cells Review Glial Cell Dysfunction in C9orf72-Related Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Mehdi Ghasemi * , Kiandokht Keyhanian and Catherine Douthwright Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01655, USA; [email protected] (K.K.); [email protected] (C.D.) * Correspondence: [email protected]; Tel.: +1-774-441-7726; Fax: +1-508-856-4485 Abstract: Since the discovery of the chromosome 9 open reading frame 72 (C9orf72) repeat expansion mutation in 2011 as the most common genetic abnormality in amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) and frontotemporal dementia (FTD), progress in understanding the signaling pathways related to this mutation can only be described as intriguing. Two major theories have been suggested—(i) loss of function or haploinsufficiency and (ii) toxic gain of function from either C9orf72 repeat RNA or dipeptide repeat proteins (DPRs) generated from repeat-associated non-ATG (RAN) translation. Each theory has provided various signaling pathways that potentially participate in the disease progression. Dysregulation of the immune system, particularly glial cell dysfunction (mainly microglia and astrocytes), is demonstrated to play a pivotal role in both loss and gain of function theories of C9orf72 pathogenesis. In this review, we discuss the pathogenic roles of glial cells in C9orf72 ALS/FTD as evidenced by pre-clinical and clinical studies showing the presence of gliosis in C9orf72 ALS/FTD, pathologic hallmarks in glial cells, including TAR DNA-binding protein 43 (TDP-43) and p62 aggregates, and toxicity of C9orf72 glial cells. A better understanding of these pathways can provide new insights into the development of therapies targeting glial cell Citation: Ghasemi, M.; Keyhanian, abnormalities in C9orf72 ALS/FTD. K.; Douthwright, C. Glial Cell Dysfunction in C9orf72-Related Keywords: C9orf72 gene; C9orf72 repeat expansion mutation; amyotrophic lateral sclerosis (ALS); Amyotrophic Lateral Sclerosis and frontotemporal dementia (FTD); astrocytes; microglia; glial cells Frontotemporal Dementia. Cells 2021, 10, 249. https://doi.org/10.3390/ cells10020249 Academic Editors: Kyoungho Suk, 1. Introduction Makoto Tsuda and Hoon Ryu Amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s disease) and fron- Received: 30 December 2020 totemporal dementia (FTD) are two devastating neurodegenerative diseases with a high Accepted: 25 January 2021 burden on society. It is currently believed that ALS and FTD are parts of a disease spec- Published: 28 January 2021 trum that share clinical, genetic, and pathological findings. Clinically, 30–50% of ALS patients have cognitive deficits, and ~15% of patients with FTD exhibit symptoms/signs Publisher’s Note: MDPI stays neutral of ALS [1,2]. Histopathological studies have also shown that >97% of ALS and ~50% of with regard to jurisdictional claims in FTD patients aggregate inclusions of the TAR DNA-binding protein 43 (TDP-43) in both published maps and institutional affil- affected neurons and glial cells [3–7]. In 2011, a trio of teams [8–10] discovered a GGGGCC iations. (G4C2) nucleotide repeat expansion mutation in the first intron of the chromosome 9 open reading frame 72 (C9orf72) gene as the most frequent genetic cause in up to 35–45% of familial ALS, 5–20% of sporadic ALS, 15–25% of familial FTD, and 6–7% of sporadic FTD patients [11]. Although the number of hexanucleotide repeats varies considerably among Copyright: © 2021 by the authors. these patients, overall, repeat numbers of <30 are considered to be non-pathogenic [12]. Licensee MDPI, Basel, Switzerland. The relationship between repeat expansion size and phenotype is still equivocal, which This article is an open access article could be due to somatic variability in expansion size [2]. C9orf72 ALS patients have a mean distributed under the terms and onset age of 57 years old and a median survival rate of 30–37 months [13]. Although more conditions of the Creative Commons frequent bulbar onset has been reported in C9orf72 ALS patients compared to ALS patients Attribution (CC BY) license (https:// without a C9orf72 repeat expansion mutation [14–16], this is still debatable, because a creativecommons.org/licenses/by/ recent multi-center prospective natural history study on C9orf72 ALS cases reported a 4.0/). Cells 2021, 10, 249. https://doi.org/10.3390/cells10020249 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 26 Cells 2021, 10, 249 2 of 25 a recent multi-center prospective natural history study on C9orf72 ALS cases reported a higher rate of limb (54%) than bulbar (39%) onset [13]. What is undebatable, however, is higher rate of limb (54%) than bulbar (39%) onset [13]. What is undebatable, however, is thatthat the the prevalence prevalence of of FTD is significantly significantly higher in C9orf72 ALS cases, accompanied by higherhigher rates of disease progression andand prominentprominent cognitive/behavioralcognitive/behavioral changes [14,17] [14,17] (Figure(Figure 11).). Co-morbidCo-morbid dementiadementia isis presentpresent inin 50%50% ofof C9orf72 ALS patients [[14].14]. C9orf72 FTDFTD patients havehave alsoalso more more common common psychotic psychotic features features and and irrational irrational behavior behavior compared com- paredto non- toC9orf72 non-C9orf72FTD cases FTD [cases18,19 ].[18,19]. FigureFigure 1. Clinical 1. Clinical findings findings at onset at onsetin the inchromosome the chromosome 9 open 9 reading open reading frame frame72 (C9orf72 72 (C9orf72)-associated)-associated amyotrophic amyotrophic lateral lateralsclerosis (ALS)/frontotemporalsclerosis (ALS)/frontotemporal dementia (FTD). dementia (FTD). AlthoughAlthough muchmuch moremore research research is neededis needed to understand to understand the normal the normal function function of C9orf72 of C9orf72in humans, in humans, the discovery the discovery of C9orf72 of C9orf72repeat repeat expansion expansion mutations mutations as the as mostthe most common com- mongenetic genetic etiology etiology in ALS/FTD in ALS/FTD has openedhas opened a new a new avenue avenue of research of research for elucidatingfor elucidating dis- diseaseease mechanisms mechanisms and, and, ultimately, ultimately, therapeutic therapeutic approaches approaches for for this this fatal fatal disease.disease. Initial observationsobservations identifiedidentified decreaseddecreased levelslevels of of C9orf72 C9orf72 protein protein in in several several brain/spinal brain/spinal cord cord re- gions [8,10,20–25], suggesting a loss of function or haploinsufficiency as a main pathogenic regions [8,10,20–25], suggesting a loss of function or haploinsufficiency as a main patho- mechanism. Using a variety of C9orf72 knock out/down animal models, several mech- genic mechanism. Using a variety of C9orf72 knock out/down animal models, several anisms related to this theory were suggested, including aberrant autophagy, disrupted mechanisms related to this theory were suggested, including aberrant autophagy, dis- endosomal/lysosomal or endoplasmic reticulum (ER)-Golgi transport systems, and excito- rupted endosomal/lysosomal or endoplasmic reticulum (ER)-Golgi transport systems, toxicity [2,26]. Although this hypothesis still explains several aspects of C9orf72 ALS/FTD and excitotoxicity [2,26]. Although this hypothesis still explains several aspects of C9orf72 pathogenesis, other investigators have proposed a gain of toxic function, through the ALS/FTD pathogenesis, other investigators have proposed a gain of toxic function, generation of toxic RNA repeats and dipeptide repeat proteins (DPRs) [2]. In recent years, through the generation of toxic RNA repeats and dipeptide repeat proteins (DPRs) [2]. In compelling evidence indicates a role for immune dysregulation, particularly related to glial recent years, compelling evidence indicates a role for immune dysregulation, particularly cell abnormalities, as an important mechanism underlying C9orf72 ALS/FTD pathogenesis. related to glial cell abnormalities, as an important mechanism underlying C9orf72 Here, we comprehensively review the current literature on the pathogenic roles of glial cells, ALS/FTD pathogenesis. Here, we comprehensively review the current literature on the focusing on microglia and astrocytes, in C9orf72 ALS/FTD as evidenced by pre-clinical and pathogenicclinical studies. roles of glial cells, focusing on microglia and astrocytes, in C9orf72 ALS/FTD as evidenced by pre-clinical and clinical studies. 2. Overview of Pathogenic Mechanisms Underlying C9orf72 Repeat Expansion 2.Mutation Overview in of ALS/FTD Pathogenic Mechanisms Underlying C9orf72 Repeat Expansion Muta- tion2.1. Lossin ALS/FTD of Function Mechanisms 2.1. LossThe ofC9orf72 Functiongene Mechanisms consists of 11 exons (including two alternate non-coding first exons— 1a andThe 1b) C9orf72 [8]. Through gene consists alternative of 11 exons splicing, (includi it canng be two transcribed alternate intonon-coding three transcript first ex- ons—1avariants and (Figure 1b) 2[8].). TheThrough (G 4C alternative2)n repeat splicin expansiong, it mutationcan be transcribed is located into in intronthree tran- 1 of scriptvariants variants 1 and (Figure 3, whereas 2). The in variant (G4C2)n 2, repeat it is located expansion within mutation the promoter is located region in intron(Figure 1 2of). variantsTherefore, 1 and the 3, repeat whereas expansions in variant are 2, it not is located