CNS-derived Cells Research Articles

CDK12/13: A Promising New Therapeutic Target in ALL

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy in children and young adults. Upfront resistance to glucocorticoids used as first-line treatment in most regimens confers a poor prognosis for patients with ALL. Of patients who relapse, only half achieve long-term survival. Here we demonstrate that inhibition of cyclin dependant kinases 12 and 13 (CDK12 & CDK13), which are modulators of RNA polymerase II, has a robust synergistic anti-leukemia effect with dexamethasone, in both B-ALL and T-ALL cell lines as well as primary B-ALL patient-derived bone marrow samples. To understand the mechanism underlying the steroid resistance in ALL, we first reanalyzed shRNA screen performed by Poulard et al. This revealed that CDK13 silencing sensitized leukemic cells to dexamethasone. CDK12 is located on chromosome 17q12, containing oncogenic features and genetic alterations in various tumors. CDK12/13 inhibitor exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft mouse model. It was shown that the inhibition resensitizes HER2+ breast cancers to HER2-tyrosine kinase inhibitors via suppressing PI3K/AKT in patient-derived organoids in vitro and cell-derived xenograft or patient-derived xenograft in vivo. Forgoing research demonstrates that CDK12/13 inhibition silences transcription of DNA damage response (DDR) genes. In our study, however, we haven't observed a rise in γH2AX, a marker of DNA damage, after CDK12/13 inhibition in ALL cell lines. What we did note was a marked enhancement expression of BIM and SGK1, steroid responsive genes and pro-apoptotic factors, after CDK12/13 inhibition (Figure A). ALL cell lines exhibited high susceptibility to CDK12/13 inhibition with two chemically divergent inhibitors, SR-4835 and THZ-531. We also showcased their synergistic interactions with dexamethasone using flow cytometry and synergyfinder ZIP score analysis (Figure B). Moreover, in the GSEA comparison of ALL cell lines with and without synergy between CDK12/13 inhibitors and dexamethasone, our data underscored significant enrichment in cholesterol biosynthesis-related genesets in cell lines manifesting this synergistic effect. On the other hand, we observed resistance to SR-4835 in cell lines with high enrichment scores in NOTCH, a signaling pathway implicated in about 80% of relapsed T cell ALL/LBL cases. Additionally, new data on mice xenotransplanted with B-ALL CNS-derived patient cells, link upregulated cholesterol biosynthetic pathways to nervous system involvement in childhood ALL. Upon administering CDK12/13 inhibitors, we observed a cell cycle phase shift resulting in G1 arrest and increased apoptosis, whereas human peripheral blood mononuclear cells (PBMCs) from healthy individuals remained unaffected by this treatment. As we continue to decode the pathways through which CDK12/13 inhibitors render ALL cells more susceptible to dexamethasone, our next step includes silencing CDK12/13 using short hairpin RNA models in cell lines and consequently their RNA sequencing. This approach may mitigate the influence of secondary pathways, paving the way for enhanced treatment strategies for relapsed ALL by unmasking the underlying molecular events.

The Effect of Leukocyte- and Platelet-Rich Fibrin on Central and Peripheral Nervous System Neurons-Implications for Biomaterial Applicability.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is a second-generation platelet concentrate that is prepared directly from the patient's own blood. It is widely used in the field of regenerative medicine, and to better understand its clinical applicability we aimed to further explore the biological properties and effects of L-PRF on cells from the central and peripheral nervous system. To this end, L-PRF was prepared from healthy human donors, and confocal, transmission, and scanning electron microscopy as well as secretome analysis were performed on these clots. In addition, functional assays were completed to determine the effect of L-PRF on neural stem cells (NSCs), primary cortical neurons (pCNs), and peripheral dorsal root ganglion (DRG) neurons. We observed that L-PRF consists of a dense but porous fibrin network, containing leukocytes and aggregates of activated platelets that are distributed throughout the clot. Antibody array and ELISA confirmed that it is a reservoir for a plethora of growth factors. Key molecules that are known to have an effect on neuronal cell functions such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were slowly released over time from the clots. Next, we found that the L-PRF secretome had no significant effect on the proliferative and metabolic activity of NSCs, but it did act as a chemoattractant and improved the migration of these CNS-derived stem cells. More importantly, L-PRF growth factors had a detrimental effect on the survival of pCNs, and consequently, also interfered with their neurite outgrowth. In contrast, we found a positive effect on peripheral DRG neurons, and L-PRF growth factors improved their survival and significantly stimulated the outgrowth and branching of their neurites. Taken together, our study demonstrates the positive effects of the L-PRF secretome on peripheral neurons and supports its use in regenerative medicine but care should be taken when using it for CNS applications.

Agent Orange Causes Metabolic Dysfunction and Molecular Pathology Reminiscent of Alzheimer's Disease.

Agent Orange, an herbicide used during the Vietnam War, contains 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Agent Orange has teratogenic and carcinogenic effects, and population-based studies suggest Agent Orange exposures lead to higher rates of toxic and degenerative pathologies in the peripheral and central nervous system (CNS). This study examines the potential contribution of Agent Orange exposures to neurodegeneration. Human CNS-derived neuroepithelial cells (PNET2) treated with 2,4-D and 2,4,5-T were evaluated for viability, mitochondrial function, and Alzheimer's disease (AD)-related proteins. Treatment with 250μg/ml 2,4-D or 2,4,5-T significantly impaired mitochondrial function, caused degenerative morphological changes, and reduced viability in PNET2 cells. Correspondingly, glyceraldehyde-3-phosphate dehydrogenase expression which is insulin-regulated and marks the integrity of carbohydrate metabolism, was significantly inhibited while 4-hydroxy-2-nonenal, a marker of lipid peroxidation, was increased. Tau neuronal cytoskeletal protein was significantly reduced by 2,4,5-T, and relative tau phosphorylation was progressively elevated by 2,4,5-T followed by 2,4-D treatment relative to control. Amyloid-β protein precursor (AβPP) was increased by 2,4,5-T and 2,4-D, and 2,4,5-T caused a statistical trend (0.05 < p<0.10) increase in Aβ. Finally, altered cholinergic function due to 2,4,5-T and 2,4-D exposures was marked by significantly increased choline acetyltransferase and decreased acetylcholinesterase expression, corresponding with responses in early-stage AD. Exposures to Agent Orange herbicidal chemicals rapidly damage CNS neurons, initiating a path toward AD-type neurodegeneration. Additional research is needed to understand the permanency of these neuropathologic processes and the added risks of developing AD in Agent Orange-exposed aging Vietnam Veterans.

Leukaemia and the CNS Niche: Insights on Bidirectional Communication from a Model of Infant Leukaemia

Infant leukaemia, an acute leukaemia within the first year of life, is a rare, aggressive entity with a poor response to intensive therapy. MLL-AF4+ infant leukaemia, the most common genetic subtype, has been shown to have: an in-utero origin, a lack of co-driver mutations, and lineage plasticity. Central nervous system (CNS) disease, typically a leukaemic infiltrate of the leptomeninges, is particularly common in these patients compared with non-infant paediatric ALL, and presents a serious clinical problem. With increasing understanding of the diverse immune cell landscape in the meninges, we sought to use our recently developed fully murine infant leukaemia model to explore the interactions between leukaemia and this microenvironment. We previously identified two microRNAs (miR128a and miR130b) that are upregulated in MLL-AF4+ infant and paediatric leukaemia patient samples. Overexpression of these microRNAs individually in mouse foetal liver (FL) Lin-Sca1+cKit+ (LSK) cells resulted in microRNA-dependent lineage-specific acute leukaemias in the context of Mll-AF4 with prominent CNS infiltration (Malouf et al. Blood 2021). To identify potential niche-specific leukaemia-propagating cells in our Mll-AF4+ mir128a+ infant proB-ALL model, we performed serial transplants using sorted leukaemia progenitor populations (LSK IL7R+ and Lin-Sca1-cKit+ (LK/CLP)) from mice that had previously succumbed to leukaemia. We observed that these leukaemia progenitors were functionally identical with indistinguishable output. This suggests the leukaemia-initiating cells in this model can emerge from different cell populations, consistent with our current understanding of ALL-initiating cells in human disease. CNS leukaemia developed in these mice with features that also mirror human disease: an immunophenotypically polyclonal infiltrate of the leptomeninges, sparing the brain parenchyma. To complement the functional data, we transcriptionally validated the infant proB-ALL model for two key features: foetal cell-of-origin and infant-specific expression signatures. Published RNA sequencing of murine FL and adult bone marrow (BM) lymphoid-primed multipotent progenitors (LMPP) - a putative cell of origin for infant leukaemia - were reanalysed (Symeonidou et al. Cell Reports 2021 data set). BM-derived leukaemia cells from our model are more similar to FL than adult BM LMPPs. Using a published MLL-AF4+ patient leukaemia data set we showed that cells from our model are also transcriptionally closer to infant than non-infant paediatric MLL-AF4+ ALL patient samples (Figure 1)(Andersson et al. Nature Genetics 2015 data set). Transcriptomic differences were identified by RNA sequencing of leukaemia progenitors from the BM and CNS niche. Statistically significant Gene Ontology terms of particular interest based on these differences are summarised in Figure 2. Genes upregulated in CNS-derived cells included cell adhesion molecules, immune modulators and cell growth factor receptors. These suggest a CNS-specific adaptive response to the microenvironment in a bidirectional manner. These novel observations may represent infant leukaemia-specific adaptations or more generic CNS leukaemia interactions uncovered through the use of an immunocompetent fully murine disease model. In either scenario, the implications of bidirectional communication between leukaemia and the CNS niche are significant for the development of much-needed novel biomarkers and therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CCR6 Expression on B Cells Is Not Required for Clinical or Pathological Presentation of MOG Protein-Induced Experimental Autoimmune Encephalomyelitis despite an Altered Germinal Center Response.

B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6+, and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation.

A Novel Methodology Using Dexamethasone to Induce Neuronal Differentiation in the CNS-Derived Catecholaminergic CAD Cells.

The Cath.a-differentiated (CAD) cell line is a central nervous system-derived catecholaminergic cell line originating from tyrosine hydroxylase (TH)-producing neurons located around the locus coeruleus area of the mouse brain. CAD cells have been used as an in vitro model for cellular and molecular studies due to their ability to differentiate under serum-free media conditions. However, the lack of serum-derived survival factors, limits the longevity for differentiated CAD cells to be maintained in healthy conditions; thereby, limiting their use in long-term culture studies. Here, we present a novel differentiation method that utilizes dexamethasone (Dex), a synthetic glucocorticoid receptor agonist. Specifically, we discovered that the addition of 100µM of Dex into the 1% fetal bovine serum (FBS)-supplemented media effectively induced neuronal differentiation of CAD cells, as characterized by neurite formation and elongation. Dex-differentiated CAD cells exited the cell cycle, stopped proliferating, extended the neurites, and expressed neuronal markers. These effects were dependent on the glucocorticoid receptors (GR) as they were abolished by GR knockdown. Importantly, Dex-differentiated CAD cells showed longer survival duration than serum-free differentiated CAD cells. In addition, RNA-sequencing and qPCR data demonstrate that several genes involved in proliferation, neuronal differentiation, and survival pathways were differentially expressed in the Dex-differentiated cells. This is the first study to reveal Dex as a novel differentiation methodology used to generate postmitotic neuronal CAD cells, which may be utilized as an in vitro neuronal model for cellular and molecular neurobiology research.

IL-11 Induces Encephalitogenic Th17 Cells in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis.

IL-11+CD4+ cells accumulate in the cerebrospinal fluid of patients with early relapsing-remitting multiple sclerosis (MS) and in active brain MS lesions. Mouse studies have confirmed a causal role of IL-11 in the exacerbation of relapsing-remitting experimental autoimmune encephalomyelitis (RREAE). Administration of IL-11 at the time of clinical onset of RREAE induced an acute exacerbation and increased clinical scores, which persisted during the entire course of the disease. IL-11 increased the numbers of spinal cord inflammatory foci, as well as the numbers of peripheral and CNS-infiltrating IL-17+CD4+ cells and IL-17A serum levels. Ag recall assays revealed that IL-11 induces IL-17A+, GM-CSF+, and IL-21+CD4+ myelin Ag-reactive cells. Passive transfer of these encephalitogenic CD4+ T cells induced severe RREAE with IL-17A+CCR6+ CD4+ and B cell accumulation within the CNS. Furthermore, passive transfer of nonmanipulated CNS-derived mononuclear cells from mice with RREAE after a single dose of IL-11 induced severe RREAE with increased accumulation of IL-17A+ and CCR6+ CD4+ cells within the CNS. These results suggest that IL-11 might serve as a biomarker of early autoimmune response and a selective therapeutic target for patients with early relapsing-remitting MS.

Differential In Vitro Infection of Neural Cells by Astroviruses.

Recent advances in unbiased pathogen discovery have implicated astroviruses as pathogens of the central nervous system (CNS) of mammals, including humans. However, the capacity of astroviruses to be cultured in CNS-derived cells in vitro has not been reported to date. Both astrovirus VA1/HMO-C (VA1; mamastrovirus 9) and classic human astrovirus 4 (HAstV4; mamastrovirus 1) have been previously detected from cases of human encephalitis. We tested the ability of primary human neurons, primary human astrocytes, and other immortalized human nervous system cell lines (SK-N-SH, U87 MG, and SW-1088) to support infection and replication of these two astrovirus genotypes. Primary astrocytes and SK-N-SH cells supported the full viral life cycle of VA1 with a >100-fold increase in viral RNA levels during a multistep growth curve, detection of viral capsid, and a >100-fold increase in viral titer. Primary astrocytes were permissive with respect to HAstV4 infection and replication but did not yield infectious virus, suggesting abortive infection. Similarly, abortive infection of VA1 was observed in SW-1088 and U87 MG cells. Elevated expression of the chemokine CXCL10 was detected in VA1-infected primary astrocytes and SK-N-SH cells, suggesting that VA1 infection can induce a proinflammatory host response. These findings establish an in vitro cell culture model that is essential for investigation of the basic biology of astroviruses and their neuropathogenic potential.IMPORTANCE Encephalitis remains a diagnostic conundrum in humans as over 50% of cases are managed without the identification of an etiology. Astroviruses have been detected from the central nervous system of mammals in association with disease, suggesting that this family of RNA viruses could be responsible for cases of some neurological diseases that are currently without an ascribed etiology. However, there are significant barriers to understanding astrovirus infection as the capacity of these viruses to replicate in nervous system cells in vitro has not been determined. We describe primary and immortalized cultured cells of the nervous system that support infection by astroviruses. These results further corroborate the role of astroviruses in causing neurological diseases and will serve as an essential model to interrogate the neuropathogenesis of astrovirus infection.

Abstract LB-239: A novel workflow for isolation and multi-omic profiling of DCCs derived from cerebrospinal fluid of patients with pediatric brain cancer

Abstract Background: Clinical management of cancers of the central nevus system (CNS) is very challenging as they often exhibit low responsiveness to radiation and chemotherapy resulting in overall poor survival. Moreover, analysis of mechanisms driving cancer progression and selection of targeted therapies in CNS tumors is hindered by the limited availability to tumor tissues accessible only though surgical biopsies. A potential source of cancer material in patients with CNS is cerebrospinal fluid (CSF). Analysis of CSF-derived disseminated cancer cells (csfDCCs) holds a promise for improvement of diagnostics and monitoring of CNS tumors. For this reason, we developed a novel workflow allowing detection, isolation and multi-omic analysis of csfDCCs. Methods: In a proof of concept study a new workflow was used to analyze CSF samples from two patients with medulloblastoma and pineoblastoma. CSF-derived cells were stained for CD45 to allow identification of infiltrating immune cells. Putative csfDCCs (CD45-negative) and control cells (CD45-positive) were subjected to a multi-omic workflow allowing parallel sequencing of genomes and transcriptomes of the same cells. Single-cell mRNA was physically separated from DNA, amplified by means of whole transcriptome amplification (Ampli1 WTA) and analyzed using endpoint PCR and a proprietary single-cell RNA-Seq approach. In parallel, DNA was subjected to whole genome amplification (Ampli1 WGA) and analyzed for the presence of copy number variations as well as point mutations (Ampli1 LowPass Kit and targeted sequencing of actionable hot-spots). Results: We analyzed seven CNS-derived single cells and five cell clusters from the medulloblastoma patient and further eight cell clusters and eight single cells obtained from the pineoblastoma patient. Transcriptome analysis revealed that expression of neural lineage markers (e.g. CD133, SYP, OTX2, MSI1, MAP2, NEUROG1 and NEUROD1) is present almost exclusively in CD45-negative cells. Only two samples collected from medulloblastoma patient co-expressed CD45 and GFAP. However, DNA analysis revealed that CD45-positive and CD45/GFAP double-positive samples showed non-aberrant genomic profiles, thus these cells were classified as non-malignant. In contrast, all CD45-negative cells harbored genetic alterations confirming their malignant origin. Conclusion: Our proof of concept study shows a novel workflow allowing identification, isolation as well as parallel genome and transcriptome analysis csfDCCs of patients with pediatric CNS tumors. Citation Format: Zbigniew T. Czyz, Giancarlo Feliciello, Miodrag Guzvic, Thomas Schamberger, Sandra Grunewald, Selim Corbacioglu, Markus J. Riemenschneider, Bernhard Polzer, Christoph A. Klein. A novel workflow for isolation and multi-omic profiling of DCCs derived from cerebrospinal fluid of patients with pediatric brain cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-239.

Carnosine modulates the Sp1-Slc31a1/Ctr1 copper-sensing system and influences copper homeostasis in murine CNS-derived cells.

Carnosine (CAR) is an endogenous dipeptide physiologically present in excitable tissues, such as central nervous system (CNS) and muscle. CAR is acknowledged as a substrate involved in many homeostatic pathways and mechanisms and, due to its biochemical properties, as a molecule intertwined with the homeostasis of heavy metals such as copper (Cu). In CNS, Cu excess and dysregulation imply oxidative stress, free-radical production, and functional impairment leading to neurodegeneration. Here, we report that CAR intercepts the regulatory routes of Cu homeostasis in nervous cells and tissues. Specifically, in a murine neuron-derived cell model, i.e., the B104 neuroblastoma cells, extracellular CAR exposure up to 24 h influenced intracellular Cu entry and affected (downregulated) the key Cu-sensing system, consisting of the gene coding for the Slc31a1 transmembrane Cu importer (alias Ctr1), and the gene coding for the Cu-responsive transcription factor Sp1 ( Sp1). Also, CAR exposure upregulated CAR biosynthesis ( Carns1), extracellular degradation ( Cndp1), and transport ( Slc15a4, alias Pht1) genes and elicited CAR intracellular accumulation, contributing to the outline of functional association between CAR and Cu within the cell. Interestingly, the same gene modulation scheme acting in vitro operates in vivo in brains of mice undergoing dietary administration of CAR in drinking water for 2 wk. Overall, our findings describe for the first time a regulatory interaction between CAR and Cu pathways in CNS and indicate CAR as a novel active molecule within the network of ligands and chaperones that physiologically regulate Cu homeostasis.

Molecular imaging mass spectrometry for probing protein dynamics in neurodegenerative disease pathology.

Recent advances in the understanding of basic pathological mechanisms in various neurological diseases depend directly on the development of novel bioanalytical technologies that allow sensitive and specific chemical imaging at high resolution in cells and tissues. Mass spectrometry-based molecular imaging (IMS) has gained increasing popularity in biomedical research for mapping the spatial distribution of molecular species insitu. The technology allows for comprehensive, untargeted delineation of insitu distribution profiles of metabolites, lipids, peptides and proteins. A major advantage of IMS over conventional histochemical techniques is its superior molecular specificity. Imaging mass spectrometry has therefore great potential for probing molecular regulations in CNS-derived tissues and cells for understanding neurodegenerative disease mechanism. The goal of this review is to familiarize the reader with the experimental workflow, instrumental developments and methodological challenges as well as to give a concise overview of the major advances and recent developments and applications of IMS-based protein and peptide profiling with particular focus on neurodegenerative diseases. This article is part of the Special Issue "Proteomics".

Frequent monoallelic or skewed expression for developmental genes in CNS-derived cells and evidence for balancing selection

Cellular mosaicism due to monoallelic autosomal expression (MAE), with cell selection during development, is becoming increasingly recognized as prevalent in mammals, leading to interest in understanding its extent and mechanism(s). We report here use of clonal cell lines derived from the CNS of adult female [Formula: see text] hybrid (C57BL/6 X JF1) mice to characterize MAE as neural stem cells (nscs) differentiate to astrocyte-like cells (asls). We found that different subsets of genes show MAE in the two populations of cells; in each case, there is strong enrichment for genes specific to the respective developmental state. Genes that exhibit MAE are 22% of nsc-specific genes and 26% of asl-specific genes. Moreover, the promoters of genes with MAE have reduced CpG dinucleotides but increased CpG differences between the two parental mouse strains. Extending the study of variability to wild populations of mice, we found evidence for balancing selection as a contributing force in evolution of those genes showing developmental specificity (i.e., expressed in either nsc or asl), not just for genes showing MAE. Furthermore, we found that genes showing skewed allelic expression (SKE) were similarly enriched among cell type-specific genes and also showed a heightened probability of balancing selection. Thus, developmental stage-specific genes and genes with MAE or SKE seem to make up overlapping classes subject to selection for increased diversity. The implications of these results for development and evolution are discussed in the context of a model with stochastic epigenetic modifications taking place only during a relatively brief developmental window.

Systemic Neutrophil Depletion Modulates the Migration and Fate of Transplanted Human Neural Stem Cells to Rescue Functional Repair.

The interaction of transplanted stem cells with local cellular and molecular cues in the host CNS microenvironment may affect the potential for repair by therapeutic cell populations. In this regard, spinal cord injury (SCI), Alzheimer's disease, and other neurological injuries and diseases all exhibit dramatic and dynamic changes to the host microenvironment over time. Previously, we reported that delayed transplantation of human CNS-derived neural stem cells (hCNS-SCns) at 9 or 30 d post-SCI (dpi) resulted in extensive donor cell migration, predominantly neuronal and oligodendrocytic donor cell differentiation, and functional locomotor improvements. Here, we report that acute transplantation of hCNS-SCns at 0 dpi resulted in localized astroglial differentiation of donor cells near the lesion epicenter and failure to produce functional improvement in an all-female immunodeficient mouse model. Critically, specific immunodepletion of neutrophils (polymorphonuclear leukocytes) blocked hCNS-SCns astroglial differentiation near the lesion epicenter and rescued the capacity of these cells to restore function. These data represent novel evidence that a host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population, and support targeting the inflammatory microenvironment in combination with cell transplantation after SCI.SIGNIFICANCE STATEMENT The interaction of transplanted cells with local cellular and molecular cues in the host microenvironment is a key variable that may shape the translation of neurotransplantation research to the clinical spinal cord injury (SCI) human population, and few studies have investigated these events. We show that the specific immunodepletion of polymorphonuclear leukocyte neutrophils using anti-Ly6G inhibits donor cell astrogliosis and rescues the capacity of a donor cell population to promote locomotor improvement after SCI. Critically, our data demonstrate novel evidence that a specific host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population.

Environmentally relevant level of aflatoxin B1 elicits toxic pro-inflammatory response in murine CNS-derived cells

Aflatoxin B1 (AFB1) is a well-known member of aflatoxins (AFs) that is considered among highly stable toxic contaminants of food, worldwide. The impact of AFB1 on neural cells and systems has poorly been understood. To assess the cellular effects of AFB1 on brain, we used murine pure primary astrocytes, sub ventricular zone-derived neural precursor cells (NPCs) and microglia cell line (BV2). Cells were exposed separately to environmentally relevant level (20ng/ml) of AFB1 for 1, 2, 3, 6, 12, 24 and 48h in culture. At each time points, total free radicals production measured by luminol-enhanced cellular chemiluminescence (CL) assay; cytokines production of IL-1β, IL-6, TNF-α and IL-10 were analyzed using Bioplex ELISA and a set of genes involved in the immediate response to danger such as TLR2, TLR4 and iNOS etc. were evaluated by multiplex qPCR. Upon AFB1 exposure production, of the total free radicals significantly increased only in microglial cells after 24h and slightly elevated in the other examined cells. AFB1 also induced secretion of pro-inflammatory cytokines (i.e. TNF-α and IL-6) on both microglial cells (more TNF-α) and astrocytes (more IL-6). mRNA expression of TLR2, TLR4, MyD88 and NF-κB were up-regulated with different timing and levels among cells. Immunotoxicologically, microglial cells, and astrocytes, but not NPCs, are capable of sensing a low level of AFB1. Thus, the pro-inflammatory effects of an environmentally relevant dose of AFB1 on CNS-derived cells in vitro could potentially explain the immune dysregulation in neurodegenerative disorders.

An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation

BackgroundCNS inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse B cell subsets. Although antibody secreting cells (ASC) within the inflamed CNS have been extensively examined, memory B cell (Bmem) characterization has been limited as they do not secrete antibody without stimulation. Moreover, unlike human Bmem, reliable surface markers for murine Bmem remain elusive. New methodUsing a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC. Comparison with existing methodsStimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial ASC loss and minimal Bmem to ASC conversion of CNS-derived cells. By varying stimulation duration, TLR activators, and culture supplements, we achieved optimal conversion by culturing cells with TLR7/8 agonist R848 in the presence of feeder cells for 2days. ResultsFlow cytometry markers CD38 and CD73 characterizing murine Bmem from lymphoid tissue showed more diverse expression patterns on corresponding CNS-derived B cell subsets. Using the optimized TLR7/8 stimulation protocol, we compared virus-specific IgG Bmem versus pre-existing ASC within the brain and spinal cord. Increasing Bmem frequencies during chronic infection mirrored kinetics of ASC. However, despite initially similar Bmem and ASC accumulation, Bmem prevailed in the brain, but were lower than ASC in the spinal cord during persistence. ConclusionSimultaneous enumeration of antigen-specific Bmem and ASC using the Bmem assay optimized for CNS-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation.

Preclinical Efficacy Failure of Human CNS-Derived Stem Cells for Use in the Pathway Study ofCervical Spinal Cord Injury.

SummaryWe previously showed the efficacy of multiple research cell lines (RCLs) of human CNS neural stem cells (HuCNS-SCs) in mouse and rat models of thoracic spinal cord injury (SCI), supporting a thoracic SCI clinical trial. Experts recommend in vivo preclinical testing of the intended clinical cell lot/line (CCL) in models with validity for the planned clinical target. We therefore tested the efficacy of two HuCNS-SC lines in cervical SCI: one RCL, and one CCL intended for use in the Pathway Study of cervical SCI in man. We assessed locomotor recovery and sensory function, as well as engraftment, migration, and fate. No evidence of efficacy of the CCL was observed; some data suggested a negative impact of the CCL on outcomes. These data raise questions about the development and validation of potency/comparability assays for clinical testing of cell products, and lack of US Food and Drug Administration requirements for in vivo testing of intended clinical cell lines.

The NRF2 transcriptional target, OSGIN1, contributes to monomethyl fumarate-mediated cytoprotection in human astrocytes

Dimethyl fumarate (DMF) is indicated for the treatment of relapsing multiple sclerosis and may exert therapeutic effects via activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway. Following oral DMF administration, central nervous system (CNS) tissue is predominantly exposed to monomethyl fumarate (MMF), the bioactive metabolite of DMF, which can stabilize NRF2 and induce antioxidant gene expression; however, the detailed NRF2-dependent mechanisms modulated by MMF that lead to cytoprotection are unknown. Our data identify a mechanism for MMF-mediated cytoprotection in human astrocytes that functions in an OSGIN1-dependent manner, specifically via upregulation of the OSGIN1-61 kDa isoform. NRF2-dependent OSGIN1 expression induced P53 nuclear translocation following MMF administration, leading to cell-cycle inhibition and cell protection against oxidative challenge. This study provides mechanistic insight into MMF-mediated cytoprotection via NRF2, OSGIN1, and P53 in human CNS-derived cells and contributes to our understanding of how DMF may act clinically to ameliorate pathological processes in neurodegenerative disease.

VEGFA- a New Therapeutic Target in CNS Leukemia

Acute lymphoblastic leukemia (ALL) is the commonest cancer to affect children. Historically, central nervous system (CNS) involvement has been considered as a feature of high risk disease. Although this risk is mitigated by current intended CNS directed treatments based on risk stratification strategies, currently 10% or more of children will die from relapsing or unresponsive disease and the CNS is still a major site of relapse.CNS directed treatments bypass the blood brain barrier (BBB) but do not specifically target CNS leukemic cells. Consequently, although effective, they are associated both with serious short-term toxicities and debilitating long-term morbidities. Understanding the biology of those ALL cells which lodge in the CNS may lead to the development of drugs to specifically target them, and this could further improve outcomes but with fewer complications.We transplanted bone marrow (BM) derived leukemic cells from children with B-ALL into non-conditioned immune-deficient mice by tail-vein injection. We confirmed BM engraftment and evidence for CNS involvement was demonstrated by microscopy and flow cytometry analyses of the CSF, and was confirmed histo-pathologically in brain tissue. The pattern of leukemic infiltration of the CNS in xenograft recipients closely mimicked that seen post mortem in children with ALL. Furthermore, the MRI scans of xenograft mice showed a meningeal enhancement typical of the leukemic meningitis seen in patients with CNS involvement. Thus by using standard clinical techniques, we were able to demonstrate that the xenograft recipients of B-ALL cells faithfully recapitulates patterns of CNS disease that are seen in human.Interestingly, human leukemic cells isolated from either the CNS or BM of individual recipient mice demonstrated different kinetics of engraftment following serial passage into secondary and quaternary recipients. This functional observation implies cell intrinsic differences between leukemic cells lodged at the two sites.We next compared the gene expression profiles of leukemic cells isolated from the CNS and BM of affected children and xenograft recipients. Using gene set enrichment analysis, we demonstrate that leukemic cells that have infiltrated the CNS, in contrast to those in the BM, show features of adaptation to hypoxia including cell cycle quiescence and downregulation of oxidative phosphorylation. Consistent with these transcriptional data, we could demonstrated that fewer of the CNS-derived leukemic cells were in S/G2/M compared with BM-derived leukemic cells and that CNS-derived leukemic cells consumed less oxygen in in vitro assays.We found that CNS derived leukemic cells had a transcriptional signatures suggestive of physiological adaptation to hypoxic microenvironment and VEGFA is one of the most up-regulated genes in CNS-derived leukemic cells. We tested therapeutic potential of targeting VEGFA using bevacizumab, a VEGFA-neutralizing antibody. Bevacizumab or normal saline (NS) was injected intraperitoneally into the xenografted recipients of B-ALL cells. In the bevacizumab group, leukemic cell numbers in the CNS were significantly less at the end of treatment, and staining of brain sections confirmed reduced leukemic burden in CNS of bevacizumab-treated mice compared with control mice. This in vivo preclinical assay shows that VEGFA could be targeted to treat the patients with ALL who have CNS involvement.In conclusion, we have shown that B-ALL cells residing in the CNS have gene expression signatures and phenotypic features that are distinct from those in the BM, and that this can be effectively targeted in preclinical model of CNS leukemia. These observations may inform not only further mechanistic research, but also future clinical trials. DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Repulsive Epithelial Cues Direct Glial Migration along the Nerve

Most glial cells show pronounced migratory abilities and generally follow axonal trajectories to reach their final destination. However, the molecular cues controlling their directional migration are largely unknown. To address this, we established glial migration onto the developing Drosophila leg imaginal disc as a model. Here, CNS-derived glial cells move along nerves containing motoaxons and sensory axons. Along their path, glial cells encounter at least three choice points where directional decisions are needed. Subsequent genetic analyses allowed uncovering mechanisms that escaped previous studies. Most strikingly, we found that glial cells require the expression of the repulsive guidance receptors PlexinA/B and Robo2 to prevent breaking away from the nerve. Interestingly, the repulsive ligands are presented by the underlying leg imaginal disc epithelium, which appears to push glial cells toward the axon fascicle. In conclusion, nerve formation not only requires neuron-glia interaction but also depends on glial-epithelial communication.

TREM2 CLEARANCE OF AMYLOID: PERIPHERAL VS. CNS-DERIVED CELLS

TREM2 CLEARANCE OF AMYLOID: PERIPHERAL VS. CNS-DERIVED CELLS