Dysregulated molecular pathways in amyotrophic lateral sclerosis-frontotemporal dementia spectrum disorder.

Published online: September 15, 2017

Review

Dysregulated molecular pathways in amyotrophic lateral sclerosis–frontotemporal dementia spectrum disorder Fen-Biao Gao*

, Sandra Almeida & Rodrigo Lopez-Gonzalez

Abstract Frontotemporal dementia (FTD), the second most common form of dementia in people under 65 years of age, is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD overlaps extensively with the motor neuron disease amyotrophic lateral sclerosis (ALS), especially at the genetic level. Both FTD and ALS can be caused by many mutations in the same set of genes; the most prevalent of these mutations is a GGGGCC repeat expansion in the first intron of C9ORF72. As shown by recent intensive studies, some key cellular pathways are dysregulated in the ALSFTD spectrum disorder, including autophagy, nucleocytoplasmic transport, DNA damage repair, pre-mRNA splicing, stress granule dynamics, and others. These exciting advances reveal the complexity of the pathogenic mechanisms of FTD and ALS and suggest promising molecular targets for future therapeutic interventions in these devastating disorders. Keywords ALS; C9ORF72; FTD; FUS; TDP-43 DOI 10.15252/embj.201797568 | Received 12 June 2017 | Revised 15 July 2017 | Accepted 30 August 2017

Introduction Dementia is one of the most daunting global health challenges of the 21st century. Millions of new cases of this age-dependent condition are diagnosed each year, and more than 100 million people worldwide will be living with dementia by 2,050 (Prince et al, 2013). The cost for the social care and treatment of these patients is increasing rapidly and is estimated to reach $1 trillion next year (Wimo et al, 2017). Unfortunately, the pathogenic mechanisms underlying different forms of dementia are still poorly understood, and effective therapies have yet to be developed. Frontotemporal dementia (FTD) is the second most common form of pre-senile dementia after Alzheimer’s disease. The various forms of FTD, such as behavioral variant FTD, semantic dementia, and progressive nonfluent aphasia, are caused by focal degeneration

of the prefrontal and/or temporal cortex, resulting in changes in personality, social behaviors, or language production, as well as other cognitive impairments (Neary et al, 1998). FTD is also associated with several other neurodegenerative disorders such as progressive supranuclear palsy, corticobasal syndrome, and amyotrophic lateral sclerosis (ALS). ALS, or Lou Gehrig’s disease, is an adult-onset neurodegenerative disorder associated with progressive motor neuron loss in the brain and spinal cord, resulting in paralysis, respiratory failure, and death mostly within 3–5 years after diagnosis (Brown, 1997). Clinical dementia in ALS patients was first reported decades ago along with notable atrophy of the frontal and temporal lobes in some cases (Hudson, 1981; Horoupian et al, 1984). With better diagnosis of FTD (Neary et al, 1998) has come greater recognition of the significant clinical overlaps between ALS and FTD (Vercelletto et al, 1999; Lomen-Hoerth et al, 2002; Strong et al, 2003). The notion that ALS and FTD are related and represent two ends of a spectrum disorder is further supported by a large body of pathological and genetic evidence accumulated during the last decade or so. In 2006, a seminal discovery was made with the finding that TDP-43, encoded by the transactive response DNA binding protein (TARDBP) gene, is a major component of the pathological aggregates in both ALS and FTD brains (Arai et al, 2006; Neumann et al, 2006). TDP-43 pathology—including cytoplasmic inclusions and nuclear depletion of TDP-43—is present in more than 95% ALS cases (Ling et al, 2013). The importance of TDP-43 in ALS is further highlighted by the identification of its disease-causing mutations (Kabashi et al, 2008; Rutherford et al, 2008; Sreedharan et al, 2008; Yokoseki et al, 2008). The term frontotemporal lobar degeneration (FTLD) refers to the predominant pathological conditions in FTD patients; about half of FTLD cases have TDP-43 pathology (FTLD-TDP) (Mackenzie et al, 2010). In addition to TDP-43, genetic mutations and pathological inclusions associated with another DNA/RNA binding protein, fused in sarcoma (FUS), are found in a small percentage of ALS and FTLD cases (FTLD-FUS) (Kwiatkowski et al, 2009; Neumann et al, 2009; Vance et al, 2009). Both TDP-43 and FUS are involved in multiple aspects of RNA metabolism, and the frequent presence of either TDP-43 or FUS pathology in both ALS and FTD strongly suggests a

Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA *Corresponding author. Tel: +1 508 856 8504; E-mail: [email protected]

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Pathogenic mechanisms in ALS and FTD

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Figure 1. Genes with genetic mutations that cause both FTD and ALS. These genes are listed chronologically by year of discovery of their involvement in FTD and ALS. Two major pathological proteins in both FTD and ALS—TDP-43 (encoded by the TARDBP gene) and FUS—are also listed here.

common pathogenic mechanism involving defective RNA metabolism (Ling et al, 2013). The overlap between ALS and FTD is most striking at the genetic level. In the year when TDP-43 was discovered as a major pathological protein in ALS and FTD (Arai et al, 2006; Neumann et al, 2006), a locus on chromosome 9p13.3–21.3 was linked to both disorders (Morita et al, 2006; Vance et al, 2006). Unlike all other ALS/FTD disease genes, the genetic mutation at this locus is a GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 (DeJesusHernandez et al, 2011; Renton et al, 2011), which turns out to be the most common genetic cause in both disorders, providing further strong evidence that they share some common pathogenic mechanisms. Remarkably, most other genes whose mutations cause FTD are also involved in ALS (Fig 1), including valosin-containing protein (VCP) (Watts et al, 2004; Johnson et al, 2010), charged multivesicular body protein 2B (CHMP2B) (Skibinski et al, 2005; Parkinson et al, 2006), ubiquilin 2 (UBQLN2) (Deng et al, 2011), sequestosome 1 (SQSTM1/P62) (Fecto et al, 2011; Rubino et al, 2012), coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) (Bannwarth et al, 2014), optineurin (OPTN) (Maruyama et al, 2010; Pottier et al, 2015), TANK-binding kinase 1 (TBK1) (Cirulli et al, 2015; Freischmidt et al, 2015; Pottier et al, 2015), cyclin F (CCNF) (Williams et al, 2016), and TIA1 (Mackenzie et al, 2017). This large array of ALS/FTD genes offers exciting entry points to dissect the pathogenic mechanisms of these diseases and raises many interesting questions: How do mutations in different genes cause the same disease? What are the common molecular pathways or neural circuits affected by different genetic mutations? And how do mutations in the same gene cause different clinical phenotypes?

Molecular pathological hallmarks in C9ORF72related ALS/FTD C9ORF72 and its protein products were completely uncharacterized when G4C2 repeat expansion was first discovered in 2011. C9ORF72

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mRNA has at least three variants that arise from alternative splicing and the use of different transcription start sites. Over the years, different names for these variants were used, which has caused some confusion in the field. Here, we use the latest nomenclature in the NCBI database. In variant 1 (NM_145005.6, V1) and variant 3 (NM_001256054.2, V3), G4C2 repeats are located in the first intron of C9ORF72. In contrast, transcription of variant 2 (NM_018325.4, V2) starts downstream of G4C2 repeats (Fig 2). The wildtype C9ORF72 allele mostly has fewer than 20–25 copies of these repeats, while in C9ORF72 ALS/FTD patients, there can be up to thousands (DeJesus-Hernandez et al, 2011; Renton et al, 2011; Gijselinck et al, 2012). G4C2 repeat expansion reduces the production of V2 mRNA in C9ORF72 patient cells or tissues (DeJesus-Hernandez et al, 2011; Almeida et al, 2013; Donnelly et al, 2013; Waite et al, 2014; van Blitterswijk et al, 2015). Some laboratories also reported a decrease in total level of V2 and V3 mRNAs that encode full-length C9ORF72 (DeJesus-Hernandez et al, 2011; Gijselinck et al, 2012; Belzil et al, 2013), which is probably primarily due to a decrease in V2, since V2 is expressed at a substantially higher level than V3 (and V1) in human neurons (Sareen et al, 2013; Tran et al, 2015). Decreased C9ORF72 protein levels in patient brain tissues have also been reported (Xiao et al, 2015). Another molecular pathological hallmark in C9ORF72 ALS/FTD is nuclear RNA foci (Fig 2). In many repeat-expansion diseases, expanded repeat RNAs form nuclear aggregates, which in principle can sequester important RNA binding proteins, resulting in misregulation of RNA processing (Mohan et al, 2014). Indeed, both sense and antisense RNAs transcribed from expanded C9ORF72 repeats form mostly nuclear RNA foci in different cell types, including brain neurons and primary fibroblasts from patients, as well as neurons and astrocytes derived from induced pluripotent stem cells (iPSCs) (DeJesus-Hernandez et al, 2011; Almeida et al, 2013; Donnelly et al, 2013; Gendron et al, 2013; Lagier-Tourenne et al, 2013; Lee et al, 2013; Mizielinska et al, 2013; Sareen et al, 2013; Zu et al, 2013; Lopez-Gonzalez et al, 2016). The third pathological hallmark in C9ORF72 ALS/FTD is the accumulation of dipeptide repeat (DPR) proteins, synthesized from both sense and antisense repeat transcripts as well as adjacent intronic sequences, mostly in the cytoplasm of patient neurons and glial cells (Ash et al, 2013; Gendron et al, 2013; Mori et al, 2013a,c; Zu et al, 2013; Mackenzie et al, 2013, 2015; Schludi et al, 2015; Vatsavayai et al, 2016; Fig 2). DPR proteins can be produced from overexpressed, engineered poly(A)+ mRNAs that contain expanded G4C2 repeats but without the AUG start codon, a process named as repeat-associated non-AUG (RAN) translation (Zu et al, 2011, 2013). In a recent study of mouse models of fragile X-associated tremor/ataxia syndrome, translation of expanded CGG repeats in their native molecular context commences at a near-cognate start codon in an upstream open reading frame (uORF) (Sellier et al, 2017), raising the intriguing possibility that at least some endogenous DPR proteins in C9ORF72 ALS/FTD can also be synthesized from spliced or unspliced intronic expanded G4C2 repeats through conventional ribosomal scanning for near-cognate start codons (Gao & Richter, 2017; Sellier et al, 2017). Both RNA foci and DPR proteins are present in several mouse models of C9ORF72 ALS/FTD (Chew et al, 2015; O’Rourke et al, 2015; Peters et al, 2015; Jiang et al, 2016; Liu et al, 2016). However, the toxic molecular species primarily responsible for disease

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Variant 1 NM_145005.6 Variant 2 NM_018325.4 Variant 3 NM_001256054.2

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Figure 2. The C9ORF72 locus and its downstream RNA and protein products. The three C9ORF72 variants are named based on the latest information in the NCBI database. Expanded G4C2 repeat-containing intron and G2C4 repeat-containing RNA generated through an unknown mechanism can form either RNA foci or be translated into DPR proteins. Unspliced pre-mRNA may also serve as the template for DPR production (not shown). C9ORF72 isoform a contains a DENN domain and interacts with SMCR8 in autophagy.

pathogenesis remain to be fully clarified (Gendron & Petrucelli, 2017; Moens et al, 2017). Nonetheless, several major downstream pathways are commonly misregulated in both ALS and FTD caused by C9ORF72 and other genetic mutations, which will be discussed in detail below.

Autophagy Macroautophagy, the most prevalent form of autophagy, is an evolutionarily conserved, highly regulated catabolic process in which some cellular contents such as misfolded proteins and damaged organelles are targeted to lysosomes for degradation. Autophagy has been implicated in many human diseases, including different forms of neurodegeneration (Nixon, 2013; Menzies et al, 2017). This cellular process requires the formation of doublemembrane phagophores triggered by the activation of the ULK1

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(also known as Atg1) complex. The elongation of phagophore membranes is dependent on two ubiquitin-like conjugation systems: the covalent conjugation of Atg5 and Atg12 and lipid conjugation of the soluble form of microtubule-associated protein light chain 3 (LC3-I) that becomes the phagophore-associated form (LC3-II). Phagophore membranes seal through a poorly understood mechanism to form autophagosomes that further mature and fuse with endosomes. Mature autophagosomes then eventually fuse with lysosomes to form autolysosomes for content degradation. Cortical neurons derived from C9ORF72 iPSCs show increased sensitivity to autophagy inhibitors, which is accompanied by an elevated level of p62, a key autophagy adaptor molecule, suggesting a compromised autophagy pathway (Almeida et al, 2013). This defect could be caused in principle by partial loss of C9ORF72, the production of DPR proteins, or both. C9ORF72 is predicted to contain a DENN (differentially expressed in normal and neoplasia) domain characteristic of Rab GDP/GTP exchange

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factors that activate Rab GTPases in various membrane trafficking events (Zhang et al, 2012; Levine et al, 2013). Indeed, C9ORF72 may regulate endocytosis and autophagy (Farg et al, 2014), and a flurry of recent studies showed that C9ORF72 physically binds to SMCR8 (Smith–Magenis syndrome chromosomal region candidate gene eight) (Amick et al, 2016; Sellier et al, 2016; Sullivan et al, 2016; Ugolino et al, 2016; Yang et al, 2016; Jung et al, 2017), which is another DENN domain-containing protein originally identified as a component of the autophagy interaction network (Behrends et al, 2010). Detailed molecular analyses from these studies suggested that C9ORF72 acts at different steps of the autophagy pathway. C9ORF72, WDR41, and SMCR8 form a multimeric protein complex that acts as a GDP/GTP exchange factor for Rab39b, a Rab GTPase with a role in autophagy (Sellier et al, 2016; Yang et al, 2016; Fig 2). C9ORF72 also directly or indirectly interacts with ULK1 (Sellier et al, 2016; Sullivan et al, 2016; Webster et al, 2016; Yang et al, 2016; Jung et al, 2017). The functional significance of the interaction of C9ORF72 with these autophagy regulators seems to depend on the specific cellular context. In one study, the C9ORF72/SMCR8 complex was phosphorylated by TBK1 or ULK1, leading to activation of Rab39b, which has little effect on basal autophagy but impairs autophagosome formation and causes p62+ aggregates to accumulate when autophagy is activated by mTOR inhibition (Sellier et al, 2016). A positive role for C9ORF72 and SMCR8 in autophagy induction is also supported by findings in knockout mice and patient neurons (Sullivan et al, 2016; Webster et al, 2016; Yang et al, 2016). In contrast, other studies reported that SMCR8 negatively regulates autophagy induction (Jung et al, 2016) and that loss of C9ORF72 increases autophagic flux and decreases the p62 level (Ugolino et al, 2016). The discrepancies between these studies may reflect different experimental conditions and cell types used. Further investigations are needed to clarify exactly how autophagy is misregulated in cell types that are directly relevant to C9ORF72 ALS/FTD. Numerous studies show that defects in the autophagy pathway cause neurodegeneration (Nixon, 2013; Menzies et al, 2017). However, neuronal cell loss was absent in C9orf72 knockout mice, although they developed splenomegaly and lymphadenopathy (Koppers et al, 2015; Atanasio et al, 2016; Burberry et al, 2016; Jiang et al, 2016; O’Rourke et al, 2016; Sullivan et al, 2016; Ugolino et al, 2016), suggesting that C9orf72 is not essential for basal autophagy in the nervous system. It is possible that partial loss of C9ORF72 in concert with toxic DPR proteins or other genetic mutations may result in more dramatic disease relevant phenotypes, consistent with the proposed “two-hit” mechanism in ALS/FTD (Pesiridis et al, 2011; Vance et al, 2013; Sellier et al, 2016) as well as the oligogenicity of at least some ALS cases (van Blitterswijk et al, 2012). Interestingly, under starvation conditions, C9ORF72 localizes to the surface of lysosomes, and loss of the mouse gene C9orf72 results in enlarged lysosomes (Amick et al, 2016; O’Rourke et al, 2016). Lysosomal defects are common in neurodegenerative diseases, such as FTD caused by progranulin haploinsufficiency (Kao et al, 2017). Like C9ORF72, tau binds to lysosomal membranes (Wang et al, 2009), and progranulin and its cleavage product granulin are localized inside the lysosome (Kao et al, 2017). It will be informative to further investigate how these disease proteins regulate lysosomal function at the molecular level.

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Many ALS/FTD disease proteins besides C9ORF72 participate directly in the autophagy pathway, suggesting a major pathogenic mechanism in these disorders (Almeida & Gao, 2016). Some disease proteins, such as p62 and optineurin, function as autophagic adaptor proteins. In addition to its function in several signaling events, p62 binds to many substrates for selective autophagy as well to LC3-II on phagophore membranes through its LC3-interacting region (LIR) (Moscat et al, 2016). Some ALS-associated mutations seem to induce a loss of function in a zebrafish model (Lattante et al, 2015), and the L341V-mutant LIR has a greatly reduced ability to bind to LC3-II (Goode et al, 2016a). Other ALS/FTD-associated mutations compromise p62’s ability to activate the signaling mediated by the transcription factor Nrf2, which is critical for cell defense against oxidative stress, especially in neurons (Goode et al, 2016b). Like p62, optineurin interacts with polyubiquitinated cargo through an LIR domain, an ubiquitin-binding domain, and a C-terminal coiledcoil domain. Loss of optineurin reduces the clearance of protein aggregates or defective mitochondria (Korac et al, 2013; Wong & Holzbaur, 2014). ALS/FTD-associated mutations in the ubiquitinbinding domain either reduce the normal function of optineurin by affecting its ability to interact with LC3 or act in a dominantnegative manner to compromise autophagic activity (Wong & Holzbaur, 2014; Shen et al, 2015). Disease mutations in optineurin such as E696K also disrupt the formation of an optineurin/TBK1 complex (Li et al, 2016). Functionally, phosphorylation of optineurin by TBK1 promotes the binding of optineurin to its cargoes such as damaged mitochondria (Heo et al, 2015; Richter et al, 2016). TBK1 also directly binds to and phosphorylates p62 (Heo et al, 2015; Matsumoto et al, 2015) and the C9ORF72/SMCR8 complex (Sellier et al, 2016). Since many disease-associated mutations in p62, optineurin, C9ORF72, and TBK1 cause partial loss of function in these genes, suboptimal autophagy induction seems to be a key pathogenic mechanism in ALS/FTD. This mechanism seems to be especially important in microglia, which are important for synaptic pruning and debris clearance throughout the nervous system (Hong et al, 2016). For instance, progranulin is highly expressed in activated microglia and suppresses aberrant microglial activation during aging (Lui et al, 2016). Moreover, C9orf72-null mice also exhibit an altered immune response in microglia (O’Rourke et al, 2016), suggesting novel molecular links between autophagy and neuroinflammation, in addition to well-documented role for TBK1 (Oakes et al, 2017). The importance of autophagy in ALS/FTD is further supported by rare disease-causing mutations in other genes that also regulate this pathway. A splicing site mutation in CHMP2B, a subunit of the endosomal sorting complexes required for transport (ESCRT-III), causes FTD linked to chromosome 3 (FTD-3) in a large Danish family (Skibinski et al, 2005). Other CHMP2B mutations have also been implicated in ALS (Parkinson et al, 2006; Cox et al, 2010). Expression of FTD-3-associated mutant protein CHMP2BIntron5, which lacks the C-terminal 36 amino acids, causes autophagosomes and multilamellar bodies to accumulation abnormally (Filimonenko et al, 2007; Lee et al, 2007). This phenotype is likely caused by dominant-negative effect of CHMP2BIntron5, since CHMP2BIntron5 binds to other ESCRT-III subunits such as Snf-7/CHMP4B more avidly than to wildtype CHMP2B (Lee et al, 2007); however, the gene itself is not required for neuronal survival or mouse viability (Lee et al, 2007; Ghazi-Noori et al, 2012). Moreover, autophagy

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requires ESCRT-III, which likely regulates the maturation of autophagosomes (Filimonenko et al, 2007; Lee et al, 2007; Rusten et al, 2007). Indeed, a strong genetic modifier of CHMP2BIntron5 toxicity, syntaxin 13, is highly localized on multilamellar bodies induced by dysfunctional ESCRT-III, further suggesting a role for ESCRT-III in autophagosome maturation (Lu et al, 2013). Mutant CHMP2B also causes lysosomal pathology (Clayton et al, 2015), which may indirectly affect the autophagy pathway. The importance of compromised autophagy is further supported by the finding that transgenic mice expressing CHMP2BIntron5 have several hallmarks of FTD-3, such as p62- and ubiquitin-positive aggregates and social behavioral deficits (Ghazi-Noori et al, 2012; Gascon et al, 2014; Vernay et al, 2016; Clayton et al, 2017). VCP, a multifunctional AAA-type ATPase, has a key role not only in ubiquitin-dependent degradation by the proteasome but also in autophagy, endosomal sorting, intracellular signaling pathways such as the NFjB pathway, and other cellular processes (Meyer et al, 2012). For instance, cells expressing FTD-associated mutant VCP are more sensitive to proteasome inhibition and accumulate ubiquitinated proteins (Ju et al, 2008). Similarly, several mutant ubiquilin-2 proteins associated with ALS fail to deliver ubiquitinated cargoes, including aggregated proteins, to proteasomes (Chang & Monteiro, 2015; Hjerpe et al, 2016; Le et al, 2016). Ubiquilin 1 and ubiquilin 2 also have a function in autophagy (N’Diaye et al, 2009; Rothenberg et al, 2010), but it is unclear how disease-related mutations in ubiquilin 2 affect this pathway. Loss of VCP or expression of FTD-associated mutant VCP results in the accumulation of immature autophagosomes, TDP-43 pathology, and reduced mTOR activity (Ju et al, 2009; Tresse et al, 2010; Ching et al, 2013). VCP is also required for autophagic clearance of damaged mitochondria (Kim et al, 2013a), stress granules (Buchan et al, 2013), and ruptured lysosomes (Papadopoulos et al, 2017). This function, too, is compromised by mutations found in ALS and FTD associated with inclusion body myopathy. Taken together, studies on different disease genes strongly suggest that defects in different steps of the autophagy pathway are major contributors to ALS/FTD pathogenesis.

Nucleocytoplasmic transport Compromised autophagy activity may lead to the formation and accumulation of large protein aggregates or inclusions. Under some circumstances, these structures can be neuroprotective, as in the case of intranuclear inclusions in Huntington’s disease (HD) (Saudou et al, 1998; Arrasate et al, 2004). On the other hand, abnormal protein aggregates adversely affect the survival of neuronal and nonneuronal cells through multiple pathways. For instance, cytoplasmic, but not nuclear, toxic aggregates, sequester and mislocalize proteins involved in nucleocytoplasmic transport (Woerner et al, 2016). Active transport of proteins and RNAs through nuclear pore complexes (NPCs) is an essential cellular process in which the small GTPase Ran regulates the interaction between transport receptors, such as the importin family of proteins, and cargo molecules (Gorlich & Kutay, 1999). Each NPC is a large macromolecular machine composed of hundreds of nuclear pore proteins belonging to about 30 different nucleoporins (NUPs) (Beck & Hurt, 2017). Some NUPs, such as the NUP107/160

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subcomplex, have an extremely long half-life in postmitotic cells and are therefore susceptible to age-related deterioration, which may result in loss of nuclear integrity (D’Angelo et al, 2009; Savas et al, 2012; Toyama et al, 2013). Thus, abnormal nucleocytoplasmic transport likely has a major role in various age-dependent neurodegenerative diseases. In one of the earliest studies suggesting a potential defect in nucleocytoplasmic transport in ALS, importin a and b family proteins were mislocalized in a transgenic mouse model expressing mutant SOD1(G93A) (Zhang et al, 2006; Fig 3). Subsequent immunostaining analysis of sporadic and familial ALS cases with SOD1 mutations suggested that some components of NPCs localize discontinuously on the nuclear membrane (Kinoshita et al, 2009). Compromise of the nuclear import pathway may contribute to the cytoplasmic accumulation of TDP-43 or FUS in patients with ALS, FTLD-TDP-43, or FTLD-FUS. For example, in cultured cells, general blockage of nuclear import (Winton et al, 2008) or knockdown of some nuclear import factors such as karyopherin-b1 and transportin 1 (Dormann et al, 2010; Nishimura et al, 2010) causes TDP-43 and FUS to accumulate in the cytoplasm. Moreover, some nucleocytoplasmic transport proteins such as transportin 1 and NPC components show decreased expression or an abnormal subcellular distribution in postmortem CNS tissues of some ALS and FTD patients (Nishimura et al, 2010; Neumann et al, 2012; Takeuchi et al, 2013; Troakes et al, 2013; Zhang et al, 2015; Shang et al, 2017). Loss of normal TDP-43 function reduces the expression of both Ran in FTD patient cells with GRN mutations (Ward et al, 2014) and Ran-binding protein 1 in SH-SY5Y cells (Stalekar et al, 2015), suggesting a potential molecular feedback loop that may in part account for disease progression. Further underscoring the importance of nucleocytoplasmic transport in ALS, an adultonset form of ALS is associated with partial loss of GLE1, an important mediator of RNA export at the nuclear pore (Kaneb et al, 2015). The molecular mechanisms underlying the involvement of nucleocytoplasmic transport in ALS/FTD pathogenesis are further elucidated by recent studies on C9ORF72 repeat expansions (Fig 3). In C9ORF72 iPSC-derived neurons, the nucleocytoplasmic Ran gradient is decreased, and nuclear import of proteins and nuclear export of RNAs are compromised (Freibaum et al, 2015; Zhang et al, 2015). mRNAs also accumulate in the nucleus in cell lines expressing expanded G4C2 repeats (Rossi et al, 2015). To explore the underlying mechanisms, genetic screens in simple model organisms such as fruit flies and yeast were carried out independently by several collaborative teams (Freibaum et al, 2015; Jovicic et al, 2015; Zhang et al, 2015; Boeynaems et al, 2016). In a fly model in which 30 copies of G4C2 repeats with a restriction enzyme site in the middle are expressed in the 50 UTR of the reporter gene GFP (Xu et al, 2013), G4C2 repeat toxicity was suppressed by increased activity of Ran GTPase-activating protein (RanGAP), which stimulates the GTPase activity of Ran and positively regulates nucleocytoplasmic transport (Zhang et al, 2015). Moreover, nuclear import was partially inhibited, and other molecular manipulations of nucleocytoplasmic transport either enhanced or suppressed (G4C2)30 toxicity in the fly (Zhang et al, 2015). Consistent with these findings, many genetic modifiers of the rough eye phenotype that encode key proteins involved in the nucleocytoplasmic transport of both proteins and RNAs, such as

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Figure 3. Nucleocytoplasmic transport defects in different neurodegenerative diseases. In C9ORF72-related FTD/ALS, expanded G4C2 repeats bind to RanGAP and disrupt the Ran gradient; poly(PR) interacts with the FG repeats of nuclear pore proteins; poly(GR) binds to importin; and poly(GA) aggregates sequester RanGAP and HR23. Cytoplasmic and nuclear aggregates formed by TDP-43 or mutant huntingtin (mHTT) sequester some NPC components and factors important for nucleocytoplasmic transport.

Nup50, Ran, and Gle1, were identified in an unbiased genetic screen in a fly model where (G4C2)58 was expressed in the context of poly(A)+ mRNA together with the GFP coding region but without the AUG start codon (Freibaum et al, 2015). The defects in nucleocytoplasmic transport in C9ORF72-related ALS/FTD are likely caused by both toxic repeat RNAs and DPR proteins. RanGAP1 directly binds to G4C2 repeat RNA in vitro, and colocalizes with G4C2 RNA foci in C9ORF72 patient cells (Zhang et al, 2015). The notion that RanGAP1 is a novel RNA binding protein is also supported by a recent eCLIP analysis that revealed a preferential binding of the protein to the CGGCGG motif (Brannan et al, 2016). Thus, G4C2 repeat RNA might compromise nucleocytoplasmic transport by interacting physically with RanGAP1. However, since poly(GR) expression alone also causes severe retinal degeneration in the fly (Mizielinska et al, 2014; Wen et al, 2014; Yang et al, 2015), poly(GR) proteins that are readily detectable in (G4C2)58 flies may be partly responsible for the observed repeat toxicity as well (Freibaum et al, 2015). Indeed, unbiased genetic screens in yeast identified many modifiers that implicate nucleocytoplasmic transport as a key target of poly(GR) and poly(PR) toxicity (Jovicic et al, 2015). A subsequent targeted RNAi screen in Drosophila confirmed that importins, NUPs, and many other

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nucleocytoplasmic transport proteins strongly modify the toxicity of arginine-rich DPRs (Boeynaems et al, 2016). In addition to these genetic studies in model organisms, interactome analysis revealed that some importins, NUPs, and the lamin B receptor bind directly to poly(GR) and poly(PR) proteins (Lee et al, 2016; Lopez-Gonzalez et al, 2016; Shi et al, 2017). In particular, poly(PR) binds to the central channel of the nuclear pore and promotes polymerization of the phenylalanine:glycine domain of NUPs, thereby reducing trafficking through the pore (Shi et al, 2017). These findings point to a specific molecular mechanism by which arginine-rich DPR proteins compromise nuclear pore function and nucleocytoplasmic transport. Poly(GA) aggregates can also sequester nucleocytoplasmic transport proteins such as HR23 and induce an abnormal distribution of RanGAP1 in mice, and overexpression of HR23 or importins or NUPs in cultured cells rescues poly(GA) toxicity (Zhang et al, 2016; Khosravi et al, 2017). Moreover, transport factors are partially sequestered and nucleocytoplasmic transport is disrupted in multiple iPSC and animal models of HD and in HD patient brains (GassetRosa et al, 2017; Grima et al, 2017), further supporting the notion that defects in this cellular pathway are common in different neurodegenerative diseases (Fig 3).

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DNA damage The integrity of genomic DNA in mammalian postmitotic neurons needs to be maintained without new replication for up to decades. DNA is constantly being damaged in many ways. In neurons, a major cause of such damage is endogenous reactive oxygen species (ROS). The production of ROS from mitochondrial respiration and other metabolic processes continuously generates numerous oxidative base modifications and changes in the sugar phosphate backbone of DNA, leading to single-strand breaks (SSBs) and, less frequently, to presumably more deleterious DNA double-strand breaks (DSBs) (Madabhushi et al, 2014). During gene transcription, DNA damage such as spontaneous deamination of dC and dU on the unpaired single-stranded DNA can also lead to DSBs (SkourtiStathaki & Proudfoot, 2014). Even normal neuronal activity increases neuronal DSBs, which is a physiological event, but may also contribute to neurodegeneration under pathological conditions (Suberbielle et al, 2013; Madabhushi et al, 2015). Mammalian neurons and other cell types have evolved sophisticated DNA damage response and repair mechanisms. In postmitotic cells such as neurons, DSBs are repaired primarily by the nonhomologous end-joining pathway, in which DSB sites are recognized and bound by the Ku70-Ku80 complex, which activates DNA-dependent protein kinase (DNA-PK) and recruits a host of factors, including another kinase, ataxia telangiectasia mutated (ATM), to complete the repair (Rulten & Grundy, 2017). ATM phosphorylates multiple proteins involved in the repair pathway, among them the histone H2AX, a widely used DNA damage marker, and p53, whose stabilization may lead to apoptotic cell death (Sancar et al, 2004). Defects in these DNA repair pathways contribute to many neurodegenerative diseases including ALS and FTD (Madabhushi et al, 2014), as highlighted by the involvement of FUS, TDP-43, and DPR proteins in DNA damage and repair. This notion is further strengthened by the identification of NEK1, a serine/threonine protein kinase involved in DNA damage repair and other cellular processes, and its interactor C21ORF2, as risk factors for ALS (Brenner et al, 2016; Kenna et al, 2016; van Rheenen et al, 2016). A well-studied example in which ALS/FTD disease mutations directly affect the DNA damage response is FUS. FUS participates in the formation of a D-loop during DNA DSB repair, suggesting a potential role in genomic stability (Baechtold et al, 1999). Indeed, FUS-deficient fibroblasts from knockout mice have increased sensitivity to ionizing irradiation along with chromosome abnormalities, such as chromosome breakage, centromeric fusion, and aneuploidy (Hicks et al, 2000; Kuroda et al, 2000). FUS has an important and specific role in DNA damage response. First, FUS is recruited within seconds to the site of laser-induced oxidative DNA damage and DSBs (Mastrocola et al, 2013; Wang et al, 2013; Britton et al, 2014; Patel et al, 2015). Second, FUS recruitment is dependent on the interaction between an arginine/glycine-rich domain of FUS and poly(ADP-ribose) polymerase, whose polymeric product is required to recruit many other DNA damage-response proteins, including FUS itself (Mastrocola et al, 2013; Rulten et al, 2014). Third, immobilization of FUS to chromatin can induce the formation cH2AX foci, and thus, FUS may be an early component of the DNA repair machinery (Wang et al, 2013). Fourth, FUS directly interacts with HDAC1, another critical component of the DNA damage response pathway, and this interaction is enhanced by the induction of DNA

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damage (Wang et al, 2013; Qiu et al, 2014). Finally, loss of FUS reduces both HDAC1 accumulation and DNA repair activity at DSBs (Mastrocola et al, 2013; Wang et al, 2013) and increases transcription-associated DNA damage (Hill et al, 2016). Consistent with a key role for FUS in DNA damage repair, the FUS mutants R224C, R514S, H517Q, and R521C show various degrees of deficiency in DNA DSB repair (Wang et al, 2013). Accordingly, the accumulation of several proteins in the DNA repair machinery at DSB sites was reduced in cells expressing some of these mutants (Wang et al, 2013). In particular, FUS-R521C interferes with the normal interaction between FUS and HDAC1 (Wang et al, 2013; Qiu et al, 2014), and FUS-R521C transgenic mice have greatly increased DNA damage in both cortex and spinal cord (Qiu et al, 2014). Although a direct link between elevated DNA damage and eventual neuronal cell death has not been firmly established, a similar phenotype has been observed in neurons from ALS/FTD patients. For instance, iPSC-derived motor neurons of an ALS patient with a frameshift FUS mutation had an age-dependent increase in DNA damage (Higelin et al, 2016), as do motor cortex neurons in ALS patients with FUS-R521C and P525L mutations (Wang et al, 2013) and brain tissues of FTLD-FUS cases where nuclear function of FUS may be reduced (Deng et al, 2014). Phosphorylation on the N-terminal domain of FUS by DNA-PKs causes its translocation into the cytoplasm after DNA damage, suggesting that DNA damage can be the trigger that leads to mislocalization of FUS (Deng et al, 2014). Thus, compromised DNA repair caused by mutant FUS is a major potential pathogenic mechanism in ALS/FTD with FUS mutations or FUS aggregates in the cytoplasm. In contrast to FUS, the potential link between TDP-43 and DNA damage is surprisingly understudied. Although nuclear depletion of TDP-43 is a hallmark of ALS and FTD, only recently have researchers begun to investigate whether loss of TDP-43 normal function increases DNA damage. Like FUS, TDP-43 colocalizes with active RNA polymerase II and the DNA damage repair protein BRCA1 at the sites of transcription-associated DNA damage in U2OS cells (Hill et al, 2016). Moreover, knockdown of TDP-43 increases sensitivity to a-amanitin, a transcription-arresting agent (Hill et al, 2016). In support of a link between TDP-43 pathology and genomic instability, overexpression of human TDP-43 (hTDP-43) in a fly model resulted in nuclear clearance of the protein, derepression of retrotransposable elements, and cell death from increased DNA damage (Krug et al, 2017). The molecular details concerning how TDP-43 contributes to the prevention or repair of transcriptionassociated or other forms of DNA remain to be further investigated. Recently, an age-dependent increase in DNA damage and oxidative stress was reported in iPSC-derived motor neurons of patients with C9ORF72 repeat expansions (Lopez-Gonzalez et al, 2016). DNA damage is also increased in spinal cord neurons of C9ORF72 ALS patients (Farg et al, 2017; Walker et al, 2017). Expression of poly(GR) in iPSC-derived control neurons or neuronal cell lines is sufficient to induce DNA damage (Lopez-Gonzalez et al, 2016; Farg et al, 2017). In addition, reduction in oxidative stress by treatment with antioxidants partially rescues DNA damage both in motor neurons from C9ORF72 carriers and in neurons expressing (GR)80, Thus, oxidative stress may be partly responsible for increased DNA damage (Lopez-Gonzalez et al, 2016). In a Drosophila model of intronic G4C2 repeat expansion, neurodegeneration is not observed but proteins with oxidoreductase activities are enriched among a

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small number of differentially expressed genes (Tran et al, 2015), suggesting that increased oxidative stress might be an early pathogenic event. It is not known whether other molecular pathways also contribute to increased DNA damage in C9ORF72 ALS/FTD.

Pre-mRNA splicing Intron removal from pre-mRNAs is highly regulated and requires the specific interactions of more than 100 proteins with cis elements such as the 50 splice site, the branch point, and the 30 splice site, as well as other regulatory elements. For the majority of pre-mRNAs, spliceosome assembly starts with the recruitment of U1 and U2 small nuclear ribonucleoproteins (snRNPs) and then other complexes. Recently, cryo-electron microscopy structural analyses provided further mechanistic insight into the workings of this highly sophisticated macromolecular machine (e.g., Rauhut et al, 2016; Yan et al, 2017). During pre-mRNA splicing, many other RNA binding proteins (RBPs) such as serine/arginine (SR) and heterogeneous nuclear RNP (hnRNP) family proteins help define the exon/intron boundaries and thereby regulate alternative splicing in a developmental stage- and cell type-specific manner. The importance of regulated pre-mRNA splicing is further underscored by widespread changes in alternative splicing and many genetic mutations in either cis elements or trans factors in various human diseases (Singh & Cooper, 2012; Chabot & Shkreta, 2016). In ALS patients with C9ORF72 repeat expansion, alternative splicing in the cerebellum and frontal cortex was extensively misregulated and more frequent than in sporadic cases (Prudencio et al, 2015). Splicing defects have also been found in lymphoblastoid cell lines derived from some C9ORF72-ALS patients (Cooper-Knock et al, 2015). Interestingly, alternative splicing events in such patients are eightfold to ninefold more frequent in the cerebellum than in the frontal cortex (Prudencio et al, 2015), which is consistent with the more pronounced DPR pathology in the cerebellum of C9ORF72 patients (Mori et al, 2013; Ash et al, 2013; Mackenzie et al, 2015). In light of these findings, it is worth highlighting recent evidence that cerebellum is also involved in cognitive processing (Wagner et al, 2017). The mis-splicing described above may be explained in part by the finding that U2 snRNP proteins are major interactors of poly (GR) and poly(PR) in nuclear extracts and that these DPR proteins block spliceosome assembly and splicing in an in vitro assay (Yin et al, 2017). These proteins are also present in the interactomes of poly(GR) and poly(PR) identified by others (Lee et al, 2016; Lin et al, 2016; Lopez-Gonzalez et al, 2016; Boeynaems et al, 2017). Moreover, some components of U2 snRNP, which normally localize to the nucleus, are mislocalized in the cytoplasm of motor neurons derived from C9ORF72 iPSCs, and nearly 50% of mis-spliced exons identified in C9ORF72 brains by Prudencio et al (2015) are U2 snRNP-dependent (Yin et al, 2017). Thus, U2 snRNP may be a major target of the toxicity of arginine-rich DPR proteins. In C9ORF72-related ALS/FTD, many proteins can bind to G4C2 repeat RNAs in vitro, and some are reported to colocalize with RNA foci in some patient cells (Almeida et al, 2013; Donnelly et al, 2013; Lee et al, 2013; Mori et al, 2013b; Reddy et al, 2013; Sareen et al, 2013; Xu et al, 2013; Cooper-Knock et al, 2014; Haeusler et al, 2014; Rossi et al, 2015; Conlon et al, 2016). Thus, sequestration of

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these proteins may alter pre-mRNA splicing. On the other hand, abundant sense RNA foci produced in a Drosophila model of intronic G4C2 repeat expansion do not induce neurodegeneration (Tran et al, 2015). Moreover, in C9ORF72 patients, sense foci are not associated with any clinicopathological features, and the number of cells with antisense foci correlates inversely with the age of disease onset, suggesting a potential beneficial role (DeJesusHernandez et al, 2017). Interestingly, expanded repeat RNAs alone can undergo gelation in vitro without other proteins through a process called liquid–liquid phase separation, which may be also responsible for RNA foci formation in human cells (Jain & Vale, 2017). Thus, it remains to be determined whether enough of any protein can be sequestered by RNA foci to affect splicing in a detrimental way in C9ORF72 patients. Long before TDP-43 was identified as an FTD/ALS pathological protein (Neumann et al, 2006) and disease-causing FUS mutations were identified in ALS and FTD (Kwiatkowski et al, 2009; Vance et al, 2009), these proteins were known to have key roles in premRNA splicing (e.g., Yang et al, 1998; Buratti et al, 2001). Indeed, TDP-43 binds to thousands of different RNAs in mammalian cell lines and the brain, from microRNAs to long introns (Polymenidou et al, 2011; Sephton et al, 2011; Tollervey et al, 2011; Xiao et al, 2011; Colombrita et al, 2012; Li et al, 2013a; Narayanan et al, 2013; Fan et al, 2014). Binding of TDP-43 to very long introns (> 100 kb) affects the stability of these pre-mRNA targets, and binding to exon– intron junctions and some intronic sequences regulates alternative splicing (Polymenidou et al, 2011; Tollervey et al, 2011; Passoni et al, 2012). This regulation is partly mediated by UG-rich sequences that interact with both RNA recognition motifs of TDP-43 (Passoni et al, 2012; Lukavsky et al, 2013). Similarly, FUS binds along the whole length of thousands of nascent RNAs and regulates alternative splicing of RNAs that are mostly distinct from those dependent on TDP-43 (Lagier-Tourenne et al, 2012; Rogelj et al, 2012). Loss of TDP-43 results in aberrant mRNAs containing cryptic exons that often lead to premature stop codons and nonsensemediated decay (Ling et al, 2015). This mode of regulation targets a large number of transcripts, and many of these nonconserved cryptic exons are cell type specific (Tan et al, 2016; Jeong et al, 2017) and may occur in Alzheimer’s brains in addition to ALS/FTD cases (Sun et al, 2017). Since the majority of ALS and FTD cases show TDP-43 pathology, including depletion of TDP-43 in the nucleus, loss of normal TDP-43 function in splicing is likely to be a key pathogenic factor in disease progression. On the other hand, mutant TDP-43 can also affect splicing in a gain-of-toxic-function manner. For instance, mutant TDP-43(Q331K) expressed in vivo can either enhance or impair splicing in an RNA target-dependent manner and cause motor neuron disease without loss of nuclear TDP-43 (Arnold et al, 2013). Mutant TDP-43(M337V) also causes mislocalization of the splicing factor PSF and NeuN in the cytosol and misregulation of PSF/NeuN-dependent splicing, which is essential for neuronal function (Wang et al, 2015). Similarly, FUS interacts with U1, U11, and U12 snRNPs, which are mislocalized to the cytosol by mutant FUS (Sun et al, 2015; Yu & Reed, 2015; Yu et al, 2015; Reber et al, 2016). Moreover, both loss of function and gain of toxic function in hnRNP A2/B1 likely contribute to defects in alternative splicing in patient fibroblasts and iPSC-derived motor neurons harboring the ALS-associated mutation D290V (Martinez et al, 2016). These

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RBPs with LCD RRM

RRM

Liquid–Liquid Phase Separation

LCD

[RNA] [Salt] Temperature pH

Gly Pro Arg Poly(GR) Poly(PR)

…GGGGGGNNGGGGGYGGGGGNYGGGNGGGGGY… Examples

• FUS • TDP-43 • hnRNP A1 • hnRNP A2B1

Dispersed proteins

• TIA1 • TIAR • G3BP1

Liquid droplets

TDP-43 toxicity i Continuous stress (e.g. mutation) Translation

Irreversible aggregates

PERK elF2α TIA1

AAA

Mutant FUS

FUS

LCD LCD

AAAA PABP

7 mG

AAA

A

AA

AA

A

A

AA

AAAA AA AA A AA

ATX-2 Mutant Ataxin-2

Individual Components

AA

A

m 7G 7G

m

© EMBO

m7G

OR

7 mG

AA AA

G3BP1

AA

AA

C

m

FUS

7 AAA m G A m7G m7 AAA G A AAA A AAAA m 7G

m 7G

A

7G

AAA A

AAAA

PUM 2

AAAA PABP

AA AA

mG 40S LCD

7

G

mG m 7G

7 mG

TDP-43

RBP

7

7 m G

LCD

A A AA A A AA

7G 7 m mG

m 7G

Poly(PR)

e or

AA

AA

LCD

“stalled”

AA

7 mG

AA

h

A

LCD

P

A

m7G

m 7G

40S

elF2α

AAAA RBP PABP

S

LCD

FMRP

m7G

l el

m7

Poly(GR)

Stress Granule (SG)

Figure 4. The roles of different FTD/ALS disease proteins in SG formation. Upper left: An artificial sequence is presented to illustrate the amino acid composition of LCDs. Upper right: A schematic representation of liquid–liquid phase separation in vitro that can be influenced by salt concentrations or RNA concentrations or genetic mutations. Center: TDP-43 aggregates or inhibition of general translation by arginine-rich DPR proteins can lead to elF2a phosphorylation which promotes SG formation. Mutant proteins in FTD/ALS such as FUS or ataxin-2 promote the formation of irreversible aggregates and thereby prevent the dissociation of SGs. Lower left: A schematic representation of some individual components of SGs. LCD: LCD-containing RBPs. Lower right: A schematic representation of an SG with a densely packed core.

results underscore the important role of misregulation of splicing by different disease proteins in ALS/FTD.

Stress granules In addition to multifunctional roles in the cellular processes mentioned above, several disease proteins in ALS/FTD are also associated with stress granules (SGs). SGs are non-membranebound, cytoplasmic ribonucleoprotein (RNP) granules composed of mRNAs, translation initiation factors, 40S ribosomes, and many other RBPs (Panas et al, 2016; Protter & Parker, 2016). SGs of up to a few micrometers can be induced by various cellular stresses that inhibit translation initiation, such as oxidative stress, glucose starvation, mitochondrial dysfunction, and viral infection. These cytoplasmic structures can either disassemble upon release of cellular stress

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or be degraded by the autophagy pathway. As a stress response mechanism, SG formation is likely to be beneficial under certain circumstances. On the other hand, since SGs may be mechanistically linked to aggregate formation in several neurodegenerative diseases, dysregulated formation or clearance of SGs may be a key pathogenic mechanism (Wolozin, 2012; Li et al, 2013b; Ramaswami et al, 2013). The recent identification of TIA1 mutations in ALS/FTD that alter SG dynamics further highlights the importance of this pathway in neurodegeneration (Mackenzie et al, 2017). One of the best-illustrated examples of the potential link between SGs and aggregate formation is FUS. In response to oxidative stress or heat shock treatment, endogenous FUS is targeted to SGs in HeLa cells (Andersson et al, 2008). Some ALSassociated mutant FUS proteins (such as R521G) show increased cytoplasmic localization and induce SG formation with or without further stress; the increase is probably dependent on the level of

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ectopic expression (Bosco et al, 2010; Gal et al, 2011; Sun et al, 2011). Indeed, in fibroblasts or iPSC-derived motor neurons containing endogenous ALS-associated FUS mutations, FUSpositive SGs accumulate only after stress treatment or neuronal aging (Japtok et al, 2015; Lenzi et al, 2015; Lim et al, 2016). The recruitment of endogenous or mutant FUS to SGs and its interaction with other SG components are dependent on RNA (Andersson et al, 2008; Bentmann et al, 2012; Daigle et al, 2013; Shelkovnikova et al, 2013; Vance et al, 2013). Another determinant of SG formation is the prionlike domain located at the N-terminus of FUS (King et al, 2012), a low-complexity domain (LCD) also present in many RBPs and other proteins that mediates liquid–liquid phase separation, a process thought to underlie the formation of many membraneless organelles such as SGs (Burke et al, 2015; Lin et al, 2015; Molliex et al, 2015; Murakami et al, 2015; Patel et al, 2015; Fig 4). ALS/FTD-associated mutations in the LC and non-LC domains of FUS further promote the transition of liquid droplets into fibrillar aggregates in vitro and therefore may prevent the proper dissociation of FUS-containing SGs in living cells (Murakami et al, 2015; Patel et al, 2015). These findings provide a plausible molecular mechanism underlying RBP aggregates in ALS/FTD. TDP-43 also associates with SGs through an interaction with the core SG protein TIA-1, and some but not other disease-related mutations in TDP-43 enhance SG formation (Freibaum et al, 2010; Liu-Yesucevitz et al, 2010; Dewey et al, 2011; McDonald et al, 2011; Bentmann et al, 2012). Similarly, disease mutations in hnRNP A2 and hnRNP A1 also increase SG formation (Kim et al, 2013b). Early observations that some TDP-43-positive inclusions in patient tissues may contain SG markers support the notion that dysregulated SG dynamics contribute to TDP-43 proteinopathies (Liu-Yesucevitz et al, 2010; Bentmann et al, 2012). Under certain experimental conditions, blocking SG assembly through G3BP knockdown increases cell death under stress, indicating a beneficial role for SG formation (Aulas et al, 2012). In contrast, many genes that affect SG formation are strong modifiers of TDP-43 toxicity in yeast and Drosophila (Kim et al, 2014). In particular, TDP-43 toxicity increases the phosphorylation of elF2a in flies, and TDP-43 toxicity is greatly mitigated by genetic reduction in PEK, which phosphorylates elF2a and is the fly homolog of mammalian PERK, or by pharmacological treatment of flies or mammalian neurons with the PERK inhibitor GSK2606414 (Kim et al, 2014). Because reduction in PERK activity and elF2a phosphorylation blocks SG formation, these results imply that prolonged SG accumulation is detrimental and that partial inhibition of this process may have therapeutic values. Consistent with this notion, either genetic reduction or antisense oligonucleotide (ASO) knockdown of the SG component ataxin-2 in the central nervous system increased the survival of TDP-43 transgenic mice (Becker et al, 2017). Recent studies have implicated abnormal SG dynamics in C9ORF72-related ALS/FTD. Arginine-rich DPR proteins suppress global translation and thus may indirectly trigger SG formation, likely reflecting their preferential direct physical interaction with ribosomal proteins (Wen et al, 2014; Kanekura et al, 2016; LopezGonzalez et al, 2016). These DPR proteins are also associated with many other RBPs (Lee et al, 2016; Lin et al, 2016; LopezGonzalez et al, 2016; Boeynaems et al, 2017; Yin et al, 2017),

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including LCD-containing proteins that mediate the formation of membraneless organelles such as SGs, suggesting a direct role in SG dynamics. Indeed, biochemical studies indicate that LCDs are direct binding targets of poly(PR) and this interaction is polymerdependent (Lin et al, 2016). Ectopic overexpression of poly(GR) or poly(PR) induces spontaneous SG assembly, a process that requires both elF2a phosphorylation and G3BP (Lee et al, 2016; Boeynaems et al, 2017). Moreover, poly(GR) and poly(PR) themselves undergo liquid–liquid phase separation in vitro and enhance the multivalent interactions of the liquid phase of SGs in living cells (Lee et al, 2016; Boeynaems et al, 2017). Since poly (PR) is not detectable in SGs (Lee et al, 2016), it will be interesting to investigate the molecular mechanism by which argininerich DPR proteins alter SG dynamics. RNA or protein products of expanded G4C2 repeats may affect the normal function of other neuronal RNP granules as well (Burguete et al, 2015). It will be important to further investigate the relationship between endogenous DPR proteins and dynamics of various RNP granules in neurons of C9ORF72 patients.

Conclusion During the last decade or so, tremendous progress has been made in understanding the common pathogenic mechanisms in both ALS and FTD. The identification of disease genes has led to breathtaking advances in knowledge about underlying molecular and cellular pathways by both translational and basic scientists in multiple related fields. As in better studied neurodegenerative diseases, it is certain that many other dysregulated common downstream pathways in the ALS-FTD spectrum disorder besides those described here will be identified in the foreseeable future. For the sake of therapy development, it is important to determine which pathway or pathways drive disease initiation and how different pathways affect each other during disease progression. Many current studies rely on overexpression of disease RNA or proteins in cellular or animal models. However, the extent to which molecular insights obtained from these studies can be translated in patient situations remains to be seen. More effort should be focused on studying the detrimental effects of disease genes expressed in their native genetic and molecular contexts. Finally, it is still not known how same mutations such as C9ORF72 repeat expansion cause distinct clinical phenotypes in different patients or why their penetrance differs in various populations. Unique genetic modifiers or environmental influences remain to be identified in years to come.

Acknowledgements We thank Li Qiu for help with EndNote. This work is supported by grants from the National Institutes of Health (R01NS057553 and R01NS079725 to F.-B.G.). F.-B.G. is also supported by the MDA Foundation, the Packard Center for ALS Research, and the Target ALS Foundation. S.A. is supported by the ALS Association, the Frick Foundation for ALS Research, and the Alzheimer’s Association (NIRG-396129).

Conflict of interest The authors declare that they have no conflict of interest.

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Dysregulated axonal RNA translation in amyotrophic lateral sclerosis.

Roots to start research in amyotrophic lateral sclerosis: molecular pathways and novel therapeutics for future.

CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia.

C9orf72 expansions in frontotemporal dementia and amyotrophic lateral sclerosis.

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Tale of two diseases: amyotrophic lateral sclerosis and frontotemporal dementia.

The emerging roles of microRNAs in the pathogenesis of frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) spectrum disorders.

Comprehensive rehabilitative care across the spectrum of amyotrophic lateral sclerosis.

The Spectrum of C9orf72-mediated Neurodegeneration and Amyotrophic Lateral Sclerosis.

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Histocompatibility antigens in amyotrophic lateral sclerosis and parkinsonism-dementia on Guam.

The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia.

Neuropsychological study of amyotrophic lateral sclerosis and parkinsonism-dementia complex in Kii peninsula, Japan.

Analysis of the CHCHD10 gene in patients with frontotemporal dementia and amyotrophic lateral sclerosis from Spain.

Cellular immunity in Guamanians with amyotrophic lateral sclerosis and Parkinsonism-dementia.

Hippocampal and neocortical ubiquitin-immunoreactive inclusions in amyotrophic lateral sclerosis with dementia.

Profiling Speech and Pausing in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).

Frontotemporal Dementia.

Depression in amyotrophic lateral sclerosis.

Neuroimaging in amyotrophic lateral sclerosis.