Gene-targeted therapy for neurofibromatosis and schwannomatosis: The path to clinical trials.

Numerous successful gene-targeted therapies are arising for the treatment of a variety of rare diseases. At the same time, current treatment options for neurofibromatosis 1 and schwannomatosis are limited and do not directly address loss of gene/protein function. In addition, treatments have mostly focused on symptomatic tumors, but have failed to address multisystem involvement in these conditions. Gene-targeted therapies hold promise to address these limitations. However, despite intense interest over decades, multiple preclinical and clinical issues need to be resolved before they become a reality. The optimal approaches to gene-, mRNA-, or protein restoration and to delivery to the appropriate cell types remain elusive. Preclinical models that recapitulate manifestations of neurofibromatosis 1 and schwannomatosis need to be refined. The development of validated assays for measuring neurofibromin and merlin activity in animal and human tissues will be critical for early-stage trials, as will the selection of appropriate patients, based on their individual genotypes and risk/benefit balance. Once the safety of gene-targeted therapy for symptomatic tumors has been established, the possibility of addressing a wide range of symptoms, including non-tumor manifestations, should be explored. As preclinical efforts are underway, it will be essential to educate both clinicians and those affected by neurofibromatosis 1/schwannomatosis about the risks and benefits of gene-targeted therapy for these conditions.

Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathy.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved "poison" exon, 20N, whose inclusion is predicted to lead to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A expression or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.

CHD7 regulates cardiovascular development through ATP-dependent and -independent activities.

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.

A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development.

BACKGROUND: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. RESULTS: We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. CONCLUSION: Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

Analysis of patient-specific NF1 variants leads to functional insights for Ras signaling that can impact personalized medicine.

We have created a panel of 29 NF1 variant complementary DNAs (cDNAs) representing missense variants, many with clinically relevant phenotypes, in-frame deletions, splice variants, and nonsense variants. We have determined the functional consequences of the variants, assessing their ability to produce mature neurofibromin and restore Ras signaling activity in NF1 null (-/-) cells. cDNAs demonstrate variant-specific differences in neurofibromin protein levels, suggesting that some variants lead to neurofibromatosis type 1 (NF1) gene or protein instability or enhanced degradation. When expressed at high levels, some variant proteins are still able to repress Ras activity, indicating that the NF1 phenotype may be due to low protein abundance. In contrast, other variant proteins are incapable of repressing Ras activity, indicating that some do not functionally engage Ras and stimulate GTPase activity. We observed that effects on protein abundance and Ras activity can be mutually exclusive. These assays allow us to categorize variants by functional effects, may help to classify variants of unknown significance, and may have future implications for more directed therapeutics.

Functional characterization and potential therapeutic avenues for variants in the NTRK2 gene causing developmental and epileptic encephalopathies.

Variants within the Neurotrophic Tyrosine Kinase Receptor Type 2 (NTRK2) gene have been discovered to play a role in developmental and epileptic encephalopathies, a group of debilitating conditions for which little is known about cause or treatment. Here, we determine the functional consequences of two variants: p.Tyr434Cys (Y434C) (located in the transmembrane domain) and p.Thr720Ile (T720I) (located in the catalytic domain). Wild-type and variant cDNAs were constructed and transfected into HEK293 cells. In cell culture, variant Y434C exhibited ligand-independent activation of tropomyosin-related kinase B (TRKB) signaling with an associated abnormal response to brain-derived neurotrophic factor (BDNF) stimulation and increased levels of phosphorylated extracellular signal-regulated kinase (ERK) and ETS like-1 protein (ELK1) activity. Expression of variant T720I resulted in decreased TRKB signaling with reduced mTor activity as determined by decreased levels of phosphorylated S6. With the deleterious mechanisms characterized, we utilized mediKanren (a novel artificial intelligence tool) to identify therapeutics to compensate for the pathological effects. Downregulation of TRKB through inhibition with mediKanren-predicted compound 1NM-PP1 led to decreased MEK activity. Upregulation of TRKB signaling by mediKanren-predicted valproic acid led to subsequent increase of mTor activity. Overall, our results provide further characterization of the pathogenicity of these two variants in the NTRK2 gene. Indeed, Y434C is the first patient-specific NTRK2 variant with demonstrated hypermorphic activity. Furthermore, we observed that variants Y434C and T720I result in distinct functional consequences that require distinct therapeutic strategies. These data suggest the possibility that unique mutations within different regions of the NTRK2 gene results in separate clinical presentations, representing distinct genetic disorders requiring unique therapeutics.

Mice lacking the epidermal retinol dehydrogenases SDR16C5 and SDR16C6 display accelerated hair growth and enlarged meibomian glands.

Retinol dehydrogenases catalyze the rate-limiting step in the biosynthesis of retinoic acid, a bioactive lipid molecule that regulates the expression of hundreds of genes by binding to nuclear transcription factors, the retinoic acid receptors. Several enzymes exhibit retinol dehydrogenase activities in vitro; however, their physiological relevance for retinoic acid biosynthesis in vivo remains unclear. Here, we present evidence that two murine epidermal retinol dehydrogenases, short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and SDR16C6, contribute to retinoic acid biosynthesis in living cells and are also essential for the oxidation of retinol to retinaldehyde in vivo Mice with targeted knockout of the more catalytically active SDR16C6 enzyme have no obvious phenotype, possibly due to functional redundancy, because Sdr16c5 and Sdr16c6 exhibit an overlapping expression pattern during later developmental stages and in adulthood. Mice that lack both enzymes are viable and fertile but display accelerated hair growth after shaving and also enlarged meibomian glands, consistent with a nearly 80% reduction in the retinol dehydrogenase activities of skin membrane fractions from the Sdr16c5/Sdr16c6 double-knockout mice. The up-regulation of hair-follicle stem cell genes is consistent with reduced retinoic acid signaling in the skin of the double-knockout mice. These results indicate that the retinol dehydrogenase activities of murine SDR16C5 and SDR16C6 enzymes are not critical for survival but are responsible for most of the retinol dehydrogenase activity in skin, essential for the regulation of the hair-follicle cycle, and required for the maintenance of both sebaceous and meibomian glands.

Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

The transition zone (TZ) is a domain at the base of the cilium that is involved in maintaining ciliary compartment-specific sensory and signaling activity by regulating cilia protein composition. Mutations in TZ proteins result in cilia dysfunction, often causing pleiotropic effects observed in a group of human diseases classified as ciliopathies. The purpose of this study is to describe the importance of the TZ component Meckel-Grüber syndrome 6 ( Mks6) in several organ systems and tissues regarding ciliogenesis and cilia maintenance using congenital and conditional mutant mouse models. Similar to MKS, congenital loss of Mks6 is embryonic lethal, displaying cilia loss and altered cytoskeletal microtubule modifications but only in specific cell types. Conditional Mks6 mutants have a variable cystic kidney phenotype along with severe retinal degeneration with mislocalization of phototransduction cascade proteins. However, other phenotypes, such as anosmia and obesity, which are typically associated with cilia and TZ dysfunction, were not evident. These data indicate that despite Mks6 being a core TZ component, it has tissue- or cell type-specific functions important for cilia formation and cilia sensory and signaling activities. Lewis, W. R., Bales, K. L., Revell, D. Z., Croyle, M. J., Engle, S. E., Song, C. J., Malarkey, E. B., Uytingco, C. R., Shan, D., Antonellis, P. J., Nagy, T. R., Kesterson, R. A., Mrug, M. M., Martens, J. R., Berbari, N. F., Gross, A. K., Yoder, B. K. Mks6 mutations reveal tissue- and cell type-specific roles for the cilia transition zone.

Multi-Omics Profiling for NF1 Target Discovery in Neurofibromin (NF1) Deficient Cells.

Loss of NF1 is an oncogenic driver. In efforts to define pathways responsible for the development of neurofibromas and other cancers, transcriptomic and proteomic changes are evaluated in a non-malignant NF1 null cell line. NF1 null HEK293 cells were created using CRISPR/Cas9 technology and they are compared to parental cells that express neurofibromin. A total of 1222 genes and 132 proteins are found to be differentially expressed. The analysis is integrated to identify eight transcripts/proteins that are differentially regulated in both analyses. Metacore Pathway analysis identifies Neurogenesis NGF/TrkA MAPK-mediated signaling alterations. Next, the data set is compared with other published studies that involve analysis of cells or tumors deficient for NF1 and it is found that 141 genes recur in the sample and others; only thirteen of these genes recur in two or more studies. Genes/proteins of interest are validated via q-RT-PCR or Western blot. It is shown that KRT8 and 14-3-3σ protein levels respond to exogenously introduced mNf1 cDNA. Hence, transcripts/proteins that respond to neurofibromin levels are identified and they can potentially be used as biomarkers.

Truncating PKHD1 and PKD2 mutations alter energy metabolism.

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.

Heterozygous Pkhd1C642* mice develop cystic liver disease and proximal tubule ectasia that mimics radiographic signs of medullary sponge kidney.

Heterozygosity for human polycystic kidney and hepatic disease 1 ( PKHD1) mutations was recently associated with cystic liver disease and radiographic findings resembling medullary sponge kidney (MSK). However, the relevance of these associations has been tempered by a lack of cystic liver or renal disease in heterozygous mice carrying Pkhd1 gene trap or exon deletions. To determine whether heterozygosity for a smaller Pkhd1 defect can trigger cystic renal disease in mice, we generated and characterized mice with the predicted truncating Pkhd1C642* mutation in a region corresponding to the middle of exon 20 cluster of five truncating human mutations (between PKHD1G617fs and PKHD1G644*). Mouse heterozygotes or homozygotes for the Pkhd1C642* mutation did not have noticeable liver or renal abnormalities on magnetic resonance images during their first weeks of life. However, when aged to ~1.5 yr, the Pkhd1C642* heterozygotes developed prominent cystic liver changes; tissue analyses revealed biliary cysts and increased number of bile ducts without signs of congenital hepatic fibrosis-like portal field inflammation and fibrosis that was seen in Pkhd1C642* homozygotes. Interestingly, aged female Pkhd1C642* heterozygotes, as well as homozygotes, developed radiographic changes resembling MSK. However, these changes correspond to proximal tubule ectasia, not an MSK-associated collecting duct ectasia. In summary, by demonstrating that cystic liver and kidney abnormalities are triggered by heterozygosity for the Pkhd1C642* mutation, we provide important validation for relevant human association studies. Together, these investigations indicate that PKHD1 mutation heterozygosity (predicted frequency 1 in 70 individuals) is an important underlying cause of cystic liver disorders and MSK-like manifestations in a human population.

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

A CreER mouse to study melanin concentrating hormone signaling in the developing brain.

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.

Neurofibromin (NF1) genetic variant structure-function analyses using a full-length mouse cDNA.

Neurofibromatosis type 1 (NF1) is caused by pathogenic variants or mutations in the NF1 gene that encodes neurofibromin. We describe here a new approach to determining the functional consequences of NF1 genetic variants. We established a heterologous cell culture expression system using a full-length mouse Nf1 cDNA (mNf1) and human cell lines. We demonstrate that the full-length murine cDNA produces a > 250 kDa neurofibromin protein that is capable of modulating Ras signaling. We created mutant cDNAs representing NF1 patient variants with different clinically relevant phenotypes, and assessed their ability to produce mature neurofibromin and restore Nf1 activity in NF1-/- cells. These cDNAs represent variants in multiple protein domains and various types of clinically relevant predicted variants. This approach will help advance research on neurofibromin structure and function, determine pathogenicity for missense variants, and allow for the development of activity assays and variant-directed 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.

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse.

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.

Leptin resistance is a secondary consequence of the obesity in ciliopathy mutant mice.

Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alström syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction.

Mitochondrial genetic background modulates bioenergetics and susceptibility to acute cardiac volume overload.

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.

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.

snRNA-seq of human cutaneous neurofibromas before and after selumetinib treatment implicates role of altered Schwann cell states, inter-cellular signaling, and extracellular matrix in treatment response.

Neurofibromatosis Type 1 (NF1) is caused by loss of function variants in the NF1 gene. Most patients with NF1 develop skin lesions called cutaneous neurofibromas (cNFs). Currently the only approved therapeutic for NF1 is selumetinib, a mitogen -activated protein kinase (MEK) inhibitor. The purpose of this study was to analyze the transcriptome of cNF tumors before and on selumetinib treatment to understand both tumor composition and response. We obtained biopsy sets of tumors both pre- and on- selumetinib treatment from the same individuals and were able to collect sets from four separate individuals. We sequenced mRNA from 5844 nuclei and identified 30,442 genes in the untreated group and sequenced 5701 nuclei and identified 30,127 genes in the selumetinib treated group. We identified and quantified distinct populations of cells (Schwann cells, fibroblasts, pericytes, myeloid cells, melanocytes, keratinocytes, and two populations of endothelial cells). While we anticipated that cell proportions might change with treatment, we did not identify any one cell population that changed significantly, likely due to an inherent level of variability between tumors. We also evaluated differential gene expression based on drug treatment in each cell type. Ingenuity pathway analysis (IPA) was also used to identify pathways that differ on treatment. As anticipated, we identified a significant decrease in ERK/MAPK signaling in cells including Schwann cells but most specifically in myeloid cells. Interestingly, there is a significant decrease in opioid signaling in myeloid and endothelial cells; this downward trend is also observed in Schwann cells and fibroblasts. Cell communication was assessed by RNA velocity, Scriabin, and CellChat analyses which indicated that Schwann cells and fibroblasts have dramatically altered cell states defined by specific gene expression signatures following treatment (RNA velocity). There are dramatic changes in receptor-ligand pairs following treatment (Scriabin), and robust intercellular signaling between virtually all cell types associated with extracellular matrix (ECM) pathways (Collagen, Laminin, Fibronectin, and Nectin) is downregulated after treatment. These response specific gene signatures and interaction pathways could provide clues for understanding treatment outcomes or inform future therapies.