This peptide mimics the negative regulation of CRMP2 activity by neurofibromin. Neurons treated with tat-CRMP2/neurofibromin regulating peptide 1 (t-CNRP1) exhibited a decreased Cav2.2 membrane localization, and uncoupling of neurofibromin-CRMP2 and CRMP2-Cav2.2 interactions. Proteomic analysis of a nanodisc-solubilized membrane protein library identified syntaxin 1A as a novel CRMP2-binding protein whose interaction with CRMP2 was strengthened in neurofibromin-depleted cells and reduced by t-CNRP1. Stimulus-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices was inhibited by t-CNRP1. Intrathecal administration of t-CNRP1 was antinociceptive in experimental models of inflammatory, postsurgical, and neuropathic pain. Our results demonstrate the utility of t-CNRP1 to inhibit CRMP2 protein-protein interactions for the potential treatment of pain.

Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.

Growth factors induce a characteristically short-lived Ras activation in cells emerging from quiescence. Extensive work has shown that transient as opposed to sustained Ras activation is critical for the induction of mitogenic programs. Mitogen-induced accumulation of active Ras-GTP results from increased nucleotide exchange driven by the nucleotide exchange factor Sos. In contrast, the mechanism accounting for signal termination and prompt restoration of basal Ras-GTP levels is unclear, but has been inferred to involve feedback inhibition of Sos. Remarkably, how GTP-hydrolase activating proteins (GAPs) participate in controlling the rise and fall of Ras-GTP levels is unknown.

Neurofibromatosis type 1 (NF1) is among the most common neurogenic disorders, characterized by loss of function mutations in the neurofibromin gene (NF1). NF1 patients are extremely susceptible to developing neurofibromas, which can transform into deadly malignant peripheral nerve sheath tumors (MPNSTs). At the center of these tumors are NF1-null Schwann cells. Here, we found that nanomedicine shows promise in the treatment of NF1-associated MPNSTs. We assessed the cytotoxicity of silver nanoparticles (AgNPs) in NF1-null NF1-associated MPNSTs, NF1-wildtype sporadic MPNST, and normal Schwann cells. Our data show that AgNP are selectivity cytotoxic to NF1-associated MPNSTs relative to sporadic MPNST and Schwann cells. Furthermore, we found that sensitivity to AgNPs is correlated with the expression levels of functional neurofibromin. The restoration of functional neurofibromin in NF1-associated MPNSTs reduces AgNP sensitivity, and the knockdown of neurofibromin in Schwann cells increases AgNP sensitivity. This finding is unique to AgNPs, as NF1 restoration does not alter sensitivity to standard of care chemotherapy doxorubicin in NF1-associated MPNSTs. Using an in vitro model system, we then found that AgNP can selectively eradicate NF1-associated MPNSTs in co-culture with Schwann cells at doses tolerable to normal cells. AgNP represents a novel therapy for the treatment of NF1-associated MPNSTs and addresses significant unmet clinical need.