Functional analysis and transcriptome profile of meninges and skin fibroblasts from human-aged donors

Abstract

The central nervous system (CNS) is surrounded by three membranes called meninges. Specialised fibroblasts, originating from the mesoderm and neural crest, primarily populate the meninges and serve as a binding agent. Our goal was to compare fibroblasts from meninges and skin obtained from the same human-aged donors, exploring their molecular and cellular characteristics related to CNS functions. We isolated meningeal fibroblasts (MFs) from brain donors and skin fibroblasts (SFs) from the same subjects. A functional analysis was performed measuring cell appearance, metabolic activity, and cellular orientation. We examined fibronectin, serpin H1, β-III-tubulin, and nestin through qPCR and immunofluorescence. A whole transcriptome analysis was also performed to characterise the gene expression of MFs and SFs. MFs appeared more rapidly in the post-tissue processing, while SFs showed an elevated cellular metabolism and a well-defined cellular orientation. The four markers were mostly similar between the MFs and SFs, except for nestin, more expressed in MFs. Transcriptome analysis reveals significant differences, particularly in cyclic adenosine monophosphate (cAMP) metabolism and response to forskolin, both of which are upregulated in MFs. This study highlights MFs’ unique characteristics, including the timing of appearance, metabolic activity, and gene expression patterns, particularly in cAMP metabolism and response to forskolin. These findings contribute to a deeper understanding of non-neuronal cells’ involvement in CNS activities and potentially open avenues for therapeutic exploration.

References

[1] JacobsonS, MarcusEM. Neuroanatomy for the Neuroscientist. Springer; 2008:325-331.doi:10.1007/978-0-387-70971-0

[2] DecimoI, BifariF, KramperaM, Fumagalli G. Neural stem cell niches in health and diseases. Curr Pharm Des. 2012;18:1755-1783.

[3] WellerRO, SharpMM, ChristodoulidesM, CarareRO, Møllgård K. The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. Acta Neuropathol. 2018;135:363-385.

[4] MercierF, Arikawa-Hirasawa E. Heparan sulfate niche for cell proliferation in the adult brain. Neurosci Lett. 2012;510:67-72.

[5] DecimoI, Fumagalli G, BertonV, KramperaM, BifariF. Meninges: From protective membrane to stem cell niche. Am J Stem Cells. 2012;1:92-105.

[6] EtcheversHC, CoulyG, VincentC, Le Douarin NM. Anterior cephalic neural crest is required for forebrain viability. Development. 1999;126:3533-3543.

[7] HalfterW, DongS, YipYP, Willem M, MayerU. A critical function of the pial basement membrane in cortical histogenesis. J Neurosci. 2002;22:6029-6040.

[8] RichtsmeierJT, Flaherty K. Hand in glove: brain and skull in development and dysmorphogenesis. Acta Neuropathol. 2013;125:469-489.

[9] BjornssonCS, Apostolopoulou M, TianY, TempleS. It takes a village: constructing the neurogenic niche. Dev Cell. 2015;32:435-446.

[10] BorrellV, Marín O. Meninges control tangential migration of hem-derived Cajal-Retzius cells via CXCL12/CXCR4 signaling. Nat Neurosci. 2006;9:1284-1293.

[11] SiegenthalerJA, Pleasure SJ. We have got you “covered”: how the meninges control brain development. Curr Opin Genet Dev. 2011;21:249-255.

[12] ZarbalisK, ChoeY, SiegenthalerJA, OroscoLA, Pleasure SJ. Meningeal defects alter the tangential migration of cortical interneurons in Foxc1hith/hith mice. Neural Dev. 2012;7:2.

[13] SiegenthalerJA, Ashique AM, ZarbalisK, et al. Retinoic acid from the meninges regulates cortical neuron generation. Cell. 2009;139:597-609.

[14] BeggsHE, Schahin-Reed D, ZangK, et al. FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies. Neuron. 2003;40:501-514.

[15] ZarbalisK, Siegenthaler JA, ChoeY, MaySR, Peterson AS, PleasureSJ. Cortical dysplasia and skull defects in mice with a Foxc1 allele reveal the role of meningeal differentiation in regulating cortical development. Proc Natl Acad Sci USA. 2007;104:14002-14007.

[16] JiangX, IsekiS, MaxsonRE, Sucov HM, Morriss-KayGM. Tissue origins and interactions in the mammalian skull vault. Dev Biol. 2002;241:106-116.

[17] BifariF, BertonV, PinoA, et al. Meninges harbor cells expressing neural precursor markers during development and adulthood. Front Cell Neurosci. 2015;9:383.

[18] LeeK, Saetern OC, NguyenA, RodriguezL, Schüle B. Derivation of leptomeninges explant cultures from postmortem human brain donors. J Vis Exp. 2017;2017:55045.

[19] DeSistoJ, O’Rourke R, JonesHE, et al. Single-cell transcriptomic analyses of the developing meninges reveal meningeal fibroblast diversity and function. Dev Cell. 2020;54:43-59.e4.

[20] BifariF, DecimoI, PinoA, et al. Neurogenic radial glia-like cells in meninges migrate and differentiate into functionally integrated neurons in the neonatal cortex. Cell Stem Cell. 2017;20:360-373.e7.

[21] PinoA, Fumagalli G, BifariF, DecimoI. New neurons in adult brain: distribution, molecular mechanisms and therapies. Biochem Pharmacol. 2017;141:4-22.

[22] PoloniTE, MediciV, CarlosAF, et al. Abbiategrasso brain bank protocol for collecting, processing and characterizing aging brains. J Vis Exp. 2020;2020:1-25.

[23] MartellaD, PaoliP, PionerJM, et al. Liquid crystalline networks toward regenerative medicine and tissue repair. Small. 2017;13:1702677.

[24] GanZ, DingL, BurckhardtCJ, et al. Vimentin intermediate filaments template microtubule networks to enhance persistence in cell polarity and directed migration. Cell Syst. 2016;3:500-501.

[25] Duarte-NevesJ, Pereira de Almeida L, CavadasC. Neuropeptide Y (NPY) as a therapeutic target for neurodegenerative diseases. Neurobiol Dis. 2016;95:210-224.

[26] ShevachEM, Stephens GL. The GITR-GITRL interaction: co-stimulation or contrasuppression of regulatory activity? Nat Rev Immunol. 2006;6:613-618.

[27] LacalPM, Petrillo MG, RuffiniF, et al. Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion. J Pharmacol Exp Ther. 2013;347:164-172.

[28] GaoJ, WangS, LiuS. The involvement of protein <scp>TNFSF18</scp> in promoting <scp>p-STAT1</scp> phosphorylation to induce coronary microcirculation disturbance in atherosclerotic mouse model. Drug Dev Res. 2021;82:115-122.

[29] KuleshovMV, JonesMR, RouillardAD, et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 2016;44:W90-W97.

[30] ColomboJA, NappMI, PuissantV. Leptomeningeal and skin fibroblasts: two different cell types? Int J Dev Neurosci. 1994;12:57-61.

[31] ChangHY, ChiJT, DudoitS, et al. Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci USA. 2002;99:12877-12882.

[32] MuellerMM, Fusenig NE. Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells. Differentiation. 2002;70:486-497.

[33] LynchMD, WattFM. Fibroblast heterogeneity: implications for human disease. J Clin Invest. 2018;128:26-35.

[34] HechtJH, Siegenthaler JA, PattersonKP, PleasureSJ. Primary cellular meningeal defects cause neocortical dysplasia and dyslamination. Ann Neurol. 2010;68:454-464.

[35] DasguptaK, JeongJ. Developmental biology of the meninges. Genesis. 2019;57:e23288.

[36] MishraS, ChoeY, PleasureSJ, Siegenthaler JA. Cerebrovascular defects in Foxc1 mutants correlate with aberrant WNT and VEGF—a pathways downstream of retinoic acid from the meninges. Dev Biol. 2016;420:148-165.

[37] HaushalterC, Schuhbaur B, DolleP, RhinnM. Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development. Biol Open. 2017;6:148-160.

[38] BoucherieC, BoutinC, JossinY, et al. Neural progenitor fate decision defects, cortical hypoplasia and behavioral impairment in Celsr1-deficient mice. Mol Psychiatry. 2018;23:723-734.

[39] HayakawaK, SnyderEY, LoEH. Meningeal multipotent cells: a hidden target for CNS repair? Neuromolecular Med. 2021;23:339-343.

[40] SinghM, SharmaAK. Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines. Vitr Cell Dev Biol Anim. 2011;47:83-88.

[41] VangipuramM, TingD, KimS, DiazR, SchüleB. Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp. 2013;77:3779.

[42] LemonsJMS, FengXJ, BennettBD, et al. Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol. 2010;8:e1000514.

[43] PérezMJ, PonceDP, Osorio-FuentealbaC, BehrensMI, Quintanilla RA. Mitochondrial bioenergetics is altered in fibroblasts from patients with sporadic Alzheimer’s disease. Front Neurosci. 2017;11:553.

[44] OlesenMA, Villavicencio-Tejo F, QuintanillaRA. The use of fibroblasts as a valuable strategy for studying mitochondrial impairment in neurological disorders. Transl Neurodegener. 2022;11:36.

[45] LeBleuVS, Neilson EG. Origin and functional heterogeneity of fibroblasts. FASEB J. 2020;34:3519-3536.

[46] MacKJ, SquierW, EastmanJT. Anatomy and development of the meninges: implications for subdural collections and CSF circulation. Pediatr Radiol. 2009;39:200-210.

[47] PatelN, KirmiO. Anatomy and imaging of the normal meninges. Semin Ultrasound CT MR. 2009;30:559-564.

[48] OrzechowskaB, Pabijan J, Wiltowska-ZuberJ, ZemłaJ, LekkaM. Fibroblasts change spreading capability and mechanical properties in a direct interaction with keratinocytes in conditions mimicking wound healing. J Biomech. 2018;74:134-142.

[49] KatohK. FAK-dependent cell motility and cell elongation. Cell. 2020;9:192.

[50] SriramG, Bigliardi PL, Bigliardi-QiM. Fibroblast heterogeneity and its implications for engineering organotypic skin models in vitro. Eur J Cell Biol. 2015;94:483-512.

[51] LothianC, Prakash N, LendahlU, WahlströmGM. Identification of both general and region-specific embryonic CNS enhancer elements in the nestin promoter. Exp Cell Res. 1999;248:509-519.

[52] BifariF, DecimoI, ChiamuleraC, et al. Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche. J Cell Mol Med. 2009;13:3195-3208.

[53] DarbyIA, Hewitson TD. Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol. 2007;257:143-179.

[54] LarigotL, Juricek L, DairouJ, CoumoulX. AhR signaling pathways and regulatory functions. Biochim Open. 2018;7:1-9.

[55] XuZ, SuS, ZhouS, et al. How to reprogram human fibroblasts to neurons. Cell Biosci. 2020;10:116.

[56] OkamotoN, MiyaF, KitaiY, et al. Homozygous ADCY5 mutation causes early-onset movement disorder with severe intellectual disability. Neurol Sci. 2021;1–4:2975-2978.

[57] PriceKM, WiggKG, FengY, et al. Genome-wide association study of word reading: overlap with risk genes for neurodevelopmental disorders. Genes Brain Behav. 2020;19:e12648.

[58] InselPA, OstromRS. Forskolin as a tool for examining adenylyl cyclase expression, regulation, and G protein signaling. Cell Mol Neurobiol. 2003;23:305-314.

[59] LepskiG, JannesCE, NikkhahG, Bischofberger J. cAMP promotes the differentiation of neural progenitor cells in vitro via modulation of voltage-gated calcium channels. Front Cell Neurosci. 2013;7:1-11.

[60] YangJ, CaoH, GuoS, et al. Small molecular compounds efficiently convert human fibroblasts directly into neurons. Mol Med Rep. 2020;22:4763-4771.

[61] AhmadF, ChungYW, TangY, et al. Phosphodiesterase 3B (PDE3B) regulates NLRP3 inflammasome in adipose tissue. Sci Rep. 2016;6:28056.

[62] KaramS, Margaria JP, BourcierA, et al. Cardiac overexpression of PDE4B blunts β-adrenergic response and maladaptive remodeling in heart failure. Circulation. 2020;142:161-174.

[63] SackettDL, OzbunL, ZudaireE, et al. Intracellular proadrenomedullin-derived peptides decorate the microtubules and contribute to cytoskeleton function. Endocrinology. 2008;149:2888-2898.

[64] TixierE, Leconte C, TouzaniO, RousselS, PetitE, BernaudinM. Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner. J Neurochem. 2008;106:1388-1403.

[65] FerreroH, Larrayoz IM, Gil-BeaFJ, MartínezA, Ramírez MJ. Adrenomedullin, a novel target for neurodegenerative diseases. Mol Neurobiol. 2018;55:8799-8814.

[66] HurtadoO, Serrano J, SobradoM, et al. Lack of adrenomedullin, but not complement factor H, results in larger infarct size and more extensive brain damage in a focal ischemia model. Neuroscience. 2010;171:885-892.

[67] Ochoa-CallejeroL, Pozo-Rodrigálvarez A, Martínez-MurilloR, MartínezA. Lack of adrenomedullin in mouse endothelial cells results in defective angiogenesis, enhanced vascular permeability, less metastasis, and more brain damage. Sci Rep. 2016;6:1-12.

[68] CarelliS, Giallongo T, ReyF, et al. Neural precursors cells expanded in a 3D micro-engineered niche present enhanced therapeutic efficacy in vivo. Nanotheranostics. 2021;5:8-26.

[69] MessaL, Barzaghini B, ReyF, et al. Neural precursor cells expanded inside the 3D micro-scaffold Nichoid present different non-coding RNAs profiles and transcript isoforms expression: possible epigenetic modulation by 3D growth. Biomedicine. 2021;9:1120.

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2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.