Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype.

Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.

Activation of endogenous retroviruses during brain development causes an inflammatory response.

Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.

Neuroblast differentiation during development and in neuroblastoma requires KIF1Bß-mediated transport of TRKA.

We recently identified pathogenic KIF1Bß mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bß in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bß is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bß mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bß-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bß. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bß independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bß loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bß mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.

Itga8-Cre-mediated deletion of YAP and TAZ impairs bladder contractility with minimal inflammation and chondrogenic differentiation.

A role of Yes1-associated transcriptional regulator (YAP) and WW domain-containing transcription regulator 1 (TAZ) in vascular and gastrointestinal contractility due to control of myocardin (Myocd) expression, which in turn activates contractile genes, has been demonstrated. Whether this transcriptional hierarchy applies to the urinary bladder is unclear. We found that YAP/TAZ are expressed in human detrusor myocytes and therefore exploited the Itga8-CreERT2 model for the deletion of YAP/TAZ. Recombination occurred in detrusor, and YAP/TAZ transcripts were reduced by >75%. Bladder weights were increased (by ≈22%), but histology demonstrated minimal changes in the detrusor, while arteries in the mucosa were inflamed. Real-time quantitative reverse transcription PCR (RT-qPCR) using the detrusor demonstrated reductions of Myocd (-79 ± 18%) and serum response factor (Srf) along with contractile genes. In addition, the cholinergic receptor muscarinic 2 (Chrm2) and Chrm3 were suppressed (-80 ± 23% and -80 ± 10%), whereas minute increases of Il1b and Il6 were seen. Unlike YAP/TAZ-deficient arteries, SRY (sex-determining region Y)-box 9 (Sox9) did not increase, and no chondrogenic differentiation was apparent. Reductions of smooth muscle myosin heavy chain 11 (Myh11), myosin light-chain kinase gene (Mylk), and Chrm3 were seen at the protein level. Beyond restraining the smooth muscle cell (SMC) program of gene expression, YAP/TAZ depletion silenced SMC-specific splicing, including exon 2a of Myocd. Reduced contractile differentiation was associated with weaker contraction in response to myosin phosphatase inhibition (-36%) and muscarinic activation (reduced by 53% at 0.3 µM carbachol). Finally, short-term overexpression of constitutively active YAP in human embryonic kidney 293 (HEK293) cells increased myocardin (greater than eightfold) along with archetypal target genes, but contractile genes were unaffected or reduced. YAP and TAZ thus regulate myocardin expression in the detrusor, and this is important for SMC differentiation and splicing as well as for contractility.NEW & NOTEWORTHY This study addresses the hypothesis that YAP and TAZ have an overarching role in the transcriptional hierarchy in the smooth muscle of the urinary bladder by controlling myocardin expression. Using smooth muscle-specific and inducible deletion of YAP and TAZ in adult mice, we find that YAP and TAZ control myocardin expression, contractile differentiation, smooth muscle-specific splicing, and bladder contractility. These effects are largely independent of inflammation and chondrogenic differentiation.

YAP and TAZ in Vascular Smooth Muscle Confer Protection Against Hypertensive Vasculopathy.

BACKGROUND: Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. We hypothesize that appropriate mechanotransduction and contractile function in vascular smooth muscle cells are crucial to maintain vascular wall integrity. The Hippo pathway effectors YAP (yes-associated protein 1) and TAZ (WW domain containing transcription regulator 1) have been identified as mechanosensitive transcriptional coactivators. However, their role in vascular smooth muscle cell mechanotransduction has not been investigated in vivo. METHODS: We performed physiological and molecular analyses utilizing an inducible smooth muscle-specific YAP/TAZ knockout mouse model. RESULTS: Arteries lacking YAP/TAZ have reduced agonist-mediated contraction, decreased myogenic response, and attenuated stretch-induced transcriptional regulation of smooth muscle markers. Moreover, in established hypertension, YAP/TAZ knockout results in severe vascular lesions in small mesenteric arteries characterized by neointimal hyperplasia, elastin degradation, and adventitial thickening. CONCLUSIONS: This study demonstrates a protective role of YAP/TAZ against hypertensive vasculopathy.

Introducing Braining-physical exercise as adjunctive therapy in psychiatric care: a retrospective cohort study of a new method.

BACKGROUND: Patients with severe mental disorders suffer from higher rates of poor somatic health and have shorter life expectancy than the average population. Physical activity can treat and prevent several diseases, e.g. cardiovascular and metabolic disorders as well as psychiatric symptoms. It is therefore of utmost importance to develop effective methods to integrate physical activity into psychiatric care. To meet this need, the physical activity intervention Braining was developed. This study aims to describe Braining, to assess the number of patients reached during the first years of pilot testing, to analyze clinical data in the group of patients participating in Braining 2017-2020 and to assess the intervention. METHODS: In this descriptive retrospective study we analyzed data from all patients participating in Braining training sessions ≥ 3 times (n = 239), the Braining Participants. Regular patients at the clinic served as a comparison. Furthermore, medical records were studied for a smaller cohort (n = 51), the Braining Pilot Cohort. Data was analyzed using Chi-square and Fisher's tests. RESULTS: During the introduction period of Braining, 580 patients attended an information meeting about Braining, or at least one training session. 239 patients participated in ≥ 3 training sessions, considered to be participants of Braining. These Braining Participants (n = 239), ages 19 to 82, males 23.4%, attended between 3 and 308 training sessions (median 9). The main diagnoses were affective and anxiety disorders. Number of diagnoses ranged from 0 to 10 (median = 2). For the subsample, the Braining Pilot Cohort (n = 51), participants attended between 3 and 208 training sessions (median = 20). Twelve percent were working full-time, and symptom severity of depression and general anxiety was moderate. Two thirds had ≥ 3 different classes of medication. Regarding metabolic morbidity, 28 had been diagnosed with hypertension, though blood lipids, blood glucose as well as blood pressure were within the normal range. Thirty-seven percent were prescribed Physical Activity on Prescription during 2017-2020. One severe adverse event was reported. CONCLUSIONS: The Braining intervention reached all age-groups and patients with a wide and representative diagnostic panorama, suggesting that Braining could be a promising and safe method for implementing physical activity in a psychiatric patient population.

Aberrant splicing in neuroblastoma generates RNA-fusion transcripts and provides vulnerability to spliceosome inhibitors.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

EglN3 hydroxylase stabilizes BIM-EL linking VHL type 2C mutations to pheochromocytoma pathogenesis and chemotherapy resistance.

Despite the discovery of the oxygen-sensitive regulation of HIFα by the von Hippel-Lindau (VHL) protein, the mechanisms underlying the complex genotype/phenotype correlations in VHL disease remain unknown. Some germline VHL mutations cause familial pheochromocytoma and encode proteins that preserve their ability to down-regulate HIFα. While type 1, 2A, and 2B VHL mutants are defective in regulating HIFα, type 2C mutants encode proteins that preserve their ability to down-regulate HIFα. Here, we identified an oxygen-sensitive function of VHL that is abolished by VHL type 2C mutations. We found that BIM-EL, a proapoptotic BH3-only protein, is hydroxylated by EglN3 and subsequently bound by VHL. VHL mutants fail to bind hydroxylated BIM-EL, regardless of whether they have the ability to bind hydroxylated HIFα or not. VHL binding inhibits BIM-EL phosphorylation by extracellular signal-related kinase (ERK) on serine 69. This causes BIM-EL to escape from proteasomal degradation, allowing it to enhance EglN3-induced apoptosis. BIM-EL was rapidly degraded in cells lacking wild-type VHL or in which EglN3 was inactivated genetically or by lack of oxygen, leading to enhanced cell survival and chemotherapy resistance. Combination therapy using ERK inhibitors, however, resensitizes VHL- and EglN3-deficient cells that are otherwise cisplatin-resistant.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

Identification of the intermediate filament protein synemin/SYNM as a target of myocardin family coactivators.

Myocardin (MYOCD) is a critical regulator of smooth muscle cell (SMC) differentiation, but its transcriptional targets remain to be exhaustively characterized, especially at the protein level. Here we leveraged human RNA and protein expression data to identify novel potential MYOCD targets. Using correlation analyses we found several targets that we could confirm at the protein level, including SORBS1, SLMAP, SYNM, and MCAM. We focused on SYNM, which encodes the intermediate filament protein synemin. SYNM rivalled smooth muscle myosin (MYH11) for SMC specificity and was controlled at the mRNA and protein levels by all myocardin-related transcription factors (MRTFs: MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2). MRTF activity is regulated by the ratio of filamentous to globular actin, and SYNM was accordingly reduced by interventions that depolymerize actin, such as latrunculin treatment and overexpression of constitutively active cofilin. Many MRTF target genes depend on serum response factor (SRF), but SYNM lacked SRF-binding motifs in its proximal promoter, which was not directly regulated by MYOCD. Furthermore, SYNM resisted SRF silencing, yet the time course of induction closely paralleled that of the SRF-dependent target gene ACTA2. SYNM was repressed by the ternary complex factor (TCF) FLI1 and was increased in mouse embryonic fibroblasts lacking three classical TCFs (ELK1, ELK3, and ELK4). Imaging showed colocalization of SYNM with the intermediate filament proteins desmin and vimentin, and MRTF-A/MKL1 increased SYNM-containing intermediate filaments in SMCs. These studies identify SYNM as a novel SRF-independent target of myocardin that is abundantly expressed in all SMCs.

EPAS1/HIF2α correlates with features of low-risk neuroblastoma and with adrenal chromaffin cell differentiation during sympathoadrenal development.

The hypoxia inducible transcription factor EPAS1/HIF2α has been described as an oncogene and a potential therapeutic target in neuroblastoma. Our analysis of several neuroblastoma tumour expression datasets does not support an oncogenic role, instead EPAS1 expression is associated with better patient outcome and characteristics of low-risk tumours. Treatment with HIF2α inhibitors did not block in vitro neuroblastoma cell proliferation nor xenograft growth. In addition, we analysed single cell sequencing data sets from the developing mouse sympathoadrenal lineage, wherein expression of Epas1 was a strong predictor of differentiated adrenal chromaffin cells and negatively correlated with progenitor characteristics. This was reflected in neuroblastoma tumours wherein genes co-expressed with Epas1 during sympathoadrenal development strongly predicts favourable patient outcome and features of low-risk tumours. Thus, our analysis suggest that with the current available data EPAS1/HIF2α should not be classified as a neuroblastoma oncogene and is less likely to represent a suitable drug target in this disease.

Loss of Vascular Myogenic Tone in miR-143/145 Knockout Mice Is Associated With Hypertension-Induced Vascular Lesions in Small Mesenteric Arteries.

OBJECTIVE: Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood. APPROACH AND RESULTS: The importance of smooth muscle-enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions. CONCLUSIONS: This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.

Reprint of: A chemical screen identifies trifluoperazine as an inhibitor of glioblastoma growth.

Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.

Maintaining differentiated cellular identity.

Various studies have demonstrated that somatic differentiated cells can be reprogrammed into other differentiated states or into pluripotency, thus showing that the differentiated cellular state is not irreversible. These findings have generated intense interest in the process of reprogramming and in mechanisms that govern the pluripotent state. However, the realization that differentiated cells can be triggered to switch to considerably different lineages also emphasizes that we need to understand how the identity of mature cells is normally maintained. Here we review recent studies on how the differentiated state is controlled at the transcriptional level and discuss how new insights have begun to elucidate mechanisms underlying the stable maintenance of mature cell identities.

Sarcospan integration into laminin-binding adhesion complexes that ameliorate muscular dystrophy requires utrophin and α7 integrin.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of the dystrophin-glycoprotein complex, a laminin receptor that connects the myofiber to its surrounding extracellular matrix. Utrophin, a dystrophin ortholog that is normally localized to the neuromuscular junction, is naturally upregulated in DMD muscle, which partially compensates for the loss of dystrophin. Transgenic overexpression of utrophin causes broad sarcolemma localization of utrophin, restoration of laminin binding and amelioration of disease in the mdx mouse model of DMD. We previously demonstrated that overexpression of sarcospan, a dystrophin- and utrophin-binding protein, ameliorates mdx muscular dystrophy. Sarcospan boosts levels of utrophin to therapeutic levels at the sarcolemma, where attachment to laminin is restored. However, understanding the compensatory mechanism is complicated by concomitant upregulation of α7ß1 integrin, which also binds laminin. Similar to the effects of utrophin, transgenic overexpression of α7 integrin prevents DMD disease in mice and is accompanied by increased abundance of utrophin around the extra-synaptic sarcolemma. In order to investigate the mechanisms underlying sarcospan 'rescue' of muscular dystrophy, we created double-knockout mice to test the contributions of utrophin or α7 integrin. We show that sarcospan-mediated amelioration of muscular dystrophy in DMD mice is dependent on the presence of both utrophin and α7ß1 integrin, even when they are individually expressed at therapeutic levels. Furthermore, we found that association of sarcospan into laminin-binding complexes is dependent on utrophin and α7ß1 integrin.

A chemical screen identifies trifluoperazine as an inhibitor of glioblastoma growth.

Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.

Loss of dystrophin and ß-sarcoglycan significantly exacerbates the phenotype of laminin α2 chain-deficient animals.

The adhesion molecule laminin α2 chain interacts with the dystrophin-glycoprotein complex, contributes to normal muscle function, and protects skeletal muscles from damage. Complete loss of the laminin α2 chain in mice results in a severe muscular dystrophy phenotype and death at approximately 3 weeks of age. However, it is not clear if the remaining members of the dystrophin-glycoprotein complex further protect laminin α2 chain-deficient skeletal muscle fibers from degeneration. Hence, we generated mice deficient in laminin α2 chain and dystrophin (dy(3K)/mdx) and mice devoid of laminin α2 chain and ß-sarcoglycan (dy(3K)/Sgcb). Severe muscular dystrophy and a lack of nourishment inevitably led to massive muscle wasting and death in double-knockout animals. The dy(3K)/Sgcb mice were generally more severely affected than dy(3K)/mdx mice. However, both double-knockout strains displayed exacerbated muscle degeneration, inflammation, fibrosis, and reduced life span (5 to 13 days) compared with single-knockout animals. However, neither extraocular nor cardiac muscle was affected in double-knockout animals. Our results suggest that, although laminin α2 chain, dystrophin, and ß-sarcoglycan are all part of the same adhesion complex, they have complementary, but nonredundant, roles in maintaining sarcolemmal integrity and protecting skeletal muscle fibers from damage. Moreover, the double-knockout mice could potentially serve as models in which to study extremely aggressive muscle-wasting conditions.

Bortezomib partially improves laminin α2 chain-deficient muscular dystrophy.

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted.