Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators.

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3, Plk1, Mycn, Dnmt1, Dnmt3b, and Tet3). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis-regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1-null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators.

Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.

shRNA-mediated PPARα knockdown in human glioma stem cells reduces in vitro proliferation and inhibits orthotopic xenograft tumour growth.

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD-SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo. PPARα-expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c-Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα-target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A, with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self-renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Applying support-vector machine learning algorithms toward predicting host-guest interactions with cucurbit[7]uril.

Machine learning is a valuable tool in the development of chemical technologies but its applications into supramolecular chemistry have been limited. Here, the utility of kernel-based support vector machine learning using density functional theory calculations as training data is evaluated when used to predict equilibrium binding coefficients of small molecules with cucurbit[7]uril (CB[7]). We find that utilising SVMs may confer some predictive ability. This algorithm was then used to predict the binding of drugs TAK-580 and selumetinib. The algorithm did predict strong binding for TAK-580 and poor binding for selumetinib, and these results were experimentally validated. It was discovered that the larger homologue cucurbit[8]uril (CB[8]) is partial to selumetinib, suggesting an opportunity for tunable release by introducing different concentrations of CB[7] or CB[8] into a hydrogel depot. We qualitatively demonstrated that these drugs may have utility in combination against gliomas. Finally, mass transfer simulations show CB[7] can independently tune the release of TAK-580 without affecting selumetinib. This work gives specific evidence that a machine learning approach to recognition of small molecules by macrocycles has merit and reinforces the view that machine learning may prove valuable in the development of drug delivery systems and supramolecular chemistry more broadly.

SVM Optimization for Brain Tumor Identification Using Infrared Spectroscopic Samples.

The work presented in this paper is focused on the use of spectroscopy to identify the type of tissue of human brain samples employing support vector machine classifiers. Two different spectrometers were used to acquire infrared spectroscopic signatures in the wavenumber range between 1200⁻3500 cm-1. An extensive analysis was performed to find the optimal configuration for a support vector machine classifier and determine the most relevant regions of the spectra for this particular application. The results demonstrate that the developed algorithm is robust enough to classify the infrared spectroscopic data of human brain tissue at three different discrimination levels.

An Intraoperative Visualization System Using Hyperspectral Imaging to Aid in Brain Tumor Delineation.

Hyperspectral imaging (HSI) allows for the acquisition of large numbers of spectral bands throughout the electromagnetic spectrum (within and beyond the visual range) with respect to the surface of scenes captured by sensors. Using this information and a set of complex classification algorithms, it is possible to determine which material or substance is located in each pixel. The work presented in this paper aims to exploit the characteristics of HSI to develop a demonstrator capable of delineating tumor tissue from brain tissue during neurosurgical operations. Improved delineation of tumor boundaries is expected to improve the results of surgery. The developed demonstrator is composed of two hyperspectral cameras covering a spectral range of 400-1700 nm. Furthermore, a hardware accelerator connected to a control unit is used to speed up the hyperspectral brain cancer detection algorithm to achieve processing during the time of surgery. A labeled dataset comprised of more than 300,000 spectral signatures is used as the training dataset for the supervised stage of the classification algorithm. In this preliminary study, thematic maps obtained from a validation database of seven hyperspectral images of in vivo brain tissue captured and processed during neurosurgical operations demonstrate that the system is able to discriminate between normal and tumor tissue in the brain. The results can be provided during the surgical procedure (~1 min), making it a practical system for neurosurgeons to use in the near future to improve excision and potentially improve patient outcomes.

oxBS-450K: a method for analysing hydroxymethylation using 450K BeadChips.

DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R(2)>0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.

Mitochondrial DNA and traumatic brain injury.

OBJECTIVE: Traumatic brain injury (TBI) is a multifactorial pathology with great interindividual variability in response to injury and outcome. Mitochondria contain their own DNA (mtDNA) with genomic variants that have different physiological and pathological characteristics, including susceptibility to neurodegeneration. Given the central role of mitochondria in the pathophysiology of neurological injury, we hypothesized that its genomic variants may account for the variability in outcome following TBI. METHODS: We undertook an analysis of mitochondrial haplogroups in a large, well-characterized cohort of 1,094 TBI patients. A proportional odds model including age, brain computed tomography characteristics, injury severity, pupillary reactivity, mitochondrial haplogroups, and APOE was applied to Glasgow Outcome Score (GOS) data. RESULTS: mtDNA had a significant association with 6-month GOS (p=0.008). Haplogroup K was significantly associated with favorable outcome (odds ratio=1.64, 95% confidence interval=1.08-2.51, p=0.02). There was also a significant interaction between mitochondrial genome and age (p=0.002), with a strong protective effect of both haplogroups T (p=0.015) and K (p=0.017) with advancing age. We also found a strong interaction between APOE and mitochondrial haplogroups (p=0.001), indicating a protective effect of haplogroup K in carriers of the APOE ε4 allele. INTERPRETATION: These findings reveal an interplay between mitochondrial DNA, pathophysiology of TBI, and aging. Haplogroups K and T, which share a common maternal ancestor, are shown as protective in TBI. The data also suggest that the APOE pathways interact with genetically regulated mitochondrial functions in the response to acute injury, as previously reported in Alzheimer disease.

Microglia and macrophages in glioblastoma: landscapes and treatment directions.

Glioblastoma is the most common primary malignant tumour of the central nervous system and remains uniformly and rapidly fatal. The tumour-associated macrophage (TAM) compartment comprises brain-resident microglia and bone marrow-derived macrophages (BMDMs) recruited from the periphery. Immune-suppressive and tumour-supportive TAM cell states predominate in glioblastoma, and immunotherapies, which have achieved striking success in other solid tumours have consistently failed to improve survival in this 'immune-cold' niche context. Hypoxic and necrotic regions in the tumour core are found to enrich, especially in anti-inflammatory and immune-suppressive TAM cell states. Microglia predominate at the invasive tumour margin and express pro-inflammatory and interferon TAM cell signatures. Depletion of TAMs, or repolarisation towards a pro-inflammatory state, are appealing therapeutic strategies and will depend on effective understanding and classification of TAM cell ontogeny and state based on new single-cell and spatial multi-omic in situ profiling. Here, we explore the application of these datasets to expand and refine TAM characterisation, to inform improved modelling approaches, and ultimately underpin the effective manipulation of function.

Dopaminergic Cell Replacement for Parkinson's Disease: Addressing the Intracranial Delivery Hurdle.

Parkinson's disease (PD) is an increasingly prevalent neurological disorder, affecting more than 8.5 million individuals worldwide. α-Synucleinopathy in PD is considered to cause dopaminergic neuronal loss in the substantia nigra, resulting in characteristic motor dysfunction that is the target for current medical and surgical therapies. Standard treatment for PD has remained unchanged for several decades and does not alter disease progression. Furthermore, symptomatic therapies for PD are limited by issues surrounding long-term efficacy and side effects. Cell replacement therapy (CRT) presents an alternative approach that has the potential to restore striatal dopaminergic input and ameliorate debilitating motor symptoms in PD. Despite promising pre-clinical data, CRT has demonstrated mixed success clinically. Recent advances in graft biology have renewed interest in the field, resulting in several worldwide ongoing clinical trials. However, factors surrounding the effective neurosurgical delivery of cell grafts have remained under-studied, despite their significant potential to influence therapeutic outcomes. Here, we focus on the key neurosurgical factors to consider for the clinical translation of CRT. We review the instruments that have been used for cell graft delivery, highlighting current features and limitations, while discussing how future devices could address these challenges. Finally, we review other novel developments that may enhance graft accessibility, delivery, and efficacy. Challenges surrounding neurosurgical delivery may critically contribute to the success of CRT, so it is crucial that we address these issues to ensure that CRT does not falter at the final hurdle.

MRI-Guided Focused Ultrasound for the Treatment of Dystonia: A Narrative Review.

Contemporary surgical management of dystonia includes neuromodulation via deep brain stimulation (DBS) or ablative techniques such as radiofrequency (RF) ablation. MRI-guided focused ultrasound (MRgFUS) is an emerging modality that uses high-intensity ultrasound to precisely ablate targets in the brain; this is incisionless, potentially avoiding the surgical risks of a burr hole and transcortical tract to reach the anatomical target. There is some evidence of efficacy in essential tremor and Parkinson's disease (PD), but, to date, there is no study aggregating the evidence of MRgFUS in dystonia. In this narrative review, we searched Medline, Embase, CINAHL, EBSCO, and ClinicalTrials.gov for primary studies and clinical trials on MRgFUS in the treatment of dystonia. Data were analyzed concerning dystonia phenotype, reported outcomes, and complications. PD-related dystonia was also included within the scope of the review. Using our search criteria, six articles on the use of MRgFUS in adult dystonia and three articles on the use of FUS in dystonia in PD were included. Four trials on the use of FUS in dystonia were also found on ClinicalTrials.gov, one of which was completed in December 2013. All included studies showed evidence of symptomatic improvement, mostly in focal hand dystonia; improvements were also found in dystonia-associated tremor, cervicobrachial dystonia, and dystonia-associated chronic neuropathic pain as well as PD-related dystonia. Reported complications included transient neurological deficits and persistent arm pain in one study. However, the evidence is limited to level-4 case series at present. MRgFUS is an emerging modality that appears to be safe and effective, particularly in focal hand dystonia, without major adverse effects. However, the quality of evidence is low at present, and long-term outcomes are unknown. High-quality prospective studies comparing MRgFUS to other surgical techniques will be useful in determining its role in the management of dystonia.

Elevated FOXG1 in glioblastoma stem cells cooperates with Wnt/ß-catenin to induce exit from quiescence.

Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/ß-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivo and that additional ß-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs.

Pilocytic astrocytoma originating at the cavernous sinus.

We report a case of histologically proven pilocytic astrocytoma arising within the cavernous sinus, confirmed radiographically and at operation. We discuss the implications in the context of previous reports of ectopic glioma origin. In particular, the possiblity of glioma development within glial cell islands in the peripheral segment of cranial nerves is explored.

Atypical presentation of delayed communicating hydrocephalus after supratentorial glioma resection with opening of the ventricles.

We report four cases of communicating hydrocephalus, requiring shunt placement, in the subset of patients whose ventricles were breached at the time of glioma resection (a total 97 cases over 3 years). The hydrocephalus in these cases presented without ventricular dilatation on computed tomography (CT) scanning, and in 3 cases without headache. Failure to progress, visual deterioration or cerebrospinal fluid (CSF) leak in the post-operative patient after tumour resection with ventricular opening should alert clinicians to the possibility of hydrocephalus, despite the absence of headache or ventriculomegaly, and lumbar puncture should be performed without delay.

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain.

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

Emergency Intraoperative Ultrasound for the Neurosurgical Trainee.

The use of intraoperative ultrasound in emergency cranial neurosurgical procedures is not well described. It may improve surgical outcomes and is useful when other neuro-navigation systems are not readily available. We provide a practical guide for neurosurgical trainees to utilize ultrasound for various emergency cranial neurosurgical procedures, including lesion localization, insertion of an external ventricular drain, and shunt revision surgery. Intraoperative ultrasound is a useful modality for urgent neurosurgical procedures.

On-chip perivascular niche supporting stemness of patient-derived glioma cells in a serum-free, flowable culture.

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC - microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells-brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.

A map of transcriptional heterogeneity and regulatory variation in human microglia.

Microglia, the tissue-resident macrophages of the central nervous system (CNS), play critical roles in immune defense, development and homeostasis. However, isolating microglia from humans in large numbers is challenging. Here, we profiled gene expression variation in primary human microglia isolated from 141 patients undergoing neurosurgery. Using single-cell and bulk RNA sequencing, we identify how age, sex and clinical pathology influence microglia gene expression and which genetic variants have microglia-specific functions using expression quantitative trait loci (eQTL) mapping. We follow up one of our findings using a human induced pluripotent stem cell-based macrophage model to fine-map a candidate causal variant for Alzheimer's disease at the BIN1 locus. Our study provides a population-scale transcriptional map of a critically important cell for human CNS development and disease.