Anne Schänzer

A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2α

Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.

Extracellular RNA Liberates Tumor Necrosis Factor-a to Promote Tumor Cell Trafficking and Progression

Extracellular RNA (eRNA) released from injured cells promotes tissue permeability, thrombosis, and inflammation in vitro and in vivo, and RNase1 pretreatment can reduce all these effects. In this study, we investigated the role of the eRNA/RNase1 system in tumor progression and metastasis. Under quiescent and stimulatory conditions, tumor cells released much higher levels of endogenous extracellular RNA (eRNA) than nontumor cells. In glioblastomas, eRNA was detected at higher levels in tumors than nontumor tissue. eRNA induced tumor cells to adhere to and migrate through human cerebral microvascular endothelial cells (HCMEC/D3), in a manner requiring activation of VEGF signaling. In addition, eRNA liberated TNF-α from macrophages in a manner requiring activation of the TNF-α-converting enzyme TACE. Accordingly, supernatants derived from eRNA-treated macrophages enhanced tumor cell adhesion to HCMEC/D3. TNF-α release evoked by eRNA relied upon signaling activation of mitogen-activated protein kinases and the NF-κB pathway. In subcutaneous xenograft models of human cancer, administration of RNase1 but not DNase decreased tumor volume and weight. Taken together, these results suggest that eRNA released from tumor cells has the capacity to promote tumor cell invasion through endothelial barriers by both direct and indirect mechanisms, including through a mechanism involving TNF-α release from tumor-infiltrating monocytes/macrophages. Our findings establish a crucial role for eRNA in driving tumor progression, and they suggest applications for extracellular RNase1 as an antiinvasive regimen for cancer treatment.

Stress-Induced Upregulation of SLC19A3 is Impaired in Biotin-Thiamine-Responsive Basal Ganglia Disease

Biotin‐thiamine‐responsive basal ganglia disease (BTBGD) is a potentially treatable disorder caused by mutations in the SLC19A3 gene, encoding the human thiamine transporter 2. Manifestation of BTBGD as acute encephalopathy triggered by a febrile infection has been frequently reported, but the underlying mechanisms are not clear. We investigated a family with two brothers being compound heterozygous for the SLC19A3 mutations p.W94R and p.Q393*fs. Post‐mortem analysis of the brain of one brother showed a mixture of acute, subacute and chronic changes with cystic and necrotic lesions and hemorrhage in the putamen, and hemorrhagic lesions in the caudate nucleus and cortical layers. SLC19A3 expression was substantially reduced in the cortex, basal ganglia and cerebellum compared with an age‐matched control. Importantly, exposure of fibroblasts to stress factors such as acidosis or hypoxia markedly upregulated SLC19A3 in control cells, but failed to elevate SLC19A3 expression in the patients fibroblasts. These results demonstrate ubiquitously reduced thiamine transporter function in the cerebral gray matter, and neuropathological alterations similar to Wernickes disease in BTBGD. They also suggest that episodes of encephalopathy are caused by a substantially reduced capacity of mutant neuronal cells to increase SLC19A3 expression, necessary to adapt to stress conditions.

A human post-mortem brain model for the standardization of multi-centre MRI studies

Multi-centre MRI studies of the brain are essential for enrolling large and diverse patient cohorts, as required for the investigation of heterogeneous neurological and psychiatric diseases. However, the multi-site comparison of standard MRI data sets that are weighted with respect to tissue parameters such as the relaxation times (T1, T2) and proton density (PD) may be problematic, as signal intensities and image contrasts depend on site-specific details such as the sequences used, imaging parameters, and sensitivity profiles of the radiofrequency (RF) coils. Water or gel phantoms are frequently used for long-term and/or inter-site quality assessment. However, these phantoms hardly mimic the structure, shape, size or tissue distribution of the human brain. The goals of this study were: (1) to validate the long-term stability of a human post-mortem brain phantom, performing quantitative mapping of T1, T2, and PD, and the magnetization transfer ratio (MTR) over a period of 18months; (2) to acquire and analyse data for this phantom and the brain of a healthy control (HC) in a multi-centre study for MRI protocol standardization in four centres, while conducting a voxel-wise as well as whole brain grey (GM) and white matter (WM) tissue volume comparison. MTR, T2, and the quotient of PD in WM and GM were stable in the post-mortem brain with no significant changes. T1 was found to decrease from 267/236ms (GM/WM) to 234/216ms between 5 and 17weeks post embedment, stabilizing during an 18-month period following the first scan at about 215/190ms. The volumetric measures, based on T1-weighted MP-RAGE images obtained at all participating centres, revealed inter- and intra-centre variations in the evaluated GM and WM volumes that displayed similar trends in both the post-mortem brain as well as the HC. At a confidence level of 95%, brain regions such as the brainstem, deep GM structures as well as boundaries between GM and WM tissues were found to be less reproducible than other brain regions in all participating centres. The results demonstrate that a post-mortem brain phantom may be used as a reliable tool for multi-centre MR studies.

Rapidly progressive hypertrophic cardiomyopathy in an infant with Noonan syndrome with multiple lentigines: Palliative treatment with a rapamycin analog

Noonan syndrome with multiple lentigines (NSML) frequently manifests with hypertrophic cardiomyopathy (HCM). Recently, it was demonstrated that mTOR inhibition reverses HCM in NSML mice. We report for the first time on the effects of treatment with a rapamycin analog in an infant with LS and malignant HCM. In the boy, progressive HCM was diagnosed during the first week of life and a diagnosis of NSML was established at age 20 weeks by showing a heterozygous Q510E mutation in PTPN11. Immunoblotting with antibodies against pERK, pAkt, and pS6RP in fibroblasts demonstrated enhanced Akt/mTOR pathway activity. Because of the patients critical condition, everolimus therapy was started at age 24 weeks and continued until heart transplantation at age 36 weeks. Prior to surgery, heart failure improved from NYHA stage IV to II and brain natriuretic peptide values decreased from 9,600 to <1,000 pg/ml, but no reversal of cardiac hypertrophy was observed. Examination of the explanted heart revealed severe hypertrophy and myofiber disarray with extensive perivascular fibrosis. These findings provide evidence that Akt/mTOR activity is enhanced in NSML with HCM and suggest that rapamycin treatment could principally be feasible for infantile NSML. The preliminary experiences made in this single patient indicate that therapy should start early to prevent irreversible cardiac remodelling.

Increased Probability of Co-Occurrence of Two Rare Diseases in Consanguineous Families and Resolution of a Complex Phenotype by Next Generation Sequencing.

Massively parallel sequencing of whole genomes and exomes has facilitated a direct assessment of causative genetic variation, now enabling the identification of genetic factors involved in rare diseases (RD) with Mendelian inheritance patterns on an almost routine basis. Here, we describe the illustrative case of a single consanguineous family where this strategy suffered from the difficulty to distinguish between two etiologically distinct disorders, namely the co-occurrence of hereditary hypophosphatemic rickets (HRR) and congenital myopathies (CM), by their phenotypic manifestation alone. We used parametric linkage analysis, homozygosity mapping and whole exome-sequencing to identify mutations underlying HRR and CM. We also present an approximate approach for assessing the probability of co-occurrence of two unlinked recessive RD in a single family as a function of the degree of consanguinity and the frequency of the disease-causing alleles. Linkage analysis and homozygosity mapping yielded elusive results when assuming a single RD, but whole-exome sequencing helped to identify two mutations in two genes, namely SLC34A3 and SEPN1, that segregated independently in this family and that have previously been linked to two etiologically different diseases. We assess the increase in chance co-occurrence of rare diseases due to consanguinity, i.e. under circumstances that generally favor linkage mapping of recessive disease, and show that this probability can increase by several orders of magnitudes. We conclude that such potential co-occurrence represents an underestimated risk when analyzing rare or undefined diseases in consanguineous families and should be given more consideration in the clinical and genetic evaluation.

MERRF-like phenotype associated with a rare mitochondrial trnaile mutation (m.4284 G>A).

Nearly all patients affected by myoclonic epilepsy with ragged-red fibres (MERRF) harbour a mutation in the mitochondrial transfer RNALys gene. We report on an 8-year-old girl with clinical and diagnostic features of MERRF. After excluding one of the common mutations associated with MERRF, a complete sequence analysis of the mitochondrial genome revealed an m.4284 G>A mutation in the mitochondrial transfer RNAIle gene. This mutation has only once been described in a family with variable clinical symptoms, but has not yet been linked to MERRF. This case extends the mutational spectrum associated with the MERRF phenotype, and demonstrates the importance of performing a comprehensive mutational analysis in patients with suspected mitochondrial disease when common mutations have been ruled out.

Endothelial Dab1 signaling orchestrates neuro-glia-vessel communication in the central nervous system

Developing the bloodbrain barrier During development, signals need to be dynamically integrated by endothelial cells, neurons, and glia to achieve functional neuro-glia-vascular units in the central nervous system. During cortical development, neuronal Dab1 and ApoER2 receptors respond to a guidance cue called reelin. Studying mice, Segarra et al. found that Dab1 and ApoER2 are also expressed in endothelial cells (see the Perspective by Thomas). The integration of reelin signaling in endothelial cells and neurons facilitates the communication between vessels, glia, and neurons that is necessary for the correct positioning of neurons during cortical development. This integration is also important for correct communication at the neurovascular unit required for blood-brain barrier integrity in the mature brain. Science, this issue p. eaao2861; see also p. 754 A single molecule, reelin, can orchestrate both neuronal migration and the vascularization of the central nervous system. INTRODUCTION The function of the brain relies on communication among the complex network of cells that constitute this organ. Vascularization of the central nervous system (CNS) ensures adequate delivery of oxygen and nutrients to build up and maintain homeostasis of neuronal networks. Thus, it is not surprising that blood vessels and neuronal cells share multiple parallelisms orchestrating their development in synchrony and in a mutually dependent manner in the CNS. Despite the essential role of the endothelium in brain function, the means by which signaling at the interface of endothelial cells, glial cells, and neurons is integrated temporally and spatially for proper brain development has remained largely unexplored. RATIONALE Integration of signaling pathways and cellular responses among endothelial cells, glial cells, and neurons is needed to ensure proper architecture of the brain. Reelin (Reln), a large secreted glycoprotein, induces Disabled 1 (Dab1)–dependent responses in neurons to guide their migration in all layered brain structures. Secretion of reelin by Cajal-Retzius cells in the marginal zone of the cortex timely coincides with active sprouting of pial vessels ingrowing perpendicularly into the marginal zone and forming a complex vascular network needed to support brain development and function. Therefore, reelin might be in the perfect position to perform a bivalent function to timely and spatially orchestrate both neuronal migration and CNS vascularization. We reasoned that blood vessels might instruct the process of neuronal migration by a cell-autonomous function of Dab1 on endothelial cells. To investigate this, we deleted the expression of vascular Dab1 in mice and investigated the effects on CNS vascularization, neuroglial organization, and neurovascular unit function. RESULTS We found that reelin/Dab1 signaling is conserved in endothelial cells and exerts potent proangiogenic effects in the developing vasculature of the CNS by controlling endothelial cell proliferation and active filopodia extension of the vascular network. The interaction of the reelin receptor ApoER2 (apolipoprotein E receptor 2) and VEGFR2 (vascular endothelial growth factor receptor 2) mediated the proangiogenic roles of Dab1 in endothelial cells. Surprisingly, deletion of Dab1 exclusively in the vascular system induced changes in the position of postmitotic pyramidal neurons in the cortical layers of the cerebral cortex. At the cellular level, depletion of vascular Dab1 reduced the docking of the radial glia processes to the pial surface at embryonic and postnatal stages and altered the differentiation of glial cells to astrocytes. The defects in neuronal migration persisted in adult mutant animals, where stereotypical attachment of the astrocytes to penetrating vessels in the glia limitans superficialis was also found to be aberrant. The functionality of the neurovascular unit [blood-brain barrier (BBB) integrity] was also affected in reelin knockout animals, and we could attribute those defects to the lack of Dab1 signaling exclusively in endothelial cells. The increased BBB permeability was again associated with an insufficient coverage of the brain vasculature by astrocytic endfeet. Mechanistically, we determined that the astroglial attachment to the vasculature is mediated by reelin-induced deposition of laminin-α4 by endothelial cells to the extracellular compartment, which in turn enables the binding of the glial processes to the CNS vasculature via the activation of integrin-β1 in glial cells. CONCLUSION Our results shed new light on the function of the vasculature in CNS development and homeostasis—in particular, how signals from the endothelium orchestrate the communication among vessels, glial cells, and neurons, and how specific changes in the molecular signature of the endothelium affect a plethora of processes such as CNS vascularization, extracellular matrix composition, neuroglial cytoarchitecture, and BBB development. Instructive functions of vascular Dab1 in the neurovascular interface. (A) ApoER2 and Dab1 control endothelial cell proliferation and tip cell filopodia extension during CNS vascularization by cross-talking to the VEGFR2 pathway. (B and C) Vascular Dab1 also instructs radial glia organization and neuronal migration in the developing cerebral cortex (B) and the development of the blood-brain barrier (C). In both cases, Dab1 signaling in the vasculature regulates the deposition of laminin-α4 and, in turn, the activation of integrin-β1 in glial cells. The architecture of the neurovascular unit (NVU) is controlled by the communication of neurons, glia, and vascular cells. We found that the neuronal guidance cue reelin possesses proangiogenic activities that ensure the communication of endothelial cells (ECs) with the glia to control neuronal migration and the establishment of the blood-brain barrier in the mouse brain. Apolipoprotein E receptor 2 (ApoER2) and Disabled1 (Dab1) expressed in ECs are required for vascularization of the retina and the cerebral cortex. Deletion of Dab1 in ECs leads to a reduced secretion of laminin-α4 and decreased activation of integrin-β1 in glial cells, which in turn control neuronal migration and barrier properties of the NVU. Thus, reelin signaling in the endothelium is an instructive and integrative cue essential for neuro-glia-vascular communication.

Novel STAC3 Mutations in the First Non-Amerindian Patient with Native American Myopathy

Native American myopathy (NAM) is an autosomal recessive congenital myopathy, up till now exclusively described in Lumbee Indians who harbor one single homozygous mutation (c.1046G>C, pW284S) in the STAC3 gene, encoding a protein important for proper excitation-contraction coupling in muscle. Here, we report the first non-Amerindian patient of Turkish ancestry, being compound heterozygous for the mutations c.862A>T (p.K288*) and c.432+4A>T (aberrant splicing with skipping of exon 4). Symptoms in NAM include congenital muscle weakness and contractures, progressive scoliosis, early ventilatory failure, a peculiar facial gestalt with mild ptosis and downturned corners of the mouth, short stature, and marked susceptibility to malignant hyperthermia. This case shows that NAM should also be considered in non-Indian patients with congenital myopathy, and suggests that STAC3 mutations should be taken into account as a potential cause of malignant hyperthermia.

Quantification of muscle pathology in infantile Pompe disease.

The effects of enzyme replacement therapy (ERT) in infantile Pompe disease are variable, necessitating the identification of biomarkers to assess the severity of disease and response to ERT. The aims of this study were to investigate whether quantification of muscle pathology in infantile Pompe disease prior to and during ERT is feasible at the light microscope, and to develop a score that summarizes the degree of muscle pathology in a comprehensive manner from PAS-stained resin sections alone. We, therefore, determined glycogen load, extent of muscle fibre disruption, and amount of autophagic vacuoles in resin-embedded muscle biopsy specimens from 11 infantile Pompe patients and 2 with early childhood phenotype by quantitative methods, correlated the findings with ultrastructural analyses, compared PAS-stained resin sections with conventional PAS-stained cryosections, and related the quantified degree of muscle damage from infantile patients to the effects of ERT. Comparison of electron and light microscopic findings demonstrated that important alterations of skeletal muscle morphology can also be depicted by examining PAS stained resin sections. Infantile patients with good response to ERT had lower muscle pathology score values prior to and during ERT than those with moderate and poor response, but the number of tissue samples available for evaluation was limited. These findings suggest that quantification of muscle pathology by analysing PAS stained resin sections is in principle feasible and useful to monitor disease progression and therapy response. These results have to be validated by investigating a larger group of patients.