PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS.

The plant homeodomain zinc-finger protein, PHF6, is a transcriptional regulator, and PHF6 germline mutations cause the X-linked intellectual disability (XLID) Börjeson-Forssman-Lehmann syndrome (BFLS). The mechanisms by which PHF6 regulates transcription and how its mutations cause BFLS remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex in the vicinity of genes involved in central nervous system development and neurogenesis. Characterization of BFLS mice harbouring PHF6 patient mutations reveals an increase in embryonic neural stem cell (eNSC) self-renewal and a reduction of neural progenitors. We identify a panel of Ephrin receptors (EphRs) as direct transcriptional targets of PHF6. Mechanistically, we show that PHF6 regulation of EphR is impaired in BFLS mice and in conditional Phf6 knock-out mice. Knockdown of EphR-A phenocopies the PHF6 loss-of-function defects in altering eNSCs, and its forced expression rescues defects of BFLS mice-derived eNSCs. Our data indicate that PHF6 directly promotes Ephrin receptor expression to control eNSC behaviour in the developing brain, and that this pathway is impaired in BFLS.

CLIC1 regulation of cancer stem cells in glioblastoma.

Chloride intracellular channel 1 (CLIC1) has emerged as a therapeutic target in various cancers. CLIC1 promotes cell cycle progression and cancer stem cell (CSC) self-renewal. Furthermore, CLIC1 is shown to play diverse roles in proliferation, cell volume regulation, tumour invasion, migration, and angiogenesis. In glioblastoma (GB), CLIC1 facilitates the G1/S phase transition and tightly regulates glioma stem-like cells (GSCs), a rare population of self-renewing CSCs with central roles in tumour resistance to therapy and tumour recurrence. CLIC1 is found as either a monomeric soluble protein or as a non-covalent dimeric protein that can form an ion channel. The ratio of dimeric to monomeric protein is altered in GSCs and depends on the cell redox state. Elucidating the mechanisms underlying the alterations in CLIC1 expression and structural transitions will further our understanding of its role in GSC biology. This review will highlight the role of CLIC1 in GSCs and its significance in facilitating different hallmarks of cancer.

Transcriptional reprogramming of skeletal muscle stem cells by the niche environment.

Adult stem cells are indispensable for tissue regeneration, but their function declines with age. The niche environment in which the stem cells reside plays a critical role in their function. However, quantification of the niche effect on stem cell function is lacking. Using muscle stem cells (MuSC) as a model, we show that aging leads to a significant transcriptomic shift in their subpopulations accompanied by locus-specific gain and loss of chromatin accessibility and DNA methylation. By combining in vivo MuSC transplantation and computational methods, we show that the expression of approximately half of all age-altered genes in MuSCs from aged male mice can be restored by exposure to a young niche environment. While there is a correlation between gene reversibility and epigenetic alterations, restoration of gene expression occurs primarily at the level of transcription. The stem cell niche environment therefore represents an important therapeutic target to enhance tissue regeneration in aging.

In situ detection of protein-protein interaction by proximity ligation assay in patient derived brain tumor stem cells.

Improper or aberrant protein-protein interactions can lead to severe human diseases including cancer. Here, we describe an adapted proximity ligation assay (PLA) protocol for the assessment of galectin-1-HOXA5 interaction in brain tumor stem cells (BTSCs). We detail the steps for culturing and preparation of BTSCs followed by PLA and detection of protein interactions in situ using fluorescent microscopy. This PLA protocol is optimized specifically for BTSCs and includes key controls for effective result analysis. For complete details on the use and execution of this protocol, please refer to Sharanek et al. (2021).

Monitoring Mitochondrial Respiration in Mouse Cerebellar Granule Neurons.

Defects in mitochondrial oxidative phosphorylation have been observed in numerous neurodegenerative disorders and are linked to bioenergetic crises leading to neuronal death. The distinct metabolic profile of neurons is predominantly oxidative, which is characterized by the oxidation of glucose or its metabolites in the mitochondria to produce ATP. This process involves the tricarboxylic acid cycle, electron transfer in the respiratory chain, and oxygen consumption. Therefore, measurement of oxygen consumption rates (OCR) can be accurately applied to assess the rate of mitochondrial respiration. In this chapter, we describe our optimized protocol for the assessment of OCR specifically in primary mouse cerebellar granule neurons (CGN). The protocol includes isolation and manipulation of mouse CGNs followed by real-time assessment of mitochondrial OCR using a Seahorse XFe96 extracellular flux analyzer.

Isolation of Mouse Embryonic Neural Stem Cells and Characterization of Neural Stem Markers by Flow Cytometry.

Neurogenesis is outlined as a process in which new neurons are generated from neural stem cells (NSCs). This process comprises proliferation and fate specification of NSCs, migration of newborn neurons, and their maturation. Defects in embryonic neurogenesis have emerged as a key mechanism underlying neurodevelopmental disorders such as autism spectrum disorders and intellectual disability. An impairment in neurogenesis has also been observed in neurodegenerative disorders such as Huntington's disease. Transgenic animal models of neurodevelopmental and neurodegenerative diseases have been developed which serve as invaluable tools to investigate the early mechanisms of disease pathogenesis. In this chapter, we describe our optimized method to obtain and maintain reproducible neurosphere cultures from transgenic or patient mouse models followed by characterization of NSCs by flow cytometry.

Isolation of Adult Mouse Neural Stem Cells and Assessment of Self-Renewal by ELDA.

The generation of new neurons in the adult brain throughout life is integral to brain plasticity and repair. Adult neural stem cells (aNSCs), present in the subventricular zone (SVZ) of the lateral ventricle wall and the subgranular zone (SGZ) of the hippocampal dentate gyrus, divide symmetrically or asymmetrically to maintain the stem cell pool or become committed progenitors and differentiate into various cell lineages. Depletion or dysregulation of aNSCs impairs proper brain connectivity and function and can contribute to several brain diseases including cognitive and neurodegenerative disorders and brain cancer. In this chapter, we present our optimized method to obtain and maintain reproducible neurosphere cultures from the adult mouse brain followed by evaluation of self-renewal using the extreme limiting dilution assay (ELDA) software. We use this assay routinely on aNSCs obtained from patient mouse models to generate log fraction plots and provide confidence intervals for all limiting dilution assay (LDA) data. At the same time, given the low number of NSCs required for the completion of the ELDA experiment, it is feasible to employ this approach to conduct high-content compound screening for therapeutic interventions aimed at enhancing the stem cell pool or combating a cohort of genetic and epigenetic disorders.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

Transcriptional control of brain tumor stem cells by a carbohydrate binding protein.

Brain tumor stem cells (BTSCs) and intratumoral heterogeneity represent major challenges in glioblastoma therapy. Here, we report that the LGALS1 gene, encoding the carbohydrate binding protein, galectin1, is a key regulator of BTSCs and glioblastoma resistance to therapy. Genetic deletion of LGALS1 alters BTSC gene expression profiles and results in downregulation of gene sets associated with the mesenchymal subtype of glioblastoma. Using a combination of pharmacological and genetic approaches, we establish that inhibition of LGALS1 signaling in BTSCs impairs self-renewal, suppresses tumorigenesis, prolongs lifespan, and improves glioblastoma response to ionizing radiation in preclinical animal models. Mechanistically, we show that LGALS1 is a direct transcriptional target of STAT3 with its expression robustly regulated by the ligand OSM. Importantly, we establish that galectin1 forms a complex with the transcription factor HOXA5 to reprogram the BTSC transcriptional landscape. Our data unravel an oncogenic signaling pathway by which the galectin1/HOXA5 complex maintains BTSCs and promotes glioblastoma.

Myf6/MRF4 is a myogenic niche regulator required for the maintenance of the muscle stem cell pool.

The function and maintenance of muscle stem cells (MuSCs) are tightly regulated by signals originating from their niche environment. Skeletal myofibers are a principle component of the MuSC niche and are in direct contact with the muscle stem cells. Here, we show that Myf6 establishes a ligand/receptor interaction between muscle stem cells and their associated muscle fibers. Our data show that Myf6 transcriptionally regulates a broad spectrum of myokines and muscle-secreted proteins in skeletal myofibers, including EGF. EGFR signaling blocks p38 MAP kinase-induced differentiation of muscle stem cells. Homozygous deletion of Myf6 causes a significant reduction in the ability of muscle to produce EGF, leading to a deregulation in EGFR signaling. Consequently, although Myf6-knockout mice are born with a normal muscle stem cell compartment, they undergo a progressive reduction in their stem cell pool during postnatal life due to spontaneous exit from quiescence. Taken together, our data uncover a novel role for Myf6 in promoting the expression of key myokines, such as EGF, in the muscle fiber which prevents muscle stem cell exhaustion by blocking their premature differentiation.

OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation.

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. Here, we report our discovery of a mitochondrial OSMR that confers resistance to IR via regulation of oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.

Characterization of a Mouse Model of Börjeson-Forssman-Lehmann Syndrome.

Mutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

Pathogenesis of Börjeson-Forssman-Lehmann syndrome: Insights from PHF6 function.

Intellectual disability encompasses a large set of neurodevelopmental disorders of cognition that are more common in males than females. Although mutations in over 100 X-linked genes associated to intellectual disability have been identified, only a few X-linked intellectual disability proteins have been intensively studied. Hence, the molecular mechanisms underlying the majority of X-linked intellectual disability disorders remain poorly understood. A substantial fraction of X-linked intellectual disability genes encode nuclear proteins, suggesting that elucidating their functions in the regulation of transcription may provide novel insights into the pathogenesis of intellectual disability. Recent studies have uncovered mechanisms by which mutations of the gene encoding plant homeodomain (PHD)-like finger protein 6 (PHF6) contribute to the pathogenesis of the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS). PHF6 plays a critical role in the migration of neurons in the mouse cerebral cortex in vivo, and patient-specific mutations disrupt the ability of PHF6 to promote neuronal migration. Interestingly, PHF6 physically associates with the PAF1 transcriptional elongation complex and thereby drives neuronal migration in the cerebral cortex. PHF6 also interacts with the NuRD chromatin remodeling complex and with the nucleolar transcriptional regulator UBF, though the biological role of these interactions remains to be characterized. In other studies, PHF6 mRNA has been identified as the target of the microRNA miR-128 in the cerebral cortex, providing new insights into regulation of PHF6 function in neuronal migration. Importantly, deregulation of PHF6 function in neuronal migration triggers the formation of white matter heterotopias that harbor neuronal hyperexcitability, which may be relevant to the pathogenesis of intellectual disability and seizures in BFLS. Collectively, these studies are beginning to provide key insights into the molecular pathogenesis of BFLS.

A focused compound screen highlights the significance of epidermal growth factor receptor signalling in chordoma pathogenesis.

Chordoma is a rare primary bone cancer with limited treatment options. Surgical resection followed by radiotherapy has proven effective; however, when, in 30-40% of patients, tumours recur and metastasize, a high level of resistance to chemotherapies leaves these patients with a dearth of treatment options. Recent work published in the Journal of Pathology by Scheipl et al describing a focused compound drug screen highlights the significance of epidermal growth factor receptor (EGFR) signalling in chordoma, and shows potential for EGFR inhibitors as a way forward for developing an effective treatment for chordoma. Importantly, combining EGFR inhibitors with a MET inhibitor induces a synergistic effect on growth inhibition of resistant chordoma cells, highlighting the significance of combined EGFR and MET inhibitors as a potential avenue to defeat chemoresistance in chordoma patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling.

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of these mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis.

CDK5 phosphorylates DRP1 and drives mitochondrial defects in NMDA-induced neuronal death.

Defects in mitochondrial fission and cyclin dependent kinase 5 (CDK5) activation are early events that precede neuronal loss following NMDA-induced neuronal death. Here, we report that the cytoplasmic CDK5 tightly regulates mitochondrial morphology defects associated with NMDA-induced neuronal injury via regulation of the mitochondrial fission protein, dynamin-related protein 1 (DRP1). We show that DRP1 is a direct target of CDK5. CDK5-mediated phosphorylation of DRP1 at a conserved Serine residue, S585, is elevated at the mitochondria and is associated with increased mitochondrial fission. Ectopic expression of a cytoplasmic CDK5 or mutant DRP1-S585D results in increased mitochondrial fragmentation in primary neurons. Conversely, expression of a dominant negative form of cytoplasmic CDK5 or mutant DRP1-S585A results in elongated mitochondria. In addition, pharmacological inhibition of CDK5 by Roscovitine inhibits DRP1 phosphorylation and mitochondrial fission associated with NMDA-induced neuronal loss. Importantly, conditional deletion of CDK5 significantly attenuates DRP1 phosphorylation at S585 and rescues mitochondrial fission defects in neurons exposed to NMDA. Our studies delineate an important mechanism by which CDK5 regulates mitochondrial morphology defects associated with neuronal injury.

The X-linked intellectual disability protein PHF6 associates with the PAF1 complex and regulates neuronal migration in the mammalian brain.

Intellectual disability is a prevalent disorder that remains incurable. Mutations of the X-linked protein PHF6 cause the intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS). However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. We report that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex in vivo, leading to the formation of white matter heterotopias displaying neuronal hyperexcitability. We find that PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype in vivo. We also identify Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5), a potential schizophrenia susceptibility gene, as a critical downstream target of PHF6 in the control of neuronal migration. These findings define PHF6, PAF1, and NGC/CSPG5 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of developmental disorders of cognition.