BV-2 Microglial Cells Overexpressing C9orf72 Hexanucleotide Repeat Expansion Produce DPR Proteins and Show Normal Functionality but No RNA Foci.

Hannah Rostalski,Dorit Hoffmann,Jussi Tohka,Rebekka Wittrahm,Tomi Hietanen,Mikko Hiltunen,Eino Solje,Teemu Natunen,Ali Abdollahzadeh,Mari Takalo,Andrea Behánová,Anne M Remes,Annakaisa Haapasalo,Petra Mäkinen,Stina Leskelä,Nadine Huber

doi:10.3389/fneur.2020.550140

Abstract

Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia.

Highlights

Microglia are resident immune cells in the brain that perform vital functions during brain development, homeostasis, and aging
We found that overexpression of the 66R but not 2R in N2a cells led to the formation of sense RNA foci (Figures 1A,C), as detected by Fluorescence in situ Hybridization (FISH) and TYE 563-(CCCCGG)3 probe targeted against the expanded GGGGCC repeats
Most of the mechanistic studies related to the chromosome 9 open-reading frame 72 (C9orf72) hexanucleotide repeat expansion (HRE) have concentrated on neurons, but there is no information on how the C9orf72 HRE influences the phenotype or function of microglia

Summary

Introduction

Microglia are resident immune cells in the brain that perform vital functions during brain development, homeostasis, and aging. Because glial cells have been pinpointed as Abbreviations: AAV, adeno-associated virus; AF, Alexa Fluor; ALS, amyotrophic lateral sclerosis; BG, background; Clec7a, C-type lectin domain family 7, member a; CNS, central nervous system; Cst, cystatin F; DAPI, 4′ ,6-diamidino2-phenylindole; DAM, disease-associated microglia; df, degrees of freedom; DPBS, Dulbecco’s phosphate-buffered saline; DPR, dipeptide repeat; FISH, fluorescence in situ hybridization; FTLD, frontotemporal lobar degeneration; HEK, human embryonic kidney; HRE, hexanucleotide repeat expansion; Hsp, heat shock protein; ICC, immunocytochemistry; IFN, interferon; IL, interleukin; kDa, kilodalton; LAMP2A, lysosome-associated membrane protein 2 a; LC3B, microtubule associated protein 1 light chain 3 beta; LNA, locked nucleic acid; LPS, lipopolysaccharide; N2a, Neuro-2a; n.d., not detectable; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; RBP, RNAbinding protein; RT, room temperature; TDP-43, TAR DNA-binding protein 43; TNF, tumor necrosis factor; Trem, triggering receptor expressed on myeloid cells 2; Tyrobp, TYRO protein tyrosine kinase binding protein; UPS, ubiquitin–proteasome system; WB, Western blotting; ZsGreen, Zoanthus sp. Because glial cells have been pinpointed as Abbreviations: AAV, adeno-associated virus; AF, Alexa Fluor; ALS, amyotrophic lateral sclerosis; BG, background; Clec7a, C-type lectin domain family 7, member a; CNS, central nervous system; Cst, cystatin F; DAPI, 4′ ,6-diamidino2-phenylindole; DAM, disease-associated microglia; df, degrees of freedom; DPBS, Dulbecco’s phosphate-buffered saline; DPR, dipeptide repeat; FISH, fluorescence in situ hybridization; FTLD, frontotemporal lobar degeneration; HEK, human embryonic kidney; HRE, hexanucleotide repeat expansion; Hsp, heat shock protein; ICC, immunocytochemistry; IFN, interferon; IL, interleukin; kDa, kilodalton; LAMP2A, lysosome-associated membrane protein 2 a; LC3B, microtubule associated protein 1 light chain 3 beta; LNA, locked nucleic acid; LPS, lipopolysaccharide; N2a, Neuro-2a; n.d., not detectable; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; NO, nitric oxide; RBP, RNAbinding protein; RT, room temperature; TDP-43, TAR DNA-binding protein 43; TNF, tumor necrosis factor; Trem, triggering receptor expressed on myeloid cells 2; Tyrobp, TYRO protein tyrosine kinase binding protein; UPS, ubiquitin–proteasome system; WB, Western blotting; ZsGreen, Zoanthus sp. green fluorescent protein 1

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