Pleiotropic role of TRAF7 in skull-base meningiomas and congenital heart disease.

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects.

Somatic PIK3CA Mutations in Sporadic Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are common sporadic and inherited vascular malformations of the central nervous system. Although familial CCMs are linked to loss-of-function mutations in KRIT1 (CCM1), CCM2, or PDCD10 (CCM3), the genetic cause of sporadic CCMs, representing 80% of cases, remains incompletely understood. METHODS: We developed two mouse models harboring mutations identified in human meningiomas with the use of the prostaglandin D2 synthase (PGDS) promoter. We performed targeted DNA sequencing of surgically resected CCMs from patients and confirmed our findings by droplet digital polymerase-chain-reaction analysis. RESULTS: We found that in mice expressing one of two common genetic drivers of meningioma - Pik3ca H1047R or AKT1 E17K - in PGDS-positive cells, a spectrum of typical CCMs develops (in 22% and 11% of the mice, respectively) instead of meningiomas, which prompted us to analyze tissue samples from sporadic CCMs from 88 patients. We detected somatic activating PIK3CA and AKT1 mutations in 39% and 1%, respectively, of lesion tissue from the patients. Only 10% of lesions harbored mutations in the CCM genes. We analyzed lesions induced by the activating mutations Pik3ca H1074R and AKT1 E17K in mice and identified the PGDS-expressing pericyte as the probable cell of origin. CONCLUSIONS: In tissue samples from sporadic CCMs, mutations in PIK3CA were represented to a greater extent than mutations in any other gene. The contribution of somatic mutations in the genes that cause familial CCMs was comparatively small. (Funded by the Fondation ARC pour la Recherche contre le Cancer and others.).

Cerebrovascular disorders associated with genetic lesions.

Cerebrovascular disorders are underlain by perturbations in cerebral blood flow and abnormalities in blood vessel structure. Here, we provide an overview of the current knowledge of select cerebrovascular disorders that are associated with genetic lesions and connect genomic findings with analyses aiming to elucidate the cellular and molecular mechanisms of disease pathogenesis. We argue that a mechanistic understanding of genetic (familial) forms of cerebrovascular disease is a prerequisite for the development of rational therapeutic approaches, and has wider implications for treatment of sporadic (non-familial) forms, which are usually more common.

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM.

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the N-bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in Drosophila glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.

Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis.

OBJECTIVE: We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue. DESIGN: Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors. RESULTS: Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth. CONCLUSIONS: Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach.

Ccm3, a gene associated with cerebral cavernous malformations, is required for neuronal migration.

Loss of function of cerebral cavernous malformation 3 (CCM3) results in an autosomal dominant cerebrovascular disorder. Here, we uncover a developmental role for CCM3 in regulating neuronal migration in the neocortex. Using cell type-specific gene inactivation in mice, we show that CCM3 has both cell autonomous and cell non-autonomous functions in neural progenitors and is specifically required in radial glia and newly born pyramidal neurons migrating through the subventricular zone, but not in those migrating through the cortical plate. Loss of CCM3 function leads to RhoA activation, alterations in the actin and microtubule cytoskeleton affecting neuronal morphology, and abnormalities in laminar positioning of primarily late-born neurons, indicating CCM3 involvement in radial glia-dependent locomotion and possible interaction with the Cdk5/RhoA pathway. Thus, we identify a novel cytoplasmic regulator of neuronal migration and demonstrate that its inactivation in radial glia progenitors and nascent neurons produces severe malformations of cortical development.

Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations.

The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers. An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development. Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WD repeat domain 62 (WDR62) as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygyria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration.

Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1(GLU7ALA)), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1(GLU7ALA), compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1(GLU7ALA) relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system.

Notch and disease: a growing field.

Signals through the Notch receptors are used throughout development to control cellular fate choices. Our intention here is to provide an overview of the involvement of Notch signaling in human disease, which, keeping pace with the known biology of the pathway, manifests itself in a pleiotropic fashion. A pathway with such broad action in normal development, a profound involvement in the biology of adult stem cells and intricate and complex controls governing its activity, poses numerous challenges. We provide an overview of Notch related pathologies identified thus far and emphasize aspects that have been modeled in experimental systems in order to understand the underlying pathobiology and, hopefully, help the definition of rational therapeutic avenues.

Loss of cerebral cavernous malformation 3 (Ccm3) in neuroglia leads to CCM and vascular pathology.

Communication between neural cells and the vasculature is integral to the proper development and later function of the central nervous system. A mechanistic understanding of the interactions between components of the neurovascular unit has implications for various disorders, including cerebral cavernous malformations (CCMs) in which focal vascular lesions form throughout the central nervous system. Loss of function mutations in three genes with proven endothelial cell autonomous roles, CCM1/krev1 interaction trapped gene 1, CCM2, and CCM3/programmed cell death 10, cause familial CCM. By using neural specific conditional mouse mutants, we show that Ccm3 has both neural cell autonomous and nonautonomous functions. Gfap- or Emx1-Cre-mediated Ccm3 neural deletion leads to increased proliferation, increased survival, and activation of astrocytes through cell autonomous mechanisms involving activated Akt signaling. In addition, loss of neural CCM3 results in a vascular phenotype characterized by diffusely dilated and simplified cerebral vasculature along with formation of multiple vascular lesions that closely resemble human cavernomas through cell nonautonomous mechanisms. RNA sequencing of the vascular lesions shows abundant expression of molecules involved in cytoskeletal remodeling, including protein kinase A and Rho-GTPase signaling. Our findings implicate neural cells in the pathogenesis of CCMs, showing the importance of this pathway in neural/vascular interactions within the neurovascular unit.

Hypomorphic Notch 3 alleles link Notch signaling to ischemic cerebral small-vessel disease.

The most common monogenic cause of small-vessel disease leading to ischemic stroke and vascular dementia is the neurodegenerative syndrome cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is associated with mutations in the Notch 3 receptor. CADASIL pathology is characterized by vascular smooth muscle cell degeneration and accumulation of diagnostic granular osmiophilic material (GOM) in vessels. The functional nature of the Notch 3 mutations causing CADASIL and their mechanistic connection to small-vessel disease and GOM accumulation remain enigmatic. To gain insight into how Notch 3 function is linked to CADASIL pathophysiology, we studied two phenotypically distinct mutations, C455R and R1031C, respectively associated with early and late onset of stroke, by using hemodynamic analyses in transgenic mouse models, receptor activity assays in cell culture, and proteomic examination of postmortem human tissue. We demonstrate that the C455R and R1031C mutations define different hypomorphic activity states of Notch 3, a property linked to ischemic stroke susceptibility in mouse models we generated. Importantly, these mice develop osmiophilic deposits and other age-dependent phenotypes that parallel remarkably the human condition. Proteomic analysis of human brain vessels, carrying the same CADASIL mutations, identified clusterin and collagen 18 α1/endostatin as GOM components. Our findings link loss of Notch signaling with ischemic cerebral small-vessel disease, a prevalent human condition. We determine that CADASIL pathophysiology is associated with hypomorphic Notch 3 function in vascular smooth muscle cells and implicate the accumulation of clusterin and collagen 18 α1/endostatin in brain vessel pathology.

L-histidine decarboxylase and Tourette's syndrome.

Tourette's syndrome is a common developmental neuropsychiatric disorder characterized by chronic motor and vocal tics. Despite a strong genetic contribution, inheritance is complex, and risk alleles have proven difficult to identify. Here, we describe an analysis of linkage in a two-generation pedigree leading to the identification of a rare functional mutation in the HDC gene encoding L-histidine decarboxylase, the rate-limiting enzyme in histamine biosynthesis. Our findings, together with previously published data from model systems, point to a role for histaminergic neurotransmission in the mechanism and modulation of Tourette's syndrome and tics.

Altered Expression of Several Molecular Mediators of Cerebrospinal Fluid Production in Hyp Mice.

Context: X-linked hypophosphatemia (XLH) is a genetic disease, causing life-long hypophosphatemia due to overproduction of fibroblast growth factor 23 (FGF23). XLH is associated with Chiari malformations, cranial synostosis, and syringomyelia. FGF23 signals through FGFR1c and requires a coreceptor, α-Klotho, which is expressed in the renal distal convoluted tubules and the choroid plexus (ChP). In the ChP, α-Klotho participates in regulating cerebrospinal fluid (CSF) production by shuttling the sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) to the luminal membrane. The sodium/potassium/chloride cotransporter 1 (NKCC1) also makes a substantial contribution to CSF production. Objective: Since CSF production has not been studied in XLH, we sought to determine if there are changes in the expression of these molecules in the ChP of Hyp mice, the murine model of XLH, as a first step toward testing the hypothesis that altered CSF production contributes to the cranial and spinal malformations seen this disease. Methods: Semi-quantitative real-time PCR was used to analyze the level of expression of transcripts for Fgfr1c, and thee key regulators of CSF production, Klotho, Atp1a1 and Slc12a2. In situ hybridization was used to provide anatomical localization for the encoded proteins. Results: Real-time polymerase chain reaction (RT-PCR) demonstrated significant upregulation of Klotho transcripts in the fourth ventricle of Hyp mice compared to controls. Transcript levels for Fgfr1c were unchanged in Hyp mice. Atp1a1 transcripts encoding the alpha-1 subunit of Na+/K+-ATPase were significantly downregulated in the third and lateral ventricles (LV). Expression levels of the Slc12a2 transcript (which encodes NKCC1) were unchanged in Hyp mice compared to controls. In situ hybridization (ISH) confirmed the presence of all 4 transcripts in the LV ChP both of WT and Hyp mice. Conclusion: This is the first study to document a significant change in the level of expression of the molecular machinery required for CSF production in Hyp mice. Whether similar changes occur in patients with XLH, potentially contributing to the cranial and spinal cord abnormalities frequently seen in XLH, remains to be determined.

Microcephaly-associated protein WDR62 shuttles from the Golgi apparatus to the spindle poles in human neural progenitors.

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions. Recessive mutations in WDR62 cause structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated WDR62 function in corticogenesis through the analysis of a C-terminal truncating mutation (D955AfsX112). Using induced Pluripotent Stem Cells (iPSCs) obtained from a patient and his unaffected parent, as well as isogenic corrected lines, we generated 2D and 3D models of human neurodevelopment, including neuroepithelial stem cells, cerebro-cortical progenitors, terminally differentiated neurons, and cerebral organoids. We report that WDR62 localizes to the Golgi apparatus during interphase in cultured cells and human fetal brain tissue, and translocates to the mitotic spindle poles in a microtubule-dependent manner. Moreover, we demonstrate that WDR62 dysfunction impairs mitotic progression and results in alterations of the neurogenic trajectories of iPSC neuroderivatives. In summary, impairment of WDR62 localization and function results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in the etiology of MCPH.

Super-enhancer hijacking drives ectopic expression of hedgehog pathway ligands in meningiomas.

Hedgehog signaling mediates embryologic development of the central nervous system and other tissues and is frequently hijacked by neoplasia to facilitate uncontrolled cellular proliferation. Meningiomas, the most common primary brain tumor, exhibit Hedgehog signaling activation in 6.5% of cases, triggered by recurrent mutations in pathway mediators such as SMO. In this study, we find 35.6% of meningiomas that lack previously known drivers acquired various types of somatic structural variations affecting chromosomes 2q35 and 7q36.3. These cases exhibit ectopic expression of Hedgehog ligands, IHH and SHH, respectively, resulting in Hedgehog signaling activation. Recurrent tandem duplications involving IHH permit de novo chromatin interactions between super-enhancers within DIRC3 and a locus containing IHH. Our work expands the landscape of meningioma molecular drivers and demonstrates enhancer hijacking of Hedgehog ligands as a route to activate this pathway  in neoplasia.

WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters.

NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.

Modeling uniquely human gene regulatory function via targeted humanization of the mouse genome.

The evolution of uniquely human traits likely entailed changes in developmental gene regulation. Human Accelerated Regions (HARs), which include transcriptional enhancers harboring a significant excess of human-specific sequence changes, are leading candidates for driving gene regulatory modifications in human development. However, insight into whether HARs alter the level, distribution, and timing of endogenous gene expression remains limited. We examined the role of the HAR HACNS1 (HAR2) in human evolution by interrogating its molecular functions in a genetically humanized mouse model. We find that HACNS1 maintains its human-specific enhancer activity in the mouse embryo and modifies expression of Gbx2, which encodes a transcription factor, during limb development. Using single-cell RNA-sequencing, we demonstrate that Gbx2 is upregulated in the limb chondrogenic mesenchyme of HACNS1 homozygous embryos, supporting that HACNS1 alters gene expression in cell types involved in skeletal patterning. Our findings illustrate that humanized mouse models provide mechanistic insight into how HARs modified gene expression in human evolution.

Interferon-γ resistance and immune evasion in glioma develop via Notch-regulated co-evolution of malignant and immune cells.

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of T cells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled by upregulation of oncogenes and downregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.