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.

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.

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.

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.

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.

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.

PPIL4 is essential for brain angiogenesis and implicated in intracranial aneurysms in humans.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

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.).

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

The Notch pathway in CNS homeostasis and neurodegeneration.

The role of the Notch signaling pathway in neural development has been well established over many years. More recent studies, however, have demonstrated that Notch continues to be expressed and active throughout adulthood in many areas of the central nervous system. Notch signals have been implicated in adult neurogenesis, memory formation, and synaptic plasticity in the adult organism, as well as linked to acute brain trauma and chronic neurodegenerative conditions. NOTCH3 mutations are responsible for the most common form of hereditary stroke, the progressive disorder cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Notch has also been associated with several progressive neurodegenerative diseases, including Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis. Although numerous studies link Notch activity with CNS homeostasis and neurodegenerative diseases, the data thus far are primarily correlative, rather than functional. Nevertheless, the evidence for Notch pathway activity in specific neural cellular contexts is strong, and certainly intriguing, and points to the possibility that the pathway carries therapeutic promise. This article is categorized under: Nervous System Development > Flies Signaling Pathways > Cell Fate Signaling Nervous System Development > Vertebrates: General Principles.

Id4 Downstream of Notch2 Maintains Neural Stem Cell Quiescence in the Adult Hippocampus.

Neural stem cells (NSCs) in the adult mouse hippocampal dentate gyrus (DG) are mostly quiescent, and only a few are in cell cycle at any point in time. DG NSCs become increasingly dormant with age and enter mitosis less frequently, which impinges on neurogenesis. How NSC inactivity is maintained is largely unknown. Here, we found that Id4 is a downstream target of Notch2 signaling and maintains DG NSC quiescence by blocking cell-cycle entry. Id4 expression is sufficient to promote DG NSC quiescence and Id4 knockdown rescues Notch2-induced inhibition of NSC proliferation. Id4 deletion activates NSC proliferation in the DG without evoking neuron generation, and overexpression increases NSC maintenance while promoting astrogliogenesis at the expense of neurogenesis. Together, our findings indicate that Id4 is a major effector of Notch2 signaling in NSCs and a Notch2-Id4 axis promotes NSC quiescence in the adult DG, uncoupling NSC activation from neuronal differentiation.

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.

Notch2 Signaling Maintains NSC Quiescence in the Murine Ventricular-Subventricular Zone.

Neurogenesis continues in the ventricular-subventricular zone (V-SVZ) of the adult forebrain from quiescent neural stem cells (NSCs). V-SVZ NSCs are a reservoir for new olfactory bulb (OB) neurons that migrate through the rostral migratory stream (RMS). To generate neurons, V-SVZ NSCs need to activate and enter the cell cycle. The mechanisms underlying NSC transition from quiescence to activity are poorly understood. We show that Notch2, but not Notch1, signaling conveys quiescence to V-SVZ NSCs by repressing cell-cycle-related genes and neurogenesis. Loss of Notch2 activates quiescent NSCs, which proliferate and generate new neurons of the OB lineage. Notch2 deficiency results in accelerated V-SVZ NSC exhaustion and an aging-like phenotype. Simultaneous loss of Notch1 and Notch2 resembled the total loss of Rbpj-mediated canonical Notch signaling; thus, Notch2 functions are not compensated in NSCs, and Notch2 is indispensable for the maintenance of NSC quiescence in the adult V-SVZ.

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.

Disruptions in asymmetric centrosome inheritance and WDR62-Aurora kinase B interactions in primary microcephaly.

Recessive mutations in WD repeat domain 62 (WDR62) cause microcephaly and a wide spectrum of severe brain malformations. Disruption of the mouse ortholog results in microcephaly underlain by reduced proliferation of neocortical progenitors during late neurogenesis, abnormalities in asymmetric centrosome inheritance leading to neuronal migration delays, and altered neuronal differentiation. Spindle pole localization of WDR62 and mitotic progression are defective in patient-derived fibroblasts, which, similar to mouse neocortical progenitors, transiently arrest at prometaphase. Expression of WDR62 is closely correlated with components of the chromosome passenger complex (CPC), a key regulator of mitosis. Wild type WDR62, but not disease-associated mutant forms, interacts with the CPC core enzyme Aurora kinase B and staining of CPC components at centromeres is altered in patient-derived fibroblasts. Our findings demonstrate critical and diverse functions of WDR62 in neocortical development and provide insight into the mechanisms by which its disruption leads to a plethora of structural abnormalities.

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.

B-Cell Depletion Reduces the Maturation of Cerebral Cavernous Malformations in Murine Models.

Cerebral cavernous malformations (CCMs) are relatively common vascular malformations, characterized by increased Rho kinase (ROCK) activity, vascular hyper-permeability and the presence of blood degradation products including non-heme iron. Previous studies revealed robust inflammatory cell infiltration, selective synthesis of IgG, in situ antigen driven B-cell clonal expansion, and deposition of immune complexes and complement proteins within CCM lesions. We aimed to evaluate the impact of suppressing the immune response on the formation and maturation of CCM lesions, as well as lesional iron deposition and ROCK activity. Two murine models of heterozygous Ccm3 (Pdcd10), which spontaneously develop CCM lesions with severe and milder phenotypes, were either untreated or received anti-mouse BR3 to deplete B cells. Brains from anti-mouse BR3-treated mice exhibited significantly fewer mature CCM lesions and smaller lesions compared to untreated mice. B cell depletion halted the progression of lesions into mature stage 2 lesions but did not prevent their genesis. Non-heme iron deposition and ROCK activity was decreased in lesions of B cell depleted mice. This represents the first report of the therapeutic benefit of B-cell depletion in the development and progression of CCMs, and provides a proof of principle that B cells play a critical role in CCM lesion genesis and maturation. These findings add biologics to the list of potential therapeutic agents for CCM disease. Future studies would characterize the putative antigenic trigger and further define the mechanism of immune response in the lesions.