Methotrexate Chemotherapy Induces Persistent Tri-glial Dysregulation that Underlies Chemotherapy-Related Cognitive Impairment.

Cell (2018)

2018 Dec 06

Gibson EM, Nagaraja S, Ocampo A, Tam LT, Wood LS, Pallegar PN, Greene JJ, Geraghty AC, Goldstein AK, Ni L, Woo PJ, Barres BA, Liddelow S, Vogel H, Monje M.
| DOI: 10.1016/j.cell.2018.10.049

Chemotherapy results in a frequent yet poorly understood syndrome of long-term neurological deficits. Neural precursor cell dysfunction and white matter dysfunction are thought to contribute to this debilitating syndrome. Here, we demonstrate persistent depletion of oligodendrocyte lineage cells in humans who received chemotherapy. Developing a mouse model of methotrexate chemotherapy-induced neurological dysfunction, we find a similar depletion of white matter OPCs, increased but incomplete OPC differentiation, and a persistent deficit in myelination. OPCs from chemotherapy-naive mice similarly exhibit increased differentiation when transplanted into the microenvironment of previously methotrexate-exposed brains, indicating an underlying microenvironmental perturbation. Methotrexate results in persistent activation of microglia and subsequent astrocyte activation that is dependent on inflammatory microglia. Microglial depletion normalizes oligodendroglial lineage dynamics, myelin microstructure, and cognitive behavior after methotrexate chemotherapy. These findings indicate that methotrexate chemotherapy exposure is associated with persistent tri-glial dysregulation and identify inflammatory microglia as a therapeutic target to abrogate chemotherapy-related cognitive impairment.

Loss of Adaptive Myelination Contributes to Methotrexate Chemotherapy-Related Cognitive Impairment.

Neuron

2019 May 10

Geraghty AC, Gibson EM, Ghanem RA, Greene JJ, Ocampo A, Goldstein AK, Ni L, Yang T, Marton RM, Paşca SP, Greenberg ME, Longo FM, Monje M.
PMID: 31122677 | DOI: 10.1016/j.neuron.2019.04.032

Activity-dependent myelination is thought to contribute to adaptive neurological function. However, the mechanisms by which activity regulates myelination and the extent to which myelin plasticity contributes to non-motor cognitive functions remain incompletely understood. Using a mouse model of chemotherapy-related cognitive impairment (CRCI), we recently demonstrated that methotrexate (MTX) chemotherapy induces complex glial dysfunction for which microglial activation is central. Here, we demonstrate that remote MTX exposure blocks activity-regulated myelination. MTX decreases cortical Bdnf expression, which is restored by microglial depletion. Bdnf-TrkB signaling is a required component of activity-dependent myelination. Oligodendrocyte precursor cell (OPC)-specific TrkB deletion in chemotherapy-naive mice results in impaired cognitive behavioral performance. A small-molecule TrkB agonist rescues both myelination and cognitive impairment after MTX chemotherapy. This rescue after MTX depends on intact TrkB expression in OPCs. Taken together, these findings demonstrate a molecular mechanism required for adaptive myelination that is aberrant in CRCI due to microglial activation

Onset of differentiation is post-transcriptionally controlled in adult neural stem cells

Nature

2019 Jan 30

Baser A, Skabkin M, Kleber S, Dang Y, Gülcüler Balta GS, Kalamakis G, Göpferich M, Ibañez DC, Schefzik R, Lopez AS, Bobadilla EL, Schultz C, Fischer B, Martin-Villalba A.
PMID: 30700908 | DOI: 10.1038/s41586-019-0888-x

Whether post-transcriptional regulation of gene expression controls differentiation of stem cells for tissue renewal remains unknown. Quiescent stem cells exhibit a low level of protein synthesis1, which is key to maintaining the pool of fully functional stem cells, not only in the brain but also in the bone marrow and hair follicles2-6. Neurons also maintain a subset of messenger RNAs in a translationally silent state, which react 'on demand' to intracellular and extracellular signals. This uncoupling of general availability of mRNA from translation into protein facilitates immediate responses to environmental changes and avoids excess production of proteins, which is the most energy-consuming process within the cell. However, when post-transcriptional regulation is acquired and how protein synthesis changes along the different steps of maturation are not known. Here we show that protein synthesis undergoes highly dynamic changes when stem cells differentiate to neurons in vivo. Examination of individual transcripts using RiboTag mouse models reveals that whereas stem cells translate abundant transcripts with little discrimination, translation becomes increasingly regulated with the onset of differentiation. The generation of neurogenic progeny involves translational repression of a subset of mRNAs, including mRNAs that encode the stem cell identity factors SOX2 and PAX6, and components of the translation machinery, which are enriched in a pyrimidine-rich motif. The decrease of mTORC1 activity as stem cells exit the cell cycle selectively blocks translation of these transcripts. Our results reveal a control mechanism by which the cell cycle is coupled to post-transcriptional repression of key stem cell identity factors, thereby promoting exit from stemness.

Neural network learning defines glioblastoma features to be of neural crest perivascular or radial glia lineages

Science advances

2022 Jun 10

Hu, Y;Jiang, Y;Behnan, J;Ribeiro, MM;Kalantzi, C;Zhang, MD;Lou, D;Häring, M;Sharma, N;Okawa, S;Del Sol, A;Adameyko, I;Svensson, M;Persson, O;Ernfors, P;
PMID: 35675414 | DOI: 10.1126/sciadv.abm6340

Glioblastoma is believed to originate from nervous system cells; however, a putative origin from vessel-associated progenitor cells has not been considered. We deeply single-cell RNA-sequenced glioblastoma progenitor cells of 18 patients and integrated 710 bulk tumors and 73,495 glioma single cells of 100 patients to determine the relation of glioblastoma cells to normal brain cell types. A novel neural network-based projection of the developmental trajectory of normal brain cells uncovered two principal cell-lineage features of glioblastoma, neural crest perivascular and radial glia, carrying defining methylation patterns and survival differences. Consistently, introducing tumorigenic alterations in naïve human brain perivascular cells resulted in brain tumors. Thus, our results suggest that glioblastoma can arise from the brains' vasculature, and patients with such glioblastoma have a significantly poorer outcome.

Mapping visual functions onto molecular cell types in the mouse superior colliculus

Neuron

2023 Apr 18

Liu, Y;Savier, EL;DePiero, VJ;Chen, C;Schwalbe, DC;Abraham-Fan, RJ;Chen, H;Campbell, JN;Cang, J;
PMID: 37086721 | DOI: 10.1016/j.neuron.2023.03.036

The superficial superior colliculus (sSC) carries out diverse roles in visual processing and behaviors, but how these functions are delegated among collicular neurons remains unclear. Here, using single-cell transcriptomics, we identified 28 neuron subtypes and subtype-enriched marker genes from tens of thousands of adult mouse sSC neurons. We then asked whether the sSC's molecular subtypes are tuned to different visual stimuli. Specifically, we imaged calcium dynamics in single sSC neurons in vivo during visual stimulation and then mapped marker gene transcripts onto the same neurons ex vivo. Our results identify a molecular subtype of inhibitory neuron accounting for ∼50% of the sSC's direction-selective cells, suggesting a genetic logic for the functional organization of the sSC. In addition, our studies provide a comprehensive molecular atlas of sSC neuron subtypes and a multimodal mapping method that will facilitate investigation of their respective functions, connectivity, and development.

The Major Risk Factors for Alzheimer’s Disease: Age, Sex, and Genes Modulate the Microglia Response to Ab Plaques.

Cell Rep.

2019 Apr 23

Sala Frigerio C, Wolfs L, Fattorelli N, Thrupp N, Voytyuk I, Schmidt I, Mancuso R, Chen WT, Woodbury ME, Srivastava G, Möller T, Hudry E, Das S, Saido T, Karran E, Hyman B, Perry VH, Fiers M, De Strooper B.
PMID: 31018141 | DOI: 10.1016/j.celrep.2019.03.099

Gene expression profiles of more than 10,000 individual microglial cells isolated from cortex and hippocampus of male and female AppNL-G-Fmice over time demonstrate that progressive amyloid-β accumulation accelerates two main activated microglia states that are also present during normal aging. Activated response microglia (ARMs) are composed of specialized subgroups overexpressing MHC type II and putative tissue repair genes (Dkk2, Gpnmb, and Spp1) and are strongly enriched with Alzheimer's disease (AD) risk genes. Microglia from female mice progress faster in this activation trajectory. Similar activated states are also found in a second AD model and in human brain. Apoe, the major genetic risk factor for AD, regulates the ARMs but not the interferon response microglia (IRMs). Thus, the ARMs response is the converging point for aging, sex, and genetic AD risk factors.

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

Neuron

2022 Sep 26

Bulstrode, H;Girdler, GC;Gracia, T;Aivazidis, A;Moutsopoulos, I;Young, AMH;Hancock, J;He, X;Ridley, K;Xu, Z;Stockley, JH;Finlay, J;Hallou, C;Fajardo, T;Fountain, DM;van Dongen, S;Joannides, A;Morris, R;Mair, R;Watts, C;Santarius, T;Price, SJ;Hutchinson, PJA;Hodson, EJ;Pollard, SM;Mohorianu, I;Barker, RA;Sweeney, TR;Bayraktar, O;Gergely, F;Rowitch, DH;
PMID: 36174572 | DOI: 10.1016/j.neuron.2022.09.002

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 (IFNβ) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNβ 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.

A genetic tool for the longitudinal study of a subset of post-inflammatory reactive astrocytes

Cell reports methods

2022 Aug 22

Agnew-Svoboda, W;Ubina, T;Figueroa, Z;Wong, YC;Vizcarra, EA;Roebini, B;Wilson, EH;Fiacco, TA;Riccomagno, MM;
PMID: 36046623 | DOI: 10.1016/j.crmeth.2022.100276

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.

Cell Populations Expressing Stemness-Associated Markers in Lung Adenocarcinoma

Life (Basel, Switzerland)

2021 Oct 18

Paterson, C;Kilmister, EJ;Brasch, HD;Bockett, N;Patel, J;Paterson, E;Purdie, G;Galvin, S;Davis, PF;Itinteang, T;Tan, ST;
PMID: 34685477 | DOI: 10.3390/life11101106

The stemness-associated markers OCT4, NANOG, SOX2, KLF4 and c-MYC are expressed in numerous cancer types suggesting the presence of cancer stem cells (CSCs). Immunohistochemical (IHC) staining performed on 12 lung adenocarcinoma (LA) tissue samples showed protein expression of OCT4, NANOG, SOX2, KLF4 and c-MYC, and the CSC marker CD44. In situ hybridization (ISH) performed on six of the LA tissue samples showed mRNA expression of OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence staining performed on three of the tissue samples showed co-expression of OCT4 and c-MYC with NANOG, SOX2 and KLF4 by tumor gland cells, and expression of OCT4 and c-MYC exclusively by cells within the stroma. RT-qPCR performed on five LA-derived primary cell lines showed mRNA expression of all the markers except SOX2. Western blotting performed on four LA-derived primary cell lines demonstrated protein expression of all the markers except SOX2 and NANOG. Initial tumorsphere assays performed on four LA-derived primary cell lines demonstrated 0-80% of tumorspheres surpassing the 50 µm threshold. The expression of the stemness-associated markers OCT4, SOX2, NANOG, KFL4 and c-MYC by LA at the mRNA and protein level, and the unique expression patterns suggest a putative presence of CSC subpopulations within LA, which may be a novel therapeutic target for this cancer. Further functional studies are required to investigate the possession of stemness traits.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation

Nature neuroscience

2021 Dec 01

Pellegrino, G;Martin, M;Allet, C;Lhomme, T;Geller, S;Franssen, D;Mansuy, V;Manfredi-Lozano, M;Coutteau-Robles, A;Delli, V;Rasika, S;Mazur, D;Loyens, A;Tena-Sempere, M;Siepmann, J;Pralong, FP;Ciofi, P;Corfas, G;Parent, AS;Ojeda, SR;Sharif, A;Prevot, V;
PMID: 34795451 | DOI: 10.1038/s41593-021-00960-z

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling.

J Cell Biol.

2017 Sep 06

Kourtidis A, Necela B, Lin WH, Lu R, Feathers RW, Asmann YW, Thompson EA, Anastasiadis PZ.
PMID: 28877994 | DOI: 10.1083/jcb.201612125

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis.

Expression of Embryonic Stem Cell Markers on the Microvessels of WHO Grade I Meningioma

Front. Surg.

2018 Oct 26

Shivapathasundram G, Wickremesekera AC, Brasch HD, Marsh R, Tan ST, Itinteang T.
PMID: - | DOI: 10.3389/fsurg.2018.00065

Aim: The presence of cells within meningioma (MG) that express embryonic stem cell (ESC) markers has been previously reported. However, the precise location of these cells has yet to be determined.

Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 11 WHO grade I MG tissue samples for the expression of the ESC markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence (IF) IHC staining was performed to investigate the localization of each of these ESC markers. NanoString and colorimetric in situ hybridization (CISH) mRNA expression analyses were performed on six snap-frozen MG tissue samples to confirm transcriptional activation of these proteins, respectively.

Results: DAB IHC staining demonstrated expression of OCT4, NANOG, SOX2, KLF4, and c-MYC within all 11 MG tissue samples. IF IHC staining demonstrated the expression of the ESC markers OCT4, NANOG, SOX2, KLF4, and c-MYC on both the endothelial and pericyte layers of the microvessels. NanoString and CISH mRNA analyses confirmed transcription activation of these ESC markers.

Conclusion: This novel finding of the expression of all aforementioned ESC markers in WHO grade I MG infers the presence of a putative stem cells population which may give rise to MG.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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