Fncel 15 661447

REVIEW
published: 05 May 2021

doi: 10.3389/fncel.2021.661447
C9ORF72: What It Is, What It Does,

and Why It Matters
Julie Smeyers 1,2 , Elena-Gaia Banchi 1 and Morwena Latouche 1,2*
1
Sorbonne Université, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière,
DMU Neuroscience 6, Paris, France, 2 PSL Research university, EPHE, Neurogenetics team, Paris, France
When the non-coding repeat expansion in the C9ORF72 gene was discovered to be the
most frequent cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis
(ALS) in 2011, this gene and its derived protein, C9ORF72, were completely unknown.
The mutation appeared to produce both haploinsufficiency and gain-of-function effects
in the form of aggregating expanded RNAs and dipeptide repeat proteins (DPRs). An
unprecedented effort was then unleashed to decipher the pathogenic mechanisms
and the functions of C9ORF72 in order to design therapies. A decade later, while the
toxicity of accumulating gain-of-function products has been established and therapeutic
strategies are being developed to target it, the contribution of the loss of function starts
to appear more clearly. This article reviews the current knowledge about the C9ORF72
Edited by:
Agnes Lumi Nishimura,
protein, how it is affected by the repeat expansion in models and patients, and what
King’s College London, could be the contribution of its haploinsufficiency to the disease in light of the most
United Kingdom recent findings. We suggest that these elements should be taken into consideration to
Reviewed by: refine future therapeutic strategies, compensating for the decrease of C9ORF72 or at
Bhuvaneish Thangaraj Selvaraj,
University of Edinburgh, least preventing a further reduction.
United Kingdom
Keywords: C9ORF72, FTD/ALS, loss-of-function, autophagy, inflammation
Thomas Moens,
VIB KU Leuven Center for Brain
Disease Research, Belgium
Nicholas Kramer, INTRODUCTION
Harvard Medical School,
United States Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are rare
*Correspondence:
neurodegenerative diseases characterized by behavioral and motor disorders, respectively.
Morwena Latouche FTD results from the degeneration of the frontal and/or temporal cortical lobes, resulting in
[email protected] cognitive and/or language impairment (Gorno-Tempini et al., 2011; Rascovsky et al., 2011). ALS
is the most frequent motor neuron disease. It is caused by the degeneration of upper motor
Specialty section: neurons and their corticospinal axonal tracts (lateral sclerosis) associated with the loss of lower
This article was submitted to motor neurons and their axons, which leads to muscle wasting (amyotrophy) and paralysis
Cellular Neuropathology, (Mitsumoto et al., 1998). Despite their different symptomatology, the report of some overlap
a section of the journal
between these diseases can be traced as far back as 1869 (Charcot, 1869) and 1892 (Marie,
Frontiers in Cellular Neuroscience
1892) and has progressively been more and more documented over the years (Lomen-Hoerth
Received: 30 January 2021
et al., 2002, 2003; Lillo and Hodges, 2009). It is now recognized that FTD and ALS are part
Accepted: 17 March 2021
of a clinical, neuropathological, and genetic continuum (Ling et al., 2013; Burrell et al., 2016).
Published: 05 May 2021
This interrelation was particularly strengthened when TDP-43 was discovered in 2006 to be
Citation:
the main ubiquitinated protein composing the major form of neuropathological aggregates in
Smeyers J, Banchi E-G and
Latouche M (2021) C9ORF72: What It
both FTD and ALS (Arai et al., 2006; Neumann et al., 2006). Moreover, 2006 was marked by the
Is, What It Does, and Why It Matters. identification, on chromosome 9p, of the main locus associated with familial forms of FTD/ALS,
Front. Cell. Neurosci. 15:661447. the defining term for the cases associating clinical features of both FTD and ALS (Morita et al.,
doi: 10.3389/fncel.2021.661447 2006; Vance et al., 2006). The quest for the corresponding causal gene and mutations lasted 5 years
Frontiers in Cellular Neuroscience | www.frontiersin.org 1 May 2021 | Volume 15 | Article 661447

Smeyers et al. C9ORF72 Protein Physiology and Pathophysiology
and ended with the identification of a hexanucleotide (from 2 to 11) and gives rise to three coding variants (Figure 1A).
(G4 C2 )n repeat expansion (HRE) in the non-coding region Variant 1, V1 (NM_145005), is a short transcript including non-
of chromosome 9 open reading frame 72, C9ORF72 (DeJesus- coding exon 1a and exons 2–5 as the coding sequence. V2
Hernandez et al., 2011; Renton et al., 2011; Gijselinck et al., 2012). (NM_018325) and V3 (NM_001256054) differ in their inclusion
Until then, this gene and the protein of the same name had not of the non-coding exon 1b or 1a, respectively, and share exons
been studied, but the need to unravel the etiopathogenesis of C9- 2–11 as the coding sequence. Alternative splicing of these
FTD/ALS has triggered a massive effort in studying the function three RNA variants results in the production of two different
of the C9ORF72 protein and its relevance in disease. Therefore, isoforms: the 222-amino acid (aa) isoform (C9-short of 24 kDa)
we focus this review on what is now known about C9ORF72. encoded by V1, while the 481-aa isoform (C9-long of 54 kDa)
The HRE in C9ORF72 is the most frequent mutation in is encoded by V2 and V3 (Figure 1A; DeJesus-Hernandez
familial FTD, ALS, and FTD/ALS (Majounie et al., 2012; van et al., 2011; Renton et al., 2011; Gijselinck et al., 2012). The
Blitterswijk et al., 2012; Gijselinck et al., 2016). Patients are C9ORF72 human gene is highly conserved in primates and across
heterozygous and eventually all carriers develop the disease, but different species commonly used as model systems, suggesting
the age at onset is highly variable, from mid-20s to the ninth that the protein(s) encoded by C9ORF72 have fundamental
decade. Therefore, the penetrance is better defined as age-related biological functions. The similarity between the sequences of the
and goes from 50 at 58 years of age to 99.5% by 83 years of orthologous genes and the human gene, expressed as a percentage
age (Murphy et al., 2017). Age at onset variability is common in of identity, is very high in chimpanzee (gene LOC465031,
repeat expansion disorders and is generally correlated to the size 99.58%), rhesus macaque (gene C15H9orf72, 99.58%), mouse
of the expansion, but in the case of C9-FTD/ALS, this is a matter (gene 311004O21Rik, 98.13%), rat (gene RGD1359108, 97.71%),
of intense debate as positive (Beck et al., 2013; Hübers et al., 2014; rabbit (gene C9orf72, 98.54%), Xenopus (gene C9orf72, 83.96%),
Waite et al., 2014; Fournier et al., 2019) and negative (Gijselinck and zebrafish (gene C13H9orf72, 75.97%). However, for the
et al., 2016) correlations as well as absence of a correlation (Dols- nematode, the similarity is very poor (Caenorhabditis elegans,
Icardo et al., 2014; Suh et al., 2015; Chen et al., 2016; Jackson gene F18A1.6, 14.71%), and there is no ortholog of C9orf72 in
et al., 2020) were reported. The non-coding G4 C2 expansion Drosophila (query > C9ORF72 human gene)1 .
has two main consequences: a loss-of-function effect causing In humans, C9ORF72 transcripts are detectable in most
C9ORF72 haploinsufficiency and a gain of function associated tissues, and notably in all brain regions and the spinal cord
with the expression of abnormal bidirectionally transcribed (DeJesus-Hernandez et al., 2011; Renton et al., 2011; Rizzu et al.,
RNAs carrying the repeat (DeJesus-Hernandez et al., 2011). 2016). Regarding cell type distribution, C9ORF72 expression
These expanded RNAs accumulate in RNA foci or happen to is particularly high in myeloid cells (in particular in CD14+
be translated into dipeptide repeat proteins (DPRs) via repeat- monocytes, eosinophils, and neutrophils) and lower in lymphoid
associated non-ATG (RAN) translation (Ash et al., 2013; Lagier- cells and other tissues (Rizzu et al., 2016). Similarly, in mice
Tourenne et al., 2013; Mizielinska et al., 2013; Mori et al., harboring a LacZ reporter instead of exons 2–6 of the endogenous
2013). Pathologically, C9ORF72 expansion carriers present TDP- C9orf72 gene, the activity of the C9orf72 promoter is found in
43 inclusions in neurons and oligodendroglial cells that do not several tissues (lung, heart, liver, spleen, and kidney muscle),
overlap with the neuronal DPRs and RNA foci in neurons and with the highest levels in the brain and spinal cord (Suzuki
glial cells (Mizielinska et al., 2013; Mackenzie et al., 2014). After a et al., 2013; Atanasio et al., 2016; Jiang et al., 2016; Langseth
decade of research and the generation of a number of informative et al., 2017). This large expression profile was confirmed by
model systems, the understanding of the contribution of the in situ hybridization for the mouse C9orf72 ortholog (Koppers
different gain-of-function and loss-of-function mechanisms in et al., 2015). LacZ expression in the central nervous system
the pathogenesis is still blurry (Balendra and Isaacs, 2018; Braems (CNS) initially appeared restricted to neurons (Suzuki et al.,
et al., 2020). However, recent results strongly suggest that the 2013), but later reports demonstrated that some expression
combination of the loss-of-function effect with some gain-of- could be found also in glial cells (Jiang et al., 2016; Langseth
function entities is essential for the development of a pathological et al., 2017). Transcriptomic analyses have further demonstrated
FTD/ALS phenotype (Shi et al., 2018; Shao et al., 2019; Lutz, that, in mice, as in humans, myeloid cells including the
2020; Zhu et al., 2020). Here, we review what is known about microglia, macrophages, and dendritic cells (CD11b+ ) show a
the C9ORF72 protein, how it is affected in C9-FTD/ALS, and high expression of C9orf72 compared to other immune cell
explore the possible contribution of haploinsufficiency to the populations such as T (CD3+ ) and B (CD19+ ) cells (Atanasio
disease pathogenesis. et al., 2016; O’Rourke et al., 2016). To understand the regulation
of C9ORF72 expression between CNS tissues and cell types,
some authors (Rizzu et al., 2016) surveyed the C9ORF72 locus
C9ORF72 PHYSIOLOGICAL FUNCTIONS using cap analysis of gene expression sequence (CAGEseq). They
described several novel transcription start sites (TSS) at the
Gene Architecture, Transcripts, and C9ORF72 human locus, both in the sense and antisense strands.
Protein Distribution These TSS have distinct modes of expression in myeloid cells,
The human chromosome 9 open reading frame 72 (C9ORF72) lymphoid cells, and tissues from the CNS (cerebellum, cortex
locus is located on the reverse strand of chromosome 9 (41 kb). It
includes two non-coding exons (1a and 1b) and 10 coding exons 1
www.ensembl.org

FIGURE 1 | C9ORF72 gene structure, transcript variants, and protein isoforms under a non-pathological (A) and a pathological (B) state. (A) The C9ORF72 human
locus includes two non-coding exons (1a and 1b) and 10 coding exons (from 2 to 11). It gives rise to three coding variants: variant 1, V1, which includes exon 1a and
exons 2–5; variant 2, V2, which includes exon 1b and exons 2–11; and variant 3, V3, which includes exon 1a and exons 2–11. Alternative splicing of these three
RNA variants results in the production of two different isoforms: the 222-amino acid (aa) isoform (C9-short of 24 kDa) encoded by V1 and the 481-aa isoform
(C9-long of 54 kDa) encoded by V2 and V3. The coding exons are indicated in light blue and the non-coding exons in dark blue. (B) In a pathological state, the G4 C2
repeat localized between the two non-coding exons (1a and 1b) is abnormally expanded and results in three possible pathogenic mechanisms. Bidirectional
transcription of the hexanucleotide repeat expansion (HRE) generates G4 C2 sense and G2 C4 antisense expanded RNAs. These HRE transcripts give rise to
G-quadruplex and hairpin structures that can form RNA foci and sequester RNA-binding proteins (RBP) (B1). Expanded RNAs are also translated through
repeat-associated non-ATG (RAN) translation, resulting in the synthesis of dipeptide protein repeats (DPRs) (B2). Finally, the presence of the HRE inhibits
transcription, leading to a decrease in the C9ORF72 protein (B3).
tissues, hippocampus, caudate, and putamen), suggesting they Other studies have reported that the distribution of C9ORF72
have cell type- and/or tissue-specific functions (Rizzu et al., 2016). is dynamic in the developing and adult mouse brain, switching
Concurrently, at least five long non-coding RNAs (lncRNAs), between predominantly cytoplasmic and a nucleocytoplasmic
two sense and three antisense lncRNAs, along the C9ORF72 gene distribution according to the developmental stage and the
have been described that arise from the same promoter region as examined isoforms (Atkinson et al., 2015; Ferguson et al., 2016).
the sense C9ORF72 coding transcripts (Rizzu et al., 2016). The C9ORF72 was detected in synaptosome preparations from mouse
functions of these C9ORF72 lncRNAs are yet unknown, but they brains and co-localized with synaptic markers in mouse brain
might participate in the transcriptional regulation of C9ORF72 tissues and human induced pluripotent stem cell (iPSC)-derived
(Douglas, 2018). motor neurons (Atkinson et al., 2015; Ferguson et al., 2016; Frick
At the protein level, it is important to highlight that the range et al., 2018), supporting a role for C9ORF72 at the synapses.
and specificity of available antibodies have dramatically evolved, Further studies using tagged C9ORF72, transfected or CRISPR
allowing the community to progress from initially confusing modified, or immunocytochemical detection of endogenous
results to a much better understanding of the C9ORF72 protein C9ORF72 in cell lines or human iPSC-derived neurons have
expression profile and subcellular localization (Atkinson et al., revealed the co-localization of C9ORF72 with various organelles:
2015; Xiao et al., 2015; Ferguson et al., 2016; Davidson et al., the Golgi apparatus (Aoki et al., 2017), stress granules (Maharjan
2018; Frick et al., 2018; Laflamme et al., 2019). Globally, the et al., 2017; Chitiprolu et al., 2018), mitochondria (Wang et al.,
C9ORF72 protein is expressed mainly in the brain, spinal cord, 2021), and, most of all, compartments of the endolysosomal
and the immune system, and at lower levels in other organs (lung, pathway (Farg et al., 2014; Sellier et al., 2016; Amick et al.,
heart, liver, kidney, and skeletal muscle), in agreement with the 2016; Frick et al., 2018; Shi et al., 2018; Wang et al., 2020).
expression profile of the transcript. The 481-aa C9-long isoform Recently, an extensive study comparing the 14 commercially
is the most abundant (Frick et al., 2018). In mouse tissues and available antibodies revealed that many of these antibodies that
the human brain, C9ORF72 appears cytoplasmic, with punctate were used in previous studies fail to properly detect C9ORF72
staining in neurites suggestive of synaptic terminals (Atkinson (Laflamme et al., 2019). Using antibodies with verified selectivity,
et al., 2015; Ferguson et al., 2016; Frick et al., 2018; Laflamme the authors demonstrated that C9ORF72 was localized in the
et al., 2019; Xiao et al., 2015). Isoform-specific antibodies were lysosome, confirming their immunocytochemical results with
developed (Xiao et al., 2015) that showed C9-long to be diffuse lysosomal immunoprecipitation. Interestingly, the authors also
in the cytoplasm with some speckle-like structures in neurites, found that, in the cell lines they used, only 15% of the C9ORF72
as previously reported. Meanwhile, C9-short appeared localized protein pool was associated with lysosomes, 25% in total being
at the nuclear membrane in postmortem human brain tissue. membrane associated and 75% remaining in the soluble/cytosolic

fraction. Moreover, in human monocyte-derived macrophages, (Figure 2F; Sivadasan et al., 2016). Actin filament assembly and
where C9ORF72 is strongly expressed, it was localized to late disassembly are necessary for axonal maintenance and synaptic
phagosomes and phagolysosomes. Although some constants start strength (Flynn et al., 2012; Huang et al., 2013); thus, the
to appear, the factors driving the subcellular localization of alteration of this dynamic may account for the axonal phenotype
C9ORF72 in the different cell types and/or the cell states remain observed in C9ORF72-depleted mouse motor neurons and iPSC-
to be elucidated. derived motor neurons from C9-ALS patients (Sivadasan et al.,
2016). Neuronal activity and synaptic function also depend
C9ORF72 Protein Partners and on the energy supply provided by mitochondrial oxidative
Interactors phosphorylation (OXPHOS) complexes (Rangaraju et al., 2019),
When the mutation of C9ORF72 was discovered in FTD and and C9ORF72 was very recently shown to regulate the activity of
ALS cases, nothing was known about the role of the C9ORF72 these complexes (Wang et al., 2021). By interacting with AIFM1,
protein. The accumulating evidence that haploinsufficiency was C9ORF72 is imported to the mitochondrial intermembrane
one of the consequences of the HRE (DeJesus-Hernandez space, where it interacts with TIMMDC1 and the prohibitin
et al., 2011; Gijselinck et al., 2012; Waite et al., 2014; (PHB) complex to stabilize OXPHOS complex I (CI) subunits to
Xiao et al., 2015, 2016; Saberi et al., 2018; Viodé et al., allow their assembly. In the absence of C9ORF72 as well as in
2018) has triggered an effort to decipher the functions of iPSC-derived motor neurons from C9-ALS patients, the level of
C9ORF72. Through the characterization of C9ORF72 subcellular mature CI and CI activity were reduced (Wang et al., 2021).
distribution, bioinformatics studies, and the identification of
protein interactors, some functions of C9ORF72 began to C9ORF72 Is a DENN Protein Regulating
be unraveled. Rab-GTPases
Bioinformatics studies revealed that C9ORF72 shows homology
Interactions With the Nuclear Membrane, to the DENN (differentially expressed in normal and neoplasia)
Cytoskeleton, Mitochondria, and Membrane-Less family of proteins (Yoshimura et al., 2010; Marat et al., 2011;
Processes Levine et al., 2013). DENN proteins comprised an N-terminal
Following the discovery of the localization of C9ORF72 longin domain, followed by DENN and C-terminal alpha
at the nuclear membrane, co-immunoprecipitation and domains (Zhang et al., 2012). These three domains encompass
immunofluorescence experiments suggested that C9ORF72 aa 23–150, 212–312, and 313–481 of the human C9-long isoform
interacted with Importin b1 and Ran-GTPase, major proteins (Xiao et al., 2016). The C9-short isoform contains only the
involved in nucleocytoplasmic import (Xiao et al., 2015). longin domain, which is associated with the regulation of
C9ORF72 also appears to regulate stress granule (SG) formation endomembrane trafficking and contains a region for binding
and degradation (Maharjan et al., 2017; Chitiprolu et al., 2018). small GTPases (De Franceschi et al., 2014). DENN domain
C9-long is recruited to SG upon stress-related stimuli. Without proteins tend to be guanosine diphosphate (GTP)–guanosine
C9ORF72, these granules cannot form and other SG-associated diphosphate (GDP) exchange factors (GEFs) for Rab GTPases,
proteins like TIA-1, G3BP1, and HuR are downregulated which are master regulators of membrane trafficking. Rab
(Maharjan et al., 2017). C9ORF72 is also necessary for cellular proteins are small GTPases that are activated when GTP-bound
recovery after the removal of stress as it associates with p62 to and inactive when GDP-bound. They interact with GEFs that
target SG for the degradation by autophagy (Chitiprolu et al., trigger the binding of GTP, which switches Rab GTPases from
2018). Through interaction with the cytosolic chaperone Hsc70 the inactive to the active state and allows them to be recruited
(heat shock cognate protein 70), C9ORF72 may also be involved to membranes (Guadagno and Progida, 2019; Yoshimura et al.,
in chaperone-mediated autophagy, or aggrephagy, a mechanism 2010). C9ORF72 has been reported to interact with several
that permits the clearance of aggregating proteins without Rabs to regulate endosomal trafficking, lysosomal biogenesis,
requiring the presence of autophagic vesicles (Sellier et al., 2016). and autophagy in cell culture models (Farg et al., 2014; Sellier
A role for C9ORF72 in axon growth was identified in et al., 2016; Tang, 2016; Webster et al., 2016; Shi et al., 2018). In
primary mouse embryonic motor neurons, where C9ORF72 neuronal cell lines and primary neuronal cultures, C9ORF72 co-
overexpression led to longer axons and increased growth cone localizes and co-immunoprecipitates with some Rabs implicated
size, whereas its knockdown resulted in reduced axonal length in autophagy and endosomal transport, including Rab1, Rab5,
and smaller growth cones (Sivadasan et al., 2016). The study of Rab7, and Rab11, and co-localizes with Rab7 and Rab11 in
C9ORF72 interactome by mass spectrometry-based proteomics human motor neurons from the spinal cord (Farg et al., 2014).
identified cofilin and other actin binding proteins such as Arp2, Depletion of C9ORF72 using siRNA inhibited endocytosis and
Arp3, and coronin as partners of C9ORF72. Cofilin is an actin caused the accumulation of autophagosomes (Farg et al., 2014).
binding protein that regulates actin dynamics, depolymerizing In induced motor neurons (iMNs) derived from the iPSCs of
the tail of the actin filaments to supply actin monomers ready for C9-FTD/ALS patients, C9ORF72 localizes to Rab5-positive early
polymerization at the growing end of the filament (Lappalainen endosomes, and neuronal death observed in patients’ iMNs
and Drubin, 1997). It is inactivated by phosphorylation by LIM was rescued when Rab5 activity was increased (Shi et al., 2018).
kinases (Lawler, 1999). Remarkably, cofilin phosphorylation was All these Rabs are at the intersection of endocytosis and
increased in the absence of C9ORF72 in mouse motor neurons macroautophagy (Nassif et al., 2017; Shi et al., 2017; Guadagno
and in brains of C9-FTD/ALS patients compared to controls and Progida, 2019). Rab1 is involved in endoplasmic reticulum

FIGURE 2 | C9ORF72 functions in neurons and myeloid cells. In both cell types, C9ORF72 regulates autophagy and vesicular trafficking at different levels: the
initiation of autophagy (A), the recruitment of substrates for autophagy degradation (B), and autophagosome maturation and closure (C). These and other functions
depend on the interaction of C9ORF72 with its partners SMCR8 and WDR1 inside a GTPase-interacting complex (D), which is also responsible for interfering with
mTORC1 signaling (G). In neurons, C9ORF72 regulates actin dynamics (F) and endosomal recycling of GluR1 at the synapse (E). In myeloid cells, C9ORF72
modulates the STING pathway (H) and lysosomal exocytosis (I).
(ER)–Golgi transport, and Rab5 is necessary for endocytosis C9ORF72 Forms a Tripartite Complex With SMCR8
and is present at early endosomes, but these two Rabs also and WDR41
regulate the initiation of autophagy (Figure 2A). Rab11 associates The interaction of C9ORF72 with the autophagy initiation
with recycling endosomes and is involved in autophagosome complex and with a number of Rabs may be dependent on
elongation, while Rab7 associates with late endosomes, is other C9ORF72 interacting partners, as there is compelling
involved in autophagosome maturation and closure, and drives evidence that C9ORF72 forms a tripartite trimer with other
the fusion of late endosomes and autophagosomes to lysosomes DENN domain-containing proteins, SMCR8 (Smith–Magenis
(Figure 2C). The interaction of C9ORF72 and Rab7L1 was also chromosome region 8) and WDR41 (WD40-repeat containing
shown to regulate extracellular vesicle secretion (Aoki et al., protein 41) (Figure 2D; Amick et al., 2016; Blokhuis et al., 2016;
2017). It was further shown that C9ORF72 interacts with the Sellier et al., 2016; Sullivan et al., 2016; Ugolino et al., 2016; Yang
Unc-51-like kinase 1 (ULK1) autophagy initiation complex and et al., 2016). This complex was reported to act as GEF for Rab8a
Rab1a and controls the initiation of autophagy by acting as (Sellier et al., 2016) and Rab39b (Sellier et al., 2016; Yang et al.,
a Rab1a effector (Webster et al., 2016). C9ORF72 binds to 2016) and to interact with the proteins of the autophagy initiation
the activated ULK1 and recruits active Rab1a to the ULK1 complex FIP200/Ulk1/ATG13/ATG101 to control autophagic
complex to promote translocation of the ULK1 complex to flux (Figure 2A; Sellier et al., 2016; Sullivan et al., 2016; Yang
the phagophore during autophagy initiation (Figure 2A). As et al., 2016). The interaction of the complex with Rab8a and
a consequence, the translocation of ULK1 is impaired when Rab39b also mediates the recruitment of p62 (Sellier et al.,
C9ORF72 is knocked down in cell lines and primary neurons 2016), the autophagy receptor that targets poly-ubiquitinated
and autophagy is markedly reduced in neurons induced from proteins for autophagy degradation (Figure 2B). Interestingly,
C9-FTD/ALS patients’ iPSCs (Webster et al., 2016). there is a significant decrease of expression of postsynaptic

Rab39b concomitant with the upregulation of GluR1 in C9orf72 which is involved in modulating inflammation and chemotaxis.
knockout mice forebrains (Xiao et al., 2019), which may be linked C9ORF72 and FIS1 could work in parallel pathways to repress the
with the role of Rab39b in coordinating AMPA receptor subunit secretion of inflammatory cytokines by preventing the binding
(GluR1–4) composition and trafficking to the postsynaptic of these ligands to RAGE (Chai et al., 2020). Interestingly, all
membrane (Figure 2E; Giannandrea et al., 2010; Mignogna et al., C9orf72 knockout mouse models strikingly develop immune
2015). Increased glutamate receptor expression has also been abnormalities and autoimmune-like diseases (Koppers et al.,
observed in postmortem cortex and spinal cord of C9-FTD/ALS 2015; Atanasio et al., 2016; Burberry et al., 2016; Jiang et al., 2016;
patients and in iMNs derived from iPSCs from C9 patients, O’Rourke et al., 2016; Sudria-Lopez et al., 2016; Sullivan et al.,
possibly leading to glutamate excitotoxicity (Selvaraj et al., 2018; 2016; Ugolino et al., 2016). Indeed, mutant mice present massive
Shi et al., 2018). macrophages and lymphocyte tissue engorgement, causing
The structure of the C9ORF72–SMCR8–WDR41 (CSW) splenomegaly, lymphadenopathy, and glomerulonephropathy,
complex was recently described by Cryo-EM analysis (Su et al., accompanied by excessive secretion of inflammatory cytokines
2020; Tang et al., 2020a,b). The complex is a homodimer of and autoantibodies (Atanasio et al., 2016; Burberry et al., 2016;
the CSW trimer, and its structure suggested that it can act as a O’Rourke et al., 2016; Sudria-Lopez et al., 2016). Depending on
GTPase-activating protein (GAP) rather than a GEF. Indeed, the the research facilities where the knockout mice were studied,
CSW complex, but not C9ORF72 or SMCR8 alone, was shown to they presented various degrees of mortality, and some of them
be a GAP for Rab8a and Rab11a (Tang et al., 2020a,b). Similarly, died prematurely (Atanasio et al., 2016; Burberry et al., 2016;
the CSW complex appears to be a GAP for ARF1 and ARF6, Sudria-Lopez et al., 2016) and presented an associated motor
previously identified as interactors of C9ORF72 (Sivadasan et al., phenotype (Atanasio et al., 2016). A comprehensive analysis of
2016), and other members of the ADP-ribosylation factor (ARF) the environmental differences that could elicit such differences
family of small GTPases (Su et al., 2020). The ARF small GTPases was undertaken by Eggan and colleagues (Burberry et al., 2020).
regulate vesicular traffic and organelle structures (D’Souza- It revealed that C9ORF72 was necessary to maintain immune cell
Schorey and Chavrier, 2006; Sztul et al., 2019). Notably, ARF6 homeostasis in response to immune-stimulating gut microbiota.
regulates actin dynamics (D’Souza-Schorey and Chavrier, 2006; All phenotypic variations between mice could be abolished
Sivadasan et al., 2016; Sztul et al., 2019), and ARF1 has been using fecal material transplantation, and lifelong suppression
reported to promote mTORC1 activation (Jewell et al., 2015). of gut microflora with antibiotics could considerably alleviate
Both processes are altered when C9ORF72 or SMCR8 is depleted inflammatory and autoimmune phenotypes in C9orf72 knockout
(Figures 2F,G; Amick et al., 2016; Sivadasan et al., 2016; Ugolino (KO) mice. The same immune defects, including immune
et al., 2016; Shao et al., 2020; Wang et al., 2020). organ hypertrophy, were observed in two mouse lines with
Altogether, the analysis of C9ORF72 functions and protein a C9ORF72 depletion specifically in myeloid cells (McCauley
partners indicates that C9ORF72 is a modulator of the activity et al., 2020), confirming that these are the main cells involved
of small GTPases that regulate membrane trafficking, autophagy, in C9ORF72 immune functions. In C9orf72fl/fl;Cx3cr1Cre or
and axonal and synaptic maintenance. Investigating whether C9orf72fl/fl;LyzMCre mice, dendritic cells, in particular among
SMCR8 and WDR41 and the GAP or GEF activities of the myeloid cells, showed the upregulation of type I interferons
CSW trimer are involved in the other functions of C9ORF72, through the STING (stimulator of interferon genes) pathway
like the regulation of mitochondrial OXPHOS complexes, will in response to cytosolic DNA (McCauley et al., 2020). STING
be necessary to complete our understanding of C9ORF72 signaling is regulated by the autophagolysosomal pathway, and
cellular functions. blocking its degradation leads to sustained IFN-I production
(Konno et al., 2013; Gonugunta et al., 2017; Prabakaran et al.,
C9ORF72 Regulation of Inflammation 2018). Due to the role of C9ORF72 in autophagy, it is possible
Since 2016, numerous reports have established that C9ORF72 that C9ORF72 modulates STING-dependent inflammation by
plays an important role in immune regulation. Transcriptomic controlling its degradation (Figure 2H; McCauley et al., 2020).
studies first showed that C9ORF72 transcripts are mostly The accumulation of LAMP1 and the abnormal levels of
expressed in myeloid cells, particularly in CD14+ monocytes in cathepsin B in the macrophages and microglia of C9orf72 KO
human and mice (Rizzu et al., 2016). In particular, C9ORF72 mice confirm that the autophagy/lysosomal pathway is impaired
expression is elevated in dendritic and microglial cells (Su in myeloid cells in the absence of C9ORF72 (Atanasio et al.,
et al., 2014; O’Rourke et al., 2016). A genome-wide synthetic 2016; Burberry et al., 2016; O’Rourke et al., 2016). Accordingly,
lethal screen was therefore conducted in human myeloid cells C9ORF72 and SMCR8 double knockout mice present more
lacking C9ORF72 to identify its genetic interactors (Chai et al., severe immune defects than C9orf72 KO only (Shao et al.,
2020). That screen identified FIS1, a mitochondrial membrane 2020). When the autolysosomal pathway was studied specifically
protein involved in mitochondrial fission and mitophagy, as in myeloid cells from C9orf72 KO, Smcr8 KO, and double
a strong genetic interactor of C9ORF72. Interestingly, this knockout mice, it was demonstrated that lysosomal activity was
interaction could be validated also for SMCR8 and WDR41 severely impaired due to the increased lysosomal exocytosis of
and was shown to be independent of FIS1 mitochondrial mature lysosomal enzymes (Figure 2I; Zhang et al., 2018; Shao
localization and role in mitochondrial fission and mitophagy. et al., 2020). Moreover, lysosomal dysfunction causes mechanistic
In fact, C9ORF72 and FIS1 were both shown to interact with target of rapamycin (mTOR) overexpression in those cells;
ligands of the receptor for advanced glycation end (RAGE), interestingly, mTOR inhibition led to a rescue in macrophage

dysfunction and peripheral immunity perturbation, including repeat increases in successive generations and is associated with
splenomegaly and lymphadenopathy. This recent study was the increased disease severity. Overall, the HRE in C9ORF72 appears
first to demonstrate the causal relationship between the role of to contract as much as it expands in blood during parent–child
C9ORF72 in the regulation of the autophagy/lysosomal pathway transmission (Gijselinck et al., 2016; Fournier et al., 2019; Jackson
and the inflammatory effects resulting from C9ORF72 loss of et al., 2020). Since familial and sporadic C9ORF72 cases have
function in vivo (Shao et al., 2020). nearly identical age at onset (Murphy et al., 2017)—but individual
age at onset correlates with parental and mean family age at
onset (Moore et al., 2020)—there are certainly a number of
genetic and environmental modifiers interacting to determine the
C9ORF72 HAPLOINSUFFICIENCY IN onset of symptoms.
FTD/ALS
Gain and Loss-of-Function Mechanisms
The C9ORF72 Mutation in FTD/ALS The non-coding HRE in C9ORF72 has been shown to generate
A Non-Coding G4 C2 Expansion in C9ORF72 Causes three major pathogenic mechanisms: two related gain-of-
C9-FTD/ALS function effects, concerning RNA and toxic protein aggregates,
When mutations in C9ORF72 were discovered in familial cases and one loss-of-function effect (Figure 1B). These mechanisms
of FTD and ALS in 2011, three independent teams provided are intrinsically linked to transcription along the complex
evidence that the disease is caused by a (G4 C2 )n HRE G4 C2 architecture of the C9ORF72 locus. In variants 1 and 3, the
in the first intronic region of C9ORF72 (Figure 2B; DeJesus- HRE is located in the first intron as transcription includes
Hernandez et al., 2011; Renton et al., 2011; Gijselinck et al., exon 1a (Figures 2A,B). The repeated sequence of pure G
2016). This mutation with an autosomal-dominant transmission and C forms highly stable DNA and RNA structures called
explains approximately 25% of familial FTD, 80% of cases with an G-quadruplex and R-loops (RNA–DNA hybrids; Figure 2B;
FTD-ALS association, and 40% of familial ALS (van Blitterswijk Fratta et al., 2012; Reddy et al., 2013; Haeusler et al., 2014). In the
et al., 2012; Majounie et al., 2012; Gijselinck et al., 2016). The HRE region, these structures cause the accumulation of aborted
size of the hexanucleotide expansion in healthy subjects ranges transcripts (Haeusler et al., 2014), and the structures formed by
between 2 and 24, with most people harboring two to eight the expanded transcripts sequester the RNA-binding proteins
repeats. An expansion above 30 is considered pathological; a involved in transcription and splicing. Consequently, these
small proportion of patients present short expansions (30–100 complexes called RNA foci affect posttranscriptional processing
units), while the vast majority have many hundreds or thousands (Fratta et al., 2012; Lee et al., 2013). These RNA foci composed
of repeats (DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012, of both sense and antisense transcripts are a consistent feature
2016; van der Zee et al., 2013; Fournier et al., 2019). In addition in the brain tissue of C9orf72 carriers (Lagier-Tourenne et al.,
to the heterogeneity of the repeat size between C9ORF72 carriers, 2013; Mizielinska et al., 2013). These foci can be found in the
there is a mosaicism caused by the somatic instability of the repeat nuclei of the motor and frontal cortex neurons, hippocampus,
number in several tissues and among central nervous system cerebellum, and the spinal cord (Lagier-Tourenne et al., 2013;
regions (Van Mossevelde et al., 2017a). Thus, in the same patient, Mizielinska et al., 2013; Cooper-Knock et al., 2014). They are
different expansion sizes can be measured in the CNS and in the mainly observed in neurons, but can occasionally be detected
blood (van Blitterswijk et al., 2013; Waite et al., 2014; Nordin in glial cells such as astrocytes, microglia, and oligodendrocytes
et al., 2015). To establish correlations between repeat expansion (Lagier-Tourenne et al., 2013; Mizielinska et al., 2013). A second
size, age at onset, and disease severity, peripheral lymphocytes gain-of-function mechanism derives from the sense and antisense
appear as an interesting non-invasive marker (Pamphlett et al., transcripts containing the HRE that induce ribosomes to translate
2013). However, in addition to the mosaicism between tissues, the them through a non-ATG (RAN) translation, resulting in the
repeat number varies in blood with age at collection and over time synthesis of dipeptide protein repeats (DPRs) that accumulate
in successive blood collections from C9ORF72 mutation carriers and form intracellular inclusions (Ash et al., 2013; Gendron et al.,
(Suh et al., 2015; Fournier et al., 2019). This mosaicism and 2013; Mori et al., 2013; Zu et al., 2013). Inclusions of DPRs
instability may be part of the mechanisms underlying the clinical can be detected by immunohistochemistry, immunoblotting, or
heterogeneity observed in patients, but conclusive evidence is still immunoassay. They are positive for p62 and negative for TDP-43
lacking (Nordin et al., 2015; Van Mossevelde et al., 2017a). The and may correspond to one or more dipeptides (Ash et al., 2013;
fact that the expansion size increases with age in the blood could Gendron et al., 2013; Mackenzie et al., 2013; Mori et al., 2013;
partly explain the conflicting results reported in the literature Zu et al., 2013; Mackenzie et al., 2014). Inclusions of DPRs are
regarding age at onset and the size of the HRE (Beck et al., 2013; mainly cytoplasmic, but can also be nuclear or found in
Dols-Icardo et al., 2014; Hübers et al., 2014; Waite et al., 2014; Suh dystrophic neurites or be present at the pre-inclusion stage
et al., 2015; Chen et al., 2016; Gijselinck et al., 2016; Fournier et al., disseminated in the cytoplasm (Mackenzie et al., 2015).
2019; Jackson et al., 2020). Other usual features of pathological Finally, the presence of the repeat causes a downregulation
repeat expansions are also unclear in C9-FTD/ALS. One study in C9ORF72 gene expression, leading to a heterozygous loss of
reported decreased age at onset from one generation to the next, function (LOF) (Figure 2B). A number of studies have explored
suggesting a mechanism of disease anticipation (Van Mossevelde why the mutation induces the haploinsufficiency of C9ORF72,
et al., 2017b). Anticipation usually occurs when the size of the focusing on epigenetic mechanisms. The repeat expansion in

C9ORF72 has been shown to bind to trimethylated histones in brain samples from C9-FTD/ALS patients, in particular in the
brain tissue from FTD/ALS patients (Belzil et al., 2013). Reduced frontal cortex and cerebellum (Belzil et al., 2013; Fratta et al.,
messenger RNA (mRNA) levels of C9ORF72 correlate with 2013; Waite et al., 2014; van Blitterswijk et al., 2015; Rizzu et al.,
the trimethylation of these histone residues in patients’ frontal 2016), as well as in the motor cortex and spinal cord (Donnelly
cortices and cerebella, while decreasing C9ORF72 association et al., 2013). V3 appeared to be decreased (Fratta et al., 2013),
with trimethylated histones restored C9ORF72 mRNA expression unaffected (van Blitterswijk et al., 2015), or increased (Jackson
in patients’ fibroblasts. Furthermore, the CpG island upstream of et al., 2020). Sense and antisense transcripts containing the HRE
the pathogenic repeat and the repeat itself are hypermethylated were globally shown to be increased in the cerebellum of patients
(Belzil et al., 2014; Xi et al., 2014, 2015; Gijselinck et al., (Mori et al., 2013). Since V3 and V2 encode the same protein, and
2016; Jackson et al., 2020), and this hypermethylation correlates the part of V1 and V3 in the total fraction of C9ORF72 mRNAs
with repeat length and decreased promoter activity. While is minor, this will nevertheless not affect the general result that
the methylation of the promoter and the expansion itself there is an overall decrease of ⇠50% of C9ORF72 transcripts and
are now clearly associated with C9ORF72 haploinsufficiency, that C9ORF72 protein levels might be correspondingly reduced
it can also result in a reduced accumulation of RNA foci in C9-FTD/ALS patients. To date, no variation in the expression
and dipeptide repeat protein aggregates in human brains (Liu of total transcripts or variants 1, 2, and 3 was observed between
et al., 2014). Opposite effects on the disease onset, duration, FTD, ALS, and FTD-ALS patients (van Blitterswijk et al., 2015).
and neurodegeneration have been reported, suggesting that
epigenetic regulation of C9ORF72 may be a potent pleiotropic Protein Expression
disease modifier. Increasing methylation states have been Two C9ORF72 protein isoforms are known to exist in humans:
associated with earlier onset ages (Gijselinck et al., 2016; Zhang a long isoform of 481 aa (C9-long of 54 kDa) encoded by V2
et al., 2017), while C9ORF72 promoter hypermethylation seems and V3 and a short isoform of 222 aa (C9-short of 24 kDa)
to slow disease progression and neurodegeneration (McMillan encoded by V1. While C9-long appears well conserved in
et al., 2015; Russ et al., 2015). evolution, the short isoform is referenced only in humans
and is not known in other non-human primates or other
C9ORF72 Expression in FTD/ALS species (see Text Foot Note 1 for further details; Gene Tree
Patients for C9orf72 ENSG00000147894). Initially available commercial
Initial analyses of C9ORF72 transcripts and protein levels have antibodies did not distinguish between the long and short
been relatively difficult and sometimes even misleading. The isoforms; their specificity had not been strongly established and
architecture of the gene and its transcripts have not been has been questioned since. A first study showed no differences
previously studied, making it difficult to get a real measure of the in C9ORF72 protein expression in lymphoblast cells or brain
expression of the different variants, and commercially available lysates from FTD or ALS patients carrying the HRE compared
antibodies for C9ORF72 have not been characterized (DeJesus- to C9ORF72 non-carrier individuals (DeJesus-Hernandez et al.,
Hernandez et al., 2011; Renton et al., 2011; Gijselinck et al., 2012). 2011). Immunohistochemistry suggested a cytoplasmic staining
Nevertheless, it is now firmly established that C9-FTD/ALS in neurons with a punctate staining of gray matter, suggestive
patients consistently present a decreased expression of C9ORF72. of synaptic localization. Another study found the protein to be
predominantly localized within the nucleus and the C9ORF72
Transcripts Expression protein levels to be reduced in fibroblast cell lines derived
From the three variants resulting from C9ORF72 transcription, from ALS patients relative to controls (Renton et al., 2011). In
V2 is by far the most abundant transcript in tissue from the following years, new antibodies have been developed that
non-mutation carriers (van Blitterswijk et al., 2015; Tran et al., have revealed a clear decrease in C9ORF72 protein levels in
2015; Rizzu et al., 2016). The HRE is located within the promoter the frontal cortex from FTD or ALS patients with C9ORF72
region of this transcript, whereas it is in intron 1 of the repeat expansion compared to controls (Waite et al., 2014; Xiao
other two transcripts, V1 and V3. Therefore, the epigenetic et al., 2015; Saberi et al., 2018). This decrease ranges between 25
mechanisms discussed above, as well as the DNA G-quadruplex and 50% according to the studies and the employed antibodies.
structures formed by the repeated GGGGCC, can variably A similar or larger decrease was also reported in other parts of
interfere with the transcription of all three variants. While a the cortex, in particular in the occipital, motor, and temporal
global reduction of C9ORF72 mRNAs, and more specifically cortices (Saberi et al., 2018; Xiao et al., 2015), while there was
of V2, was consistently observed since the discovery of the generally only a small (20%) (Frick et al., 2018) or no decrease
mutation (DeJesus-Hernandez et al., 2011; Gijselinck et al., 2012), in the cerebellum or spinal cord (Waite et al., 2014; Xiao et al.,
the expressions of V1 and V3 in patients have been more 2015; Saberi et al., 2018). In all cases but one, C9-short levels
debated. The total C9ORF72 mRNA reduction was around 34% in were too low to be detected, and therefore this reduction concerns
lymphoblast cells from blood samples and was 38–50% in frontal only C9-long isoform. When C9-short could be detected, it
cortex samples (DeJesus-Hernandez et al., 2011; Gijselinck et al., appeared increased in the temporal and frontal cortices of C9-
2012). We observed a similar 50% decrease in frontal cortex and FTD/ALS patients (Xiao et al., 2015). This is in contradiction to
lymphoblasts from a group of patients in a French cohort (Ciura the observed decrease in V1 transcript and bears no explanation
et al., 2013). A decrease in total C9ORF72 mRNAs, and often also so far, but it must be reminded that only three C9-ALS cases could
specifically of V2 and V1, was further reported in postmortem be examined in that study and that the abundance of C9-short is

generally admitted to be very low (Waite et al., 2014; Frick et al., in one model (Jiang et al., 2016). These mammalian models
2018; Saberi et al., 2018). A sensitive and robust antibody-free made it clear that C9ORF72 deficiency is not the sole or more
mass spectrometry assay was recently used to measure the level potent trigger of neurodegeneration in C9-FTD/ALS, but its
of C9ORF72 isoforms, thus permitting to alleviate the antibody exact contribution to the phenotype has not yet been fully
issue (Viodé et al., 2018). It was conducted on the frontal cortex understood. Pure gain-of-function mouse models, on the other
of C9-FTD patients (with or without ALS) and demonstrated hand, have also fallen short of recapitulating FTD/ALS. When
a decrease of 42% for total C9ORF72 and C9-long. C9-short- overexpressing the HRE (Chew et al., 2015; Herranz-Martin
specific peptides were too low to be detected, confirming the et al., 2017) or specific DPRs (Zhang et al., 2016; Schludi
scarcity of this isoform in human frontal cortex. et al., 2017; Choi et al., 2019; Hao et al., 2019; LaClair et al.,
2020), both histopathology and some behavioral modifications
Evidence of the C9ORF72 related to FTD/ALS were observed. However, bacterial artificial
chromosome (BAC) transgenic mice mainly reproduced RNA
Haploinsufficiency Involvement in the foci formation and DPR inclusions, whereas TDP-43 pathology,
Development of C9-FTD/ALS neurodegeneration, or FTD/ALS phenotypes were rare or
Over the past decade, massive effort has been undertaken inconstant (Peters et al., 2015; O’Rourke et al., 2016; Jiang et al.,
to decipher the mechanisms of C9-FTD/ALS pathogenesis. 2016; Liu Y. et al., 2016; Mordes et al., 2020; Nguyen et al.,
The three major hypotheses, toxicity of RNA foci or DPRs 2020). Spatial learning and anxiety abnormalities accompanied
or C9ORF72 deleterious haploinsufficiency, have been by loss of hippocampal neurons were observed by Jiang et al.,
investigated with some passion, and there is evidence for but no motor deficits, neuromuscular unit alterations, or
all three mechanisms. Many new experimental models have differences in social interaction, social recognition, or social
been constructed, and studies in animal models and human communication (Jiang et al., 2016). Out of the four groups that
patient samples in particular, including iPSC-derived neurons, generated C9ORF72 BAC mice, only one reported a progressive
have provided unprecedented insights into the pathogenic disease with a motor phenotype leading to hind limb paralysis
mechanisms. The study of gain-of-function effects has benefited associated with hippocampal and cortical neuron degeneration,
from the extensive knowledge already existing for other though occurring only in a subset of mice (Liu Y. et al., 2016).
nucleotide repeat diseases (Braems et al., 2020). Because of the Recently, the phenotype of these mice has been debated, as
specific nature of the mutation and the complete absence of two different research groups did not succeed in reproducing
knowledge about the C9ORF72 protein, the loss-of-function these observations (Mordes et al., 2020) while two others did
effect was initially less explored. However, the tide has turned (Nguyen et al., 2020). An environmental factor was evoked
during the last 5 years, and not only C9ORF72 haploinsufficiency since the experiments were carried out in different laboratories.
in C9-FTD/ALS patients is firmly established now, but its Nonetheless, one of the major confounding effects could be
contribution to disease pathogenesis has been re-evaluated and the genetic background, as the used FVB/N background is
has recently become unquestionable. known to present inherent seizure activity (Nguyen et al.,
2020). Intriguingly, in the study of Mordes et al., even though
In vivo Loss-of-Function and Gain-of-Function DPR inclusions and C9ORF72 RNA foci were present at
Models levels similar to those found in human C9-FTD/ALS brain,
To better understand the consequences of C9ORF72 the mice did not show any phenotype. This supports the
haploinsufficiency on FTD-ALS, numerous C9ORF72 loss- idea that the gain-of-function effect alone is not sufficient to
of-function in vivo models have been developed. In zebrafish induce neurodegeneration. Altogether, the gain-of-function
and Caenorhabditis elegans, C9ORF72 loss of function resulted models suggested that gain-of-function entities can cause
in locomotor phenotypes and motoneuron degeneration (Ciura neurodegeneration when sufficiently accumulated, but that
et al., 2013; Therrien et al., 2013). Yet, the reduction of C9ORF72 additional insults are necessary when they are present at
in genetic mouse models or with antisense oligonucleotides physiological levels.
did not mimic FTD-ALS (Balendra and Isaacs, 2018; Braems
et al., 2020). Instead, reducing C9ORF72 to 40% with antisense Synergistic Models
oligonucleotide (ASO) resulted in an upregulation of the In human iMNs derived from cells of C9-FTD/ALS patients,
microglial activation genes in the spinal cord (Lagier-Tourenne neurodegeneration was shown to depend on C9ORF72
et al., 2013), while C9orf72 KO mice predominantly developed haploinsufficiency that rendered iMNs hypersensitive to
an inflammatory phenotype, sometimes associated with a glutamate (Shi et al., 2018). Although little is known about
shortened life span, but no neurodegeneration or motor neuron the excitotoxicity mechanisms in FTD, in ALS, it is one of the
disease (Koppers et al., 2015; Atanasio et al., 2016; Burberry major contributing mechanisms involved in the degeneration
et al., 2016; Jiang et al., 2016; O’Rourke et al., 2016; Sudria- of motor neurons, and there is evidence that it is involved in
Lopez et al., 2016; Sullivan et al., 2016; Ugolino et al., 2016). C9-FTD/ALS (Starr and Sattler, 2018). An increase of glutamate
Mild motor deficits were nevertheless noticed in two studies receptors on the surface of iMNs was observed, leading to
(Atanasio et al., 2016; Jiang et al., 2016), and although non- increased susceptibility to excitotoxicity. This neurodegenerative
motor behavioral phenotypes were rarely assessed in C9orf72 phenotype could be reproduced by deleting or decreasing
KO mice, early social recognition deficits were documented C9ORF72 in control iMNs and could be fully rescued in C9-iMNs

by re-expressing C9ORF72 (long or short). At the same time, 2013). Secondly, only the non-coding HRE and not simple LOF
the re-expression of C9ORF72 also reduced the accumulation mutations in C9ORF72 have been shown to cause C9-FTD/ALS
of DPRs in C9-iMNs, which might have participated in rescuing (Harms et al., 2013). These are important arguments to consider,
the survival of C9ORF72 patient iMNs in response to glutamate but they may not necessarily impede an essential role of the LOF
treatment. Interestingly, a similar synergistic effect between in C9-FTD/ALS. The two homozygous cases are very different.
C9ORF72 loss of function and excitotoxicity was very recently The first one was a man with two large expansions inherited from
shown to cause ALS-like disease in rats (Dong et al., 2020a). KO his parents who had both developed early-onset dementia (Fratta
rats did not spontaneously develop an ALS phenotype, but like et al., 2013). This patient presented a rapidly progressive severe
the C9orf72 KO mouse models, they developed splenomegaly dementia and died within 3 years after onset. The clinical and
and lymphadenopathy (O’Rourke et al., 2016; Dong et al., neuropathological characteristics of this homozygous patient are
2020a). When treated with a subclinical dose of kainic acid to not beyond the range of the reported heterozygous cases, but
promote glutamate excitotoxicity, motor deficits appeared in nevertheless, they are at the most severe end of the spectrum.
KO animals, associated with a loss of motor neurons (Dong The second patient was a woman with one large expansion and a
et al., 2020a). Motor neurons also presented some abnormalities much shorter one of 50 ± 5 repeats (Cooper-Knock et al., 2013).
such as fragmented Golgi complex and abnormal vesicular The severity of her disease was unremarkable. Interestingly, the
trafficking. Moreover, Abo-Rady et al. showed a decreased axonal level of C9ORF72 transcripts was reduced by ± 75% in the
trafficking of lysosomes in iMNs derived from C9-FTD/ALS frontal cortex of the first homozygous patient, while it was
patient cells that was exacerbated by additionally knocking reduced to ± 50% of its normal level in the blood of the second
out C9ORF72 in these cells (Abo-Rady et al., 2020). In this patient, similarly to the average of heterozygous cases. It is
model, significant apoptosis of iMNs occurred only in cells worth noticing that, even in the case of two large expansions,
presenting the combination of the HRE and the C9ORF72 KO, there remains some C9ORF72 expression which is comparable
while the knockout did not cause any effect by itself. These to the lowest expression level that can sometimes be observed in
recent studies support the hypothesis that a total deletion of heterozygous patients (Fratta et al., 2013; van Blitterswijk et al.,
the C9ORF72 protein on its own is not sufficient to cause a 2015). Therefore, within these two cases, severity appears in
motor phenotype, but it favors the appearance of the phenotype agreement with the extent of C9ORF72 decrease, while remaining
when combined with another defective or abnormal biological within the range of disease severity and partial loss of function
effect. In line with this, two other studies have now shown that that have been reported in cohorts of heterozygous patients. It
the combination of gain-of-function mechanisms on top of is, however, not to be ignored that C9ORF72 coding mutations
the loss of function is efficiently leading to the development of are strikingly lacking in all C9-FTD/ALS cohorts that have been
an FTD/ALS-like disease in mice (Shao et al., 2019; Zhu et al., examined over the years. One splice mutation was reported in
2020). In both studies, reduction or loss of C9ORF72 provoked an ALS patient (Liu F. et al., 2016) from a Chinese cohort of 276
or exacerbated motor deficits in the background of C9-BAC patients where mutations in 24 other ALS known causing genes
mice. In the study by Zhu et al., they additionally documented had been excluded. This mutation resulted in a premature stop
that the same was true for cognitive deficits, neurodegeneration, codon decreasing the level of the mutant messenger RNA, but
glial activation, and accumulation of DPRs (Zhu et al., 2020). the patient had no family history of the disease and autopsy
Finally, a knock-in (KI) model was made in rats demonstrating could not be performed, which makes the interpretation of
the need of combining loss- and gain-of-function effects to this case difficult. One might wonder if this mutation was not
cause FTD/ALS (Dong et al., 2020b). Eighty G4 C2 repeats associated with a particular additional environmental trigger or
with flanking fragments of human exons 1a and 1b were to another variation in a potent modifier or causal gene that was
inserted in the rat C9orf72 locus, resulting in a 40% decrease not identified so far. Studies aiming to analyze whether the level
of C9ORF72 protein expression in the brain and spinal cord of of C9ORF72 associates with clinical characteristics in sufficiently
KI rats. These rats developed a strong progressive motor deficit large cohorts would greatly help in understanding the importance
accompanied by a 47% loss of spinal motoneurons. Comparable of haploinsufficiency in the disease. So far, one study conducted
phenotypes had never occurred in KO rats of the same genetic on 56 C9-FTD/ALS patients found a positive correlation between
background or in BAC mice expressing larger HRE at a similar survival after onset and the level of V1 transcript in the frontal
or a higher level. All these most recent models strongly argue cortex and the cerebellum (van Blitterswijk et al., 2015). No
in favor of a disease mechanism in C9-FTD/ALS resulting further association with a clinical phenotype could be detected
from reduced C9ORF72, synergizing with repeat-dependent for the level of cerebellar C9ORF72 protein from 17 patients
gain of toxicity. (Frick et al., 2018) or for the transcript levels in the blood of 75
C9ORF72 cases (Jackson et al., 2020). Interestingly, the C9ORF72
Patient-Based Evidence of the Contribution of protein levels were consistently more reduced in the frontal
C9ORF72 Haploinsufficiency to the Disease cortex than in the cerebellum of patients (Waite et al., 2014;
Two strong arguments that are often invoked against a primary Xiao et al., 2015; Saberi et al., 2018), highlighting the possibility
LOF mechanism in the disease concern the genetics of C9- that it will be important to quantify C9ORF72 transcripts and
FTD/ALS. Firstly, homozygosity does not strikingly increase protein specifically from the affected brain regions to see whether
the clinical severity of the disease, although only two cases it correlates with some clinical characteristics. The case reported
were reported so far (Cooper-Knock et al., 2013; Fratta et al., by McGoldrick et al. was of particular interest in this regard

(McGoldrick et al., 2018). Siblings severely affected by ALS and modifier of survival after onset (Dickson et al., 2019), while
carrying a long expansion in C9ORF72 had inherited it from neuronal transcriptome of the mid-frontal neocortex from seven
their mosaic father (unaffected at age 90) who carried a 70-repeat postmortem C9ORF72 expansion carriers showed that global C9-
allele in blood but larger expansions in the CNS. Remarkably, FTD/ALS-associated transcriptome changes appear driven by the
the father never developed the disease or TDP-43 pathology, loss of function of the C9ORF72 protein (Liu et al., 2020).
but he had similar RNA foci and DPR burdens compared to
his affected daughter, while increased expression and protein
level of C9ORF72 could be detected in his blood and CNS
tissues. Considering all the evidence now gained from the various
CONCLUSION
in vitro and in vivo models, it seems more and more established Increasing knowledge about the functions of C9ORF72 has
that C9-FTD/ALS pathogenesis needs the combination of revealed that this protein is part of a GTPase-interacting complex
C9ORF72 haploinsufficiency and the accumulation of toxic which is a potent regulator at the crossroad of autophagy
DPRs or expanded RNA. Maybe the question should now and inflammation. Interestingly, C9ORF72 also emerges as a
be considered differently: could C9ORF72 loss-of-function new protein linking neurodegeneration, inflammation, and the
mutations be pathogenic by themselves or are they more likely regulation of our immune interaction with the environment.
to be a risk factor? Whether one or the other, in which Remarkably, there is considerable genetic evidence linking the
type of conditions are they likely to play a role? Considering autophagy/lysosomal pathway to FTD/ALS (Deng et al., 2017;
the role of C9ORF72 in autophagy and immune regulation, Stamatakou et al., 2020), as proteins encoded by SQSTM1, OPTN,
as well as its function to regulate the inflammatory reaction TBK1, VCP, UBQLN2, and CHMP2B, all genes involved in ALS
to gut microbiota, maybe coding LOF mutations are more and FTD, also function in this pathway. This and the generation
likely to be found in patients suffering from the dysregulation of new complex models, as well as deeper analysis of patients’
of these functions? For example, LOF mutations could be data, have contributed to re-evaluating the role of C9ORF72
explored in systemic lupus erythematosus and rheumatoid haploinsufficiency in the disease. It must now be considered
arthritis, where it was recently shown that there is an increased when developing therapies that C9-FTD/ALS results from the
proportion of C9ORF72 intermediate expansions (Fredi et al., detrimental combination of impaired cellular homeostasis caused
2019). Alternatively, as the pro-inflammatory effects of C9ORF72 by C9ORF72 partial loss of function and the accumulation of
LOF lead to increased antitumor activity in mice (McCauley toxic gain-of-function entities (expanded RNAs, DPRs, or both).
et al., 2020), it would be interesting to investigate whether While several other strategies targeting the various mechanisms
C9ORF72 germinal or somatic variants can be associated with of the disease are currently under study (Balendra and Isaacs,
differences in survival or disease evolution in patients suffering 2018; Malik and Wiedau, 2020; Ghasemi et al., 2021), ASO
from glioma or other types of cancers. Interestingly, there is therapies are clearly the most advanced, with an ongoing phase
evidence of decreased incidence of some cancers (Fang et al., I trial of BIIB078 lead by Biogen (clinicaltrials.gov Identifier:
2013; Gibson et al., 2016) and overrepresentation of autoimmune NCT03626012). BIIB078 selectively targets C9ORF72 transcript
disorders in patients with ALS (Miller et al., 2013, 2016; Turner variants 1 and 3 that carry the expansion. At first, while the
et al., 2013). In particular, patients with C9-FTD/ALS present contribution of the loss of function to the disease seemed
an abnormally high prevalence of inflammatory arthritides, uncertain, ASOs depleting all C9ORF72 transcripts or selectively
cutaneous conditions, and gastrointestinal disorders (Miller expansion-containing transcripts were developed in parallel
et al., 2016). Finally, further evidence that C9-FTD/ALS patients (Donnelly et al., 2013; Sareen et al., 2013; Lagier-Tourenne
bear the mark of C9ORF72 haploinsufficiency and that its et al., 2013; Jiang et al., 2016; Gendron et al., 2017). Now, the
pathological consequences are part of the disease comes importance of preserving C9ORF72 protein expression has been
from neuropathological examination and RNA sequencing emphasized in a new ASO development (Liu et al., 2021), but
studies. C9orf72 KO mice, C9ORF72 patients iPSC-derived, and strategies aiming to restore control levels of the C9ORF72 protein
C9ORF72± iMNs share similar gene expression changes to the are still missing.
postmortem tissue and blood from C9-FTD/ALS patients, in
particular regarding the immune and endolysosomal pathways
(O’Rourke et al., 2016; Shi et al., 2018; McCauley et al., 2020).
Reactive microglia are frequent in the spinal cords of ALS AUTHOR CONTRIBUTIONS
cases, but microglia with large accumulations of lysosomes were
only observed in the C9-ALS cases (O’Rourke et al., 2016), JS and ML wrote the manuscript. E-GB designed the figures and
strengthening the causal link between immune dysregulation edited the manuscript. All authors contributed to the article and
and endolysosomal impairment in the pathogenesis. Moreover, approved the submitted version.
ubiquitin- and p62-positive inclusion bodies are characteristic of
C9-FTD/ALS cases (Al-Sarraj et al., 2011; Mackenzie et al., 2014),
supporting the strong involvement of the autophagy pathway FUNDING
in the disease. Recently, machine learning applied to RNA
sequencing from the frontal cortex of 34 C9ORF72 expansion JS was supported by a fellowship from PSL Research University,
carriers identified the vesicular transport module as a potent EPHE, Paris, France. Research of the authors is supported by

the European Union Joint Programme on Neurodegenerative ANR-10-IAIHU-06, and the E-Rare Research Program, which is
Disease (JPND) consortium InCure (funding code 01ED1505A), a transnational R&D program jointly funded by national funding
the Fondation Alzheimer, the program “Investissements d’avenir” organizations within the framework of the ERA-Net E-Rare 3.
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10.1002/ana.24469 use, distribution or reproduction is permitted which does not comply with these terms.

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C9ORF72: What It Is, What It Does,

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