Correspondence: M. J. Strong, Rm C7-120, London Health Sciences Centre – University Hospital, 339 Windermere Road, London, ON N6A 5A5, Canada. E-mail: Michael.Strong@Schulich.uwo.ca

(Received 3 November 2013 ; accepted 6 January 2014 )

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2014; Early Online: 1–16

ISSN 2167-8421 print/ISSN 2167-9223 online © 2014 Informa HealthcareDOI: 10.3109/21678421.2014.881377

(encoding Senataxin), VCP (encoding Valosin-Containing Protein), PFN1 (encoding Profi lin 1) , ARHGEF28 (encoding Rho Guanine Nucleotide Exchange Factor; RGNEF), HNRNPA1 (encoding Heterogeneous Nuclear Ribonucleoprotein A1) (3 – 15) or with massive hexanucleotide repeats expansions (i.e. C9orf7 2 encoding C9orf72 protein) (16,17).

While the common thread underlying this listing is a putative role for each in RNA metabolism, thus contributing to the hypothesis that ALS is to a sig-nifi cant extent a disorder of RNA metabolism, it is the presence of both neuronal and glial intracellular inclusions that is considered to be a key neuropatho-logical hallmark of ALS (1,18). Linking these obser-vations is the fi nding that a signifi cant number of the proteins that are commonly seen to form intracel-lular inclusions in ALS are RNA-binding proteins or neuronal cytoskeletal proteins such as neurofi lament (Figure 1). The former includes TDP-43, FUS/TLS, TAF-15, RBM45 and RGNEF (19 – 25). This listing has recently been expanded to include RNA foci generated by the pathological expansion of hexanu-

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neuro-degenerative disease caused by the loss of both upper and lower motor neurons. Affected individuals develop progressive muscle weakness with death due to respiratory failure typically within 3 – 5 years after symptom onset (1). While most cases are clinically sporadic ALS (SALS) with approximately 5% being familial (FALS), this boundary is becoming increas-ingly blurred and new genetic mutations associated with FALS are also being observed in otherwise spo-radic cases (2). Hence, as the number of genetic mutations associated with FALS increases (Table I), so too does our understanding of the pathogenesis of SALS. Among these are genes with point muta-tions in SOD1 (encoding superoxide dismutase 1; SOD1), TARDBP (encoding TAR DNA-Binding Protein 43; TDP-43), FUS (encoding Fused in Sarcoma/Translocated in Liposarcoma; FUS/TLS), UBQLN2 (encoding Ubiqulin 2), OPTN (encoding Optineurin), PGRN (encoding Progranulin), SETX

REVIEW ARTICLE

RNA metabolism in ALS: When normal processes become pathological

CRISTIAN A. DROPPELMANN 1 , DANAE CAMPOS-MELO 1 , MUHAMMAD ISHTIAQ 1 , KATHRYN VOLKENING 1,2 & MICHAEL J. STRONG 1,2

1 Molecular Medicine Group, Robarts Research Institute, Western University, London, Ontario, and 2 Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons. While the exact molecular and cellular basis for motor neuron death is not yet fully understood, the current conceptualization is that multiple aberrant biological processes contribute. Among these, one of the most compelling is based on alterations of RNA metabolism. In this review, we examine how the normal process of cellular response to stress leading to RNA stress granule formation might become pathological, resulting in the formation of stable protein aggregates. We discuss the emerging roles of post-translational modifi cations of RNA binding proteins in the genesis of these aggregates. We also review the contemporary literature regarding the potential role for more widespread alterations in RNA metabolism in ALS, including alterations in miRNA biogenesis, spliceosome integrity and RNA editing. A hypothesis is presented in which aberrant RNA processing, modulated through pathological stress granule formation as a refl ection of either muta-tions within intrinsically disordered or prion-like domains of critical RNA binding proteins, or the post-translational modifi cation of RNA binding proteins, contributes directly to motor neuron death.

Key words: ALS , RNA metabolism , stress granule , neuronal cytoplasmic inclusions , post-translational modifi cations

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2 C. A. Droppelmann et al.

cleotide repeats in C9orf72, postulated to give rise to RAN independent transcription and to serve as binding partners of RNA binding proteins (26,27). RNA binding proteins are multifunctional proteins playing an important role in RNA metabolism throughout its fate from transcription to mRNA maturation and degradation (28). Figure 2 illustrates the RNA metabolism pathway under non-patholog-ical conditions and indicates the steps at which sev-eral of the aforementioned ALS-related RNA binding proteins can act. Specifi cally, TDP-43, FUS/TLS, TAF-15 and RGNEF have been documented to play a role in transcriptional regulation, RNA stability,

microRNA (miRNA) processing and RNA splicing (24,29 – 35). Furthermore, TDP-43, FUS/TLS and TAF15 are capable of interacting with mRNA and form stress granules in the event of cellular stress (36,37).

The focus of this review is to discuss how the normal cellular stress response in ALS, including the formation of stress granules, could become aberrant under certain pathological circumstances, thus inducing the formation of protein aggregates. We examine the role of post-translational modifi ca-tions of RNA binding proteins in the genesis of insoluble protein aggregates and the evidence that

Table I. Functional groupings of genes known to be associated with ALS.

Gene Locus Inheritance Alternative phenotype Reference

EnzymesFIG4 6q21 Unknown PLS (197)SOD1 21q22.11 Dominant * FTLD, PMA (3)

Expanded repeatsATXN2 12q24.1 Dominant PMA (89)C9orf72 9p21.2 Dominant FTLD (16, 17)

RNA metabolism-related proteinsANG 14q11.1-q11.2 Dominant FTLD (198)ARHGEF28 1 5q13.2 Unknown None (14)EWSR1 22q12.2 Unknown None (199)FUS 16q12 Both dominant

and recessiveFTLD (6, 7)

HNRNPA1 12q13.1 Dominant None (15)SETX 9q34.13 Dominant CMT or dHMN (200)TAF15 17q11.1-q11.2 Unknown None (22)TARDBP 1p36.22 Dominant FTLD (5, 201)

Cytoskeleton proteinsNFH 22q12.2 Uncertain None (202)PRPH 12q12-q13 Sporadic None (203)PFN1 17p13.3 Dominant None (11)

Intracellular transport proteinsCHMP2B 3p11.2 Dominant FTLD (204)DCTN1 2p13 Dominant None (205)VAPB 20q13.33 Dominant PMA, PLS (206)VCP 9p13.3 Dominant FTLD (13)

Ubiquitin-related proteinsQSTM1 2 5q35 Dominant FTLD (207)UBQLN2 Xp11.21 Dominant FTLD (8)

Multifunctional proteinsOPTN 10p13 Dominant FTLD (9)

OthersALS2 1 2q33.1 Recessive PLS (208, 209)DAO 12q24 Dominant None (210)EPHA4 2q36.1 Unknown None (211)SPG11 15q21.1 Recessive None (212)Unknown 18q21 Dominant None (213)Unknown 15q15.1 – 21.1 Recessive None (214)Unknown 20p13 Dominant None (215)

Gene abbreviations: ANG: Angiogenin; ARHGEF28: Rho Guanine Nucleotide Exchange Factor, RGNEF; ATXN2: Ataxin 2; C9orf72: Chromosome 9 Open Reading Frame 72; CHMP2B: Charged Multivesicular Body Protein 2b; CMT; Charcot-Marie-Toothe; DAO: D - Amino-Acid Oxidase; DCTN1: Dynactin 1; dHMN: Distal Hereditary Motor Neuropathy; EPHA4: Ephrin Type-A Receptor 4; EWSR1: Ewing Sarcoma Breakpoint Region 1; FIG4: Phosphatidylinositol 3,5 - bisphosphate 5 - phosphatase; FUS: Fused in Sarcoma/Translocated in Liposarcoma; FTLD: Frontotemporal Lobar Degeneration; HNRNPA1: Heterogeneous Nuclear Ribonucleoprotein A1; NFH: Neurofi lament Heavy Chain; OPTN: Optineurin; p62 /SQSTM1: Sequestosome-1; PFN1: Profi lin 1; PLS: Primary Lateral Sclerosis; PMA: Progressive Muscular Atrophy; PRPH: Peripherin; SETX: Senataxin; SOD1: Superoxide Dismutase 1; SPG11: Spatacsin; TAF15: TATA-Binding Protein Associated Factor 15; TARDBP: TAR DNA-Binding Protein 43; UBQLN2: Ubiqulin 2; VAPB: Vesicle-Associated Membrane Protein-Associated Protein B and C; VCP: Valosin-Containing Protein. 1 Could also be classifi ed as guanine-nucleotide exchange factors. 2 Could also be classifi ed as a multifunctional protein. * D90A recessive in Scandinavian population.

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RNA metabolism in ALS 3

a broad range of alterations in RNA metabolism contribute to the pathogenesis of ALS, including alterations in miRNA biogenesis, spliceosome integrity and RNA editing. Taken together, these

observations suggest that RNA metabolism is dysregulated at multiple levels in ALS and that the formation of pathological intracellular inclusions is a critical determinant of motor neuron loss. We

Figure 1. Protein aggregates in motor neurons of ALS patients. Representative images of protein aggregates observed in motor neuron of ALS patients. Arrows point to pathological protein structures including skeins and aggregates that are formed by TDP-43, FUS/TLS, RGNEF and phosphorylated high molecular weight neurofi lament protein (pNFH). The nucleus of the motor neuron immunolabelled using pNFH antibody is marked with asterisk. Immunohistochemistry was performed as described in Keller et al. (2012) (25) using rabbit polyclonal anti-TDP-43 antibody (Proteintech; 1:500 dilution), rabbit polyclonal anti-FUS/TLS antibody (Proteintech; 1:90 dilution), goat polyclonal anti-RGNEF (MediMabs; 1:500 dilution) and mouse monoclonal SMI31 (pNFH) antibody (Sternberger Monoclonals; 1:30,000 dilution). Scale bar � 10 μ m.

Figure 2. Schematic of the RNA metabolism pathway in non-pathological conditions and involvement of ALS related RNA binding proteins. RNA binding proteins of interest in ALS (TDP-43, FUS/TLS, TAF-15 and RGNEF) infl uence critical steps from transcription to splicing, miRNA processing, stress granule (SG) formation and mRNA stability.

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also present a putative pathogenic mechanism that integrates this information.

Stress granules

In response to stressful conditions, eukaryotic cells require a rapid adaptation for survival; they need to produce cytoprotective proteins and conserve energy. The formation of stress granules (SGs) in a series of reversible steps is crucial in the lattermost process. These granules facilitate cell survival by translational arrest of non-essential transcripts and by sequester-ing pro-apoptotic proteins, including Receptor for Activated C Kinase 1 (RACK1), 2-Oxoglutarate and Iron-Dependent Oxygenase Domain Containing 1 (OGFOD1) (38,39), and regulators of cell growth, such as Angiogenin and Caprin-1 (40 – 43).

SGs are cytoplasmic, non-membrane enclosed particles containing different classes of components (reviewed in (44)). The fi rst class consists of initia-tion complexes and includes mRNA transcripts, Translation Initiation Factor 3 (eIF3), Translation Initiation Factor 4B (eIF4B), Translation Initiation Factor 4F (eIF4F), Poly-A Binding Protein 1 (PABP-1) and small ribosomal subunits (44,45). The second class of components consists of RNA-binding proteins with some members of this group linked to translational silencing, such as T-Cell-Restricted Intracellular Antigen-1 (TIA1 Cytotoxic Granule-Associated RNA-Binding Protein or TIA-1) and TIA1 Cytotoxic Granule-Associated RNA-Binding Protein-Like 1 (TIAR) (46), Cytoplasmic Polyadenylation Element-Binding Protein (CPEB) (47), and Ataxin-2 (48). Others are linked to RNA decay, including Tristetraprolin (TTP) (49), Z-DNA Binding Protein 1 (ZBP1) (50), Argonaute proteins (51). Still others are RNA-binding proteins not related to RNA translation or decay, including GTPase Activating Protein (SH3 Domain) Binding Protein (G3BP) (52) and Metastatic Lymph Node 51 (MLN51) (53). The third class of SG compo-nents interacts with core components through ‘ piggyback ’ interactions, such as Steroid Receptor Coactivator-3 (SRC3) (54) and Polysomal RNase 1(PMR1) (55).

SGs are closely related to another class of RNA granules – processing bodies (P-bodies) (42). SGs and P-bodies are both cytoplasmic RNA-protein granules without a membrane (56): both are assem-bled in response to stress (57); growth in size depends on retrograde transport along microtu-bules (58). They are assembled on untranslated mRNAs from disassembled polysomes (59,60), and both share a large number of proteins including eIF4E (43,61), Fas-Activated Serine/Threonine Phosphoprotein (FAST) (43) and 5 ’ -3 ’ Exoribonu-clease 1 (XRN1) (43,62). However, SGs and P-bodies differ in several ways: 1) SGs are larger than P-bodies and appear as more irregular, looser and more granular structures that often contain

regions of cytoplasm, while P-bodies are compact, dense structures (56). 2) SGs only emerge in response to stress (46,59), whereas a small number of P-bodies are observed in unstressed cells (63). 3) SGs, but not P-bodies, usually require eIF2a phosphorylation for assembly upon stress (43). 4) SGs are defi ned by translation initiation factors, including the non-canonical 48S pre-initiation complex (i.e. eIF3 and PABP-1(43,45,46)), while P-bodies are defi ned by components of mRNA decay machinery (i.e. Decapping Factors 1a (DCP1a) and 2 (DCP2) (43,63)). 5) Although SGs and P-bodies can contain the same species of RNAs, SGs contain mRNAs that are polyadenylated (46,59) while in P-bodies, mRNAs lack a poly(A)-tail (43,44,64,65).

SGs and P-bodies are highly dynamic and highly connected structures (66). In S. cerevisiae, the forma-tion of SGs is dependent on the presence of P-bodies (67). In mammalian cells, SGs and P-bodies are formed independently, since abrogation of SGs does not hinder P-bodies assembly and vice versa (43,68). However, P-bodies intermittently and transiently dock with SGs (43), with proteins such as TTP, Transcription Factor IIIB 90 KDa Subunit (BRF1) and Cytoplasmic Polyadenylation Element Binding Protein 1(CPEB1) affecting the duration of contact between granules (43,47). This raises the possibility that messenger ribonucleoprotein particles (mRNPs) may be exchanged between both granules.

Association of ALS-related RNA-binding proteins to SGs

TDP-43 and FUS/TLS are nuclear proteins that rapidly shuttle to the cytoplasm in response to stress and colocalize with SGs (69,70). However, the role of TDP-43 and FUS/TLS in the formation of SGs is unclear. Some studies have suggested that TDP-43 overexpression itself induces SG de novo formation (71,72). However, others have shown that TDP-43 overexpression is not suffi cient for SG formation; rather, an additional stress such as oxidative and osmotic stress or heat shock is required (73,74). Cel-lular models support this latter observation in the presence of normal TDP-43 expression (36,75).

It has been shown that TDP-43 knockdown in HeLa cells decreases the number of cells containing SGs formed after oxidative stress, as well as the size of SGs (76). However, it has also been reported that no change in SG formation is induced by TDP-43 knockdown (71). The same controversial results have been found in the case of TDP-43 mutants. Liu-Yesucevitz et al. (2010) (71) found enhancement of cytoplasmic translocation and SG formation caused by ALS-associated TARBDP mutants (G294A, A315T, Q331K, Q343R), whereas Bentmann at al. (2012) (77) found that TARBDP mutants (A315T, M337V, and G348C) did not affect nuclear localiza-tion and SG formation.

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RNA metabolism in ALS 5

The most comprehensive study on the functional role of TDP-43 in SGs and cellular stress response has been performed by McDonald et al. (2011) (75). These authors showed that TDP-43 and its binding partner hnRNP A2 are components of SGs formed in response to oxidative stress. Importantly, TDP-43 infl uences the stoichiometry of other SG protein components, including TIA-1 and G3BP and con-tributes to SG formation and maintenance. These results place TDP-43 as an active player in the cellular stress response (75).

Furthermore, it has been shown that accumula-tion of TDP-43, HuR RNA Binding Protein (HuR) and Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) in SGs is controlled by Extracellular Signal-Regulated Kinases (ERKs), and that c-Jun N-Terminal Kinases (JNK) specifi cally modulate accumulation of TDP-43 in SGs induced by oxida-tive stress in different cell lines. JNK could directly modulate TDP-43 localization into SGs by acting as a scaffolding protein, or indirectly through phospho-rylation of SG hnRNPs that interact with TDP-43 (74). Finally, it has been observed that 35kDa TDP-43 isoforms generated by alternate in-frame translation start sites leads to the formation of SG in human cells (78) showing that full length TDP-43 is not necessary for its participation in SG formation.

Endogenous and overexpressed FUS/TLS show a re-distribution from the nucleus to the cytoplasm in response to oxidative and osmotic stress, where it colocalizes to SGs (37,79,80) in a manner reminis-cent of TDP-43. However, FUS/TLS knockdown in HeLa, SK-N-SH (76) or HEK293 cells (37) has no impact on the formation of SGs in response to oxida-tive stress, indicating that SG formation does not require endogenous FUS/TLS. The role for FUS/TLS mutants in SG formation is less clear, with con-fl icting reports (81 – 84). The overexpression of FUS/TLS mutants R521G or C-terminal D32 results in SG formation (81). Conversely, others have reported that cellular stress is an essential requirement for SG formation in the presence of a wide range of FUS/TLS mutations (R495X, G515X, R521G, R522G, R524S and P525L) (77,82,83). Recently, Baron et al. (2013) (84) found that R495X FUS/TLS alters the dynamic properties of SGs, delaying their assem-bly, making them more dynamic, larger and, when formed, more abundant. The authors also observed that disassembly was accelerated once the oxidative stress was removed. These results support the gain-of-toxicity mechanism for mutant FUS/TLS in SG assembly and cellular stress response.

These observations highlight the controversial nature of determining the exact role of either TDP-43 or FUS/TLS in SG formation. However, to some degree this controversy relates to the experimental paradigms in which either endoge-nous proteins or overexpressed wild-type or mutated proteins are expressed. In the latter two, and con-sistent with the tenet that we are advancing, these

proteins have an intrinsic capacity to form homo- and hetero-polymers through intrinsically disordered domains. Thus, the consequences of overexpression of such proteins, and then their response to an exogenous stress suffi cient to trigger SG formation, may not mirror fully the physiologi-cal response. The further confounding variable of introducing mutations within such domains or in the RNA binding domains could be predicted to alter intra-granule structure, protein-protein or protein-nucleic acid associations. Caution should thus be exercised in interpreting what are seemingly divergent results.

TDP-43 and FUS/TLS are not the only ALS-associated proteins that have been related to SGs. TAF15, another DNA/RNA-binding protein, colo-calizes with SGs under oxidative stress conditions (37,85,86). However, the absence of TAF15 does not preclude the formation of SGs (37). In addition, heat shock induces the accumulation in SGs of the polyglutamine (polyQ) protein Ataxin-2 (87), itself a predicted RNA-binding protein (88,89). Depletion of Ataxin-2 decreases the number and size of SGs upon oxidative stress, suggesting a possible role for Ataxin-2 in SG formation. Conversely, SG forma-tion does not seem to be affected by an elevated Ataxin-2 concentration but potentially affects the localization of TIA-1. As Ataxin-2 also interferes with P-bodies, it has been proposed that Ataxin-2 impairs the correct ribosome assembly granted by RNA helicases, which is required for the induction of SGs (48,90,91).

Regardless of the putative mechanism, these preceding observations provide strong evidence suggesting that several ALS-related RNA-binding proteins participate to some extent in the formation and/or function of SGs. It is important to note that all RNA-binding proteins mentioned above also form pathological intracellular inclusions in ALS affected spinal cord, suggesting a direct connection between SGs and the formation of pathological inclusions in ALS.

SGs as precursors of pathological aggregates in ALS

Under non-pathological conditions, TDP-43 and FUS/TLS localize into SGs that assemble and disassemble in a fully reversible manner. However, under pathological conditions the normal nuclear-cytoplasmic shuttling of TDP-43 and FUS/TLS is interrupted and formation of insoluble cytoplasmic inclusions of these proteins is observed. This raises an interesting question about this process: does a dysregulation of SGs precede the formation of pathological aggregates in ALS?

Some evidence points to SGs as precursors of pathological inclusions. As already discussed, ALS-related RNA-binding proteins that form cytoplasmic aggregates, including TDP-43 and FUS/TLS, have been shown to associate with SGs. Moreover, it has

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6 C. A. Droppelmann et al.

been shown that endogenous TDP-43 localized to SGs can subsequently form ubiquitin-positive pro-tein aggregates (92). In addition, SG markers such as TIA-1 and PABP-1 have been detected as com-ponents of TDP-43 immunoreactive spinal cord motor neuron cytoplasmic inclusions (NCIs) (71,77,93) suggesting that these aggregates may be derived from SG or from accumulations of SG.

ALS-linked pathological proteins, such as TDP-43 and FUS/TLS, have the tendency to form stable, insoluble protein aggregates. This is because they possess domains within the protein that are intrinsi-cally disordered, capable of converting from a soluble form into a self-propagating aggregate-prone confor-mation, analogous to the domain described in yeast and called ‘ prion-like domains ’ by several authors (94 – 97) (Figure 3). This aggregative behaviour can be enhanced by the presence of mutations, indepen-dent of the type of amino acid generated, in the intrinsically disordered domains (15,71,82,83,98,99). Consequently, aggregation-prone mutations might

lead to an alteration of the SGs thus driving the formation of insoluble aggregates because of an excessive accumulation or over-representation of proteins that are not abundant in the normal consti-tution of the SGs. These aggregation-prone proteins would have the capability to attract more aggrega-tion-prone proteins or even wild-type proteins, as has been described for mutant FUS/TLS attracting wild-type FUS/TLS to SGs after oxidative stress in human neuronal cells (100). It remains to be determined whether such inclusions can also serve to sequester translationally competent mRNA.

Finally, pathological aggregation could also be infl uenced by chronic stress caused by environmen-tal factors such as heavy metals (101), exogenous and endogenous retroviruses (102), as well as oxida-tive stress associated with aging (103), which could even directly affect RNA species (104). This leads then to a complex scenario in which multiple sources of stress could contribute to the formation of insol-uble aggregates from SGs, irreversibly interfering

Figure 3. Putative phosphorylation and SUMOylation sites for TDP-43, FUS/TLS, TAF-15 and RGNEF. Phosphorylation sites were determined using NetPhos 2.0 software. Each line represents a potential phosphorylation site where phospho-serines are indicated in blue, phospho-threonines in green and phospho-tyrosines in red. Putative SUMOylation sites were determined using SUMOsp and SUMOplot softwares. SUMOylation sites are shown with an arrow and a number indicating their amino acid position. Only sites identifi ed using both prediction programmes are indicated. The prion-like regions of TDP-43, FUS/TLS and TAF-15 are indicated as predicted based on the description by King et al. (2010) (96). RGNEF lacks prion-like domains according PAPA software but intrinsically disordered regions according FoldIndex software are indicated.

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RNA metabolism in ALS 7

with the normal cell response to stress and, fi nally, with normal neuronal function.

Post-translational modifi cations as an emerging mechanism related to aggregate formation in ALS

Besides the presence of mutations in intrinsically disordered or prion-like domains and the chronic exposure to cellular stress factors as putative causes of aggregate formation in ALS (vide supra), it is now becoming apparent that alterations in post-translational modifi cations could be critical in the genesis and/or regulation of aggregate formation. In this section, we discuss how diverse post-translational modifi cations could provide an additional layer of complexity in the mechanism of formation of cytoplasmic inclu-sions in motor neurons of ALS patients.

Phosphorylation

The incorporation of phosphate groups into specifi c amino acids through protein kinases (phosphoryla-tion) is one of the most common mechanisms for regulating cellular function, working through a direct and subtle control of proteins involved in different roles. While phosphorylation provides an incredibly specifi c control over cellular functions, its dysregula-tion has been associated with several pathologies.

Among neurodegenerative diseases, a paradig-matic example of altered phosphorylation associated with pathology is tau protein (105). Hyperphospho-rylated tau aggregates (the pathological hallmark of the ‘ tauopathies ’ ) have been found in Alzheimer ’ s disease (106), Pick ’ s disease (107), dementia with Lewy bodies (108), corticobasal degeneration (109), supranuclear palsy (109) and ALS with cognitive impairment (110). These examples, in which hyper-phosphorylated tau forms highly condensed aggre-gates, clearly suggest a pathological role for this protein. It has been determined that hyperphospho-rylation induces self-assembly of tau (111), which is consistent with the aggregate formation observed in the tauopathies. Also, it has been observed that phos-phorylation modulates tau ’ s alpha-helical structure, affecting its polymerization (112) and providing a biochemical mechanism behind pathological tau aggregation. The importance of phosphorylation in tau polymerization and function has been subse-quently confi rmed by experiments using pseudo-hyperphosphorylated tau proteins (113,114).

Similarly, an abnormal phosphorylation of TDP-43 has been observed in frontotemporal lobar degeneration (FTLD) and ALS at the amino acids S379, S403/404 and S409/410 (115,116), with the most common being phosphorylation of S409/410 (115,117). Phosphorylation of S409/410 has been observed to mediate neurotoxicity in a C. elegans model (118) and to increase TDP-43 resistance to degradation and enhance its accumulation into insoluble aggregates in a human neuronal cell line

(119). At a mechanistic level, the role of phospho-rylation in TDP-43 aggregate formation is still not completely understood. In vitro experiments, using human and mouse cells, show that the amount of TDP-43 phosphorylation correlates with the amount of aggregates observed (120). Additionally, in vivo evidence, based in ALS pathology, suggests that phosphorylation of TDP-43 is a requirement for protein aggregate formation (121). It has also been suggested that TDP-43 phosphorylation may simply refl ect an attempt to reduce aggregation or prevent formation of large aggregates, possibly by electrostatic repulsion (120,122). It is probable that the mechanism will be more complex in that, while phosphorylated TDP-43 fragments are resistant to degradation and are preferentially accumulated within protein aggregates, such inclusions are actu-ally reversible and can be cleared through the ubiq-uitin proteasome system in neuronal cells (119). This suggests a dual role for TDP-43 phosphoryla-tion in regulating both TDP-43 aggregation and clearance . Recently, casein kinase I ε (CKI ε ) was identifi ed as a potent TDP-43 kinase that promotes S409/410 phosphorylation, aggregate formation and neurotoxicity (123). Taken together, these data are highly supportive of the hypothesis that phos-phorylation is a mechanism directly involved in the regulation of TDP-43 aggregation observed in ALS pathology.

Considering the relevance of phosphorylation in ALS pathology, we performed an analysis of phos-phorylation sites for different RNA-binding proteins involved in ALS. Using the software NetPhos 2.0 (124) (http://www.cbs.dtu.dk/services/NetPhos), we searched for predicted phosphorylation sites in TDP-43, FUS/TLS, TAF15 and RGNEF. Figure 3 shows this analysis and suggests an enormous poten-tial for phosphorylation of each of these ALS-related proteins. It is particularly striking that a high number of putative phosphorylation sites exist in RGNEF. This observation might suggest that phosphorylation could be a major regulator of RGNEF pathological aggregation in ALS. It is important to note the absence of prion-like domains within RGNEF according PAPA software (http://combi.cs.colostate.edu/supplements/papa/) (125), but the presence of intrinsically disordered regions in its structure accord-ing FoldIndex software (http://bip.weizmann.ac.il/fl dbin/fi ndex) (126) (Figure 3). These intrinsically disordered regions would be predicted to contribute to the aggregation of RGNEF observed in ALS.

SUMOylation

SUMOylation is a post-translational modifi cation characterized by the covalent attachment of small ubiquitin-like modifi ers (SUMOs) to lysine resi-dues in proteins and preventing their degradation by the proteasome. This modifi cation is dynamic and reversible, and SUMOs are capable of forming

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8 C. A. Droppelmann et al.

polySUMO chains (polySUMOylation). In general, SUMOylation can regulate the function of target proteins by affecting protein-protein interactions, therefore resulting in altered subcellular localiza-tion and/or activity (127,128). Nuclear and extra-nuclear roles of SUMOylation appear to be crucial in neuronal function in normal conditions (129). In addition, activation of SUMO conjugation is proposed to be an endogenous, neuroprotective mechanism against stress (130).

It has been described that SOD1 (131 – 133) can be SUMOylated, thereby affecting its stability and increasing its aggregation (134). Moreover, it was observed that overexpression of SUMO1 in human cells leads to SUMO-positive inclusions that colocal-ize with SOD1 (134). Likewise, an overexpressed TDP-43 splice variant lacking the C-terminal domain is located within SUMO-positive nuclear inclusions in human cells and murine primary hip-pocampal neurons (135). In response to heat shock, SUMO polymerizes into polySUMO chains and redistributes among a large numbers of proteins, including TDP-43 in human cells (136).

It is important to highlight that FUS encodes a protein with SUMO1 E3 ligase activity for Ebp1 p42, a protein participating in cell proliferation and differentiation (137). The E3 ligase activity is part of the SUMO conjugation pathway participating in most of the SUMOylations that occur in cells (128). This suggests that an alteration to FUS/TLS func-tion could directly affect normal SUMOylation activity in the cell.

Interestingly, it has been observed that phos-phorylation of several substrates negatively affects their SUMOylation. Phosphorylation of c-jun, Promyelocytic Leukemia Protein (PML), and I κ B α has been correlated to reduced SUMO covalent attachment in human cells (138 – 140). These obser-vations show a cross-regulation between phospho-rylation and SUMOylation, suggesting that both modifi cations could be signifi cant for aggregate formation in ALS.

We used SUMOsp (141) (http://sumosp.biocuckoo.org/) and SUMOplot software (http://www.abgent.com/sumoplot) to predict SUMOylation sites in ALS-related RNA-binding proteins (Figure 3). The prediction shows one SUMOylation site in TDP-43, FUS/TLS and TAF15 and four SUMOylation sites in RGNEF. While the actual SUMOylation profi le of these proteins remains to be tested, these fi ndings sup-port the idea that SUMOylation could have an impor-tant role in regulating ALS-related proteins, a process which may be cross-regulated by phosphorylation.

Methylation

Arginine methylation is a common post-translational modifi cation in eukaryotes. At least three types of protein-arginine methyltransferases (PRMTs) have been described which can transfer methyl groups

from S-adenosyl-L-methionine to the guanidino group of arginine residues (142,143). PRMT1 is the predominant type I enzyme in tissues and contributes to most of the type I protein-arginine methyltransferase activity in mammalian cells (144). Among the substrates described for PRMT1 are RNA-binding proteins (145,146) and transcription factors (147).

FUS/TLS and TAF15 are both substrates of PRMT1. It has been observed that arginine methyla-tion by PRMT1 at arginine-glycine-glycine (RGG) repeat motifs regulates the nuclear-cytoplasmic localization of both RNA-binding proteins (79,148 – 150). Interestingly, in vitro studies using human and murine cells determined that ALS-associated FUS/TLS mutants accumulate in cytoplasmic inclu-sions when they are methylated and in the nucleus when they are hypomethylated (148,149). However, methylation of mutant FUS/TLS at RGG domains does not seem to be required for its assembly into reversible stress granules (84), suggesting that meth-ylation is only required for aggregate formation of mutant FUS/TLS. Additionally, wild-type FUS/TLS was described to be mainly nuclear under normal and hypomethylated conditions (79,148,149), but showing an increased cytoplasmic localization under higher levels of arginine methylation in human cells (79). Similarly, TAF15 accumulates into SGs together with TIA-1 upon inhibition of methylation in human cells (150). All these data suggest that the control of arginine methylation has an important role in regulating the fate of mutant FUS/TLS and TAF15, retaining these proteins in the cytoplasm and possibly contributing to cytoplasmic inclusion formation.

New evidence of alterations in RNA metabolism in ALS

In the last decade, several aspects of RNA metabolism have been extensively described as being altered in ALS. Besides alterations involving SGs (discussed in this review), alterations in mRNA stability and tran-scription regulation have also been described (19). However, new aspects of RNA metabolism alteration in ALS have emerged. In this section we discuss how the pathway of miRNAs is altered in ALS, how TDP-43 aggregate formation affects its role in splic-ing, and how RNA editing is altered in ALS, an aspect closely related to TDP-43 pathology.

miRNA pathway in ALS

Among many other structural and functional simi-larities, TDP-43 and FUS/TLS have the ability to play key roles in canonical miRNA biogenesis (151) (see Figure 2). This property is primarily achieved by their ability to bind nascent and precursor miRNA transcripts (31,32,152). Both TDP-43 and FUS/TLS have been shown by mass spectrometry to bind

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RNA metabolism in ALS 9

Drosha, which mediates the fi rst step in miRNA maturation (153). FUS/TLS is recruited to chroma-tin at sites of miRNA transcription and binds to corresponding primary miRNAs (32). TDP-43 also interacts with Dicer in the cytoplasm to further cleave the precursor miRNA (31). Thus, TDP-43 can infl uence miRNA biogenesis at both a primary and precursor level in the nucleus and cytoplasm, while FUS/TLS only mediates a nuclear miRNA maturation step. Finally, the biogenesis of miRNAs is signifi cantly reduced when TDP-43 or FUS/TLS levels are reduced (32,152), confi rming the function of these two ALS-related RNA-binding proteins in miRNA processing. This new role described for TDP-43 and FUS/TLS is highly important for ALS since both proteins regulate the biogenesis of miR-NAs relevant for neuronal function, differentiation and synapse formation (32,154 – 158). Recently, TAF-15 has been shown to play a role in miRNA biogenesis regulating the expression of the onco-miR-17 locus (35). Although non-canonical miRNA biogenesis pathways are now being increasingly doc-umented (159,160), the role of TDP-43 and FUS/TLS in these pathways is not yet known.

The participation of miRNAs in ALS has only recently been documented. The fi rst clue about miR-NAs involvement in ALS came from studies in mice models with Dicer ablation. Dicer enzyme is part of the miRNA biogenesis and maturation machinery, and its deletion in various cell types of the nervous system has led to neurodegeneration in vivo, indicat-ing that global loss of miRNA biogenesis is detri-mental for neuronal survival (161 – 163). In the context of ALS, our group recently published the fi rst comprehensive examination of a miRNA expres-sion profi le of spinal cord tissues from patients. We have demonstrated a profound dysregulation of miRNAs in spinal cord in SALS compared to con-trols, suggesting that this expression pattern could be a characteristic of this neurodegenerative disorder (164). In addition, altered miRNA expression has been observed in leukocytes (165,166), frontal cor-tex (167), and in both serum and CSF (168) of ALS patients. Supporting a key role for altered target miRNA expression in ALS, the up-regulated expres-sion of a skeletal muscle specifi c miRNA, miR-206, in the SOD1-G93A model of ALS has been associ-ated with a slower rate of disease progression while its absence leads to an accelerated disease progres-sion (169). Consistent with the hypothesis that alter-ations in miRNA will most likely be mediated through a broad array of changes, it has been recently shown in the same model that miR-9 is up-regulated in ven-tral horn cells (170) while the inhibition of miR-155 extends survival (171).

Defect in spliceosome integrity

TDP-43 has critical roles in the regulation of RNA transcription and pre-mRNA splicing, interacting

with proteins such as hnRNP-H and Poly-A Binding Protein Cytoplasmic 1(PABPC-1) (72), or partici-pating in the regulation of mRNAs levels and splicing events observed in mouse brain (172) and human neuronal cells (173). The loss of TDP-43 from the nucleus and abnormal formation of cyto-plasmic aggregates in neurons and glial cells as a pathological hallmark in sporadic ALS suggests an impact on both mRNA and motor neuron survival. Recently, two different groups found that ALS pathogenesis is associated with dysregulation of pre-mRNA splicing (174,175). Both showed that the number of Gemini of coiled bodies (GEMs) decreases in spinal motor neurons of ALS patients. GEMs are nuclear bodies that contribute to the biogenesis of uridine-rich small nuclear RNA (U snRNA), a com-ponent of splicing machinery. TDP-43 associates with GEMs in cultured cells (176), suggesting that TDP-43 contributes to GEMs formation or func-tion. Interestingly, TDP-43 localizes to GEMs through an association with the Survival of Motor Neuron (SMN)-FUS/TLS complex (175). In addi-tion, both groups reported an alteration in snRNA levels in spinal cord of ALS patients. While Ishihara et al. (2013) (174) showed a decrease in the level of U12snRNA in different ALS affected tissues, Tsuiji et al. (2013) (175) found an increase in several U snRNAs including U12snRNA in ALS spinal cord. Differences in the snRNA levels found by the two groups could be explained by the larger number of spinal cord samples used by Ishihara et al. (2013). This group also found a decrease in U11/12-type snRNP (59K) in spinal motor neurons of ALS patients (174). In contrast, Tsuiji et al. (2013) showed an extensive accumulation of spliceosomal Sm-proteins in motor neuron nuclei. Taken together, these data demonstrate a profound alteration in the spliceosome integrity of motor neurons in ALS patients, which could be related to the formation of TDP-43 pathological aggregates in spinal cord in ALS. Interestingly, alteration in TDP-43 splicing function is also a consequence of TDP cross-linking and decreased solubility generated by oxidative stress (177).

Down-regulation of ADAR2

RNA-specifi c adenosine deaminases (ADARs) are enzymes participating in ‘ RNA editing ’ , whose effect is to change the nucleotide sequence of a mature RNA species relative to the encoding DNA sequence through the conversion of Adenosine (A) to Inosine (I) (A to I RNA editing) (178,179) This mechanism involves the regulation of protein coding and non-coding RNAs (179). There are four members of the ADAR family in mammals, ADARs1 – 3 and testis-expressed RNA-binding protein (TENR). ADAR1 and ADAR2 expression is highest in the CNS. ADAR3, which is catalytically inactive, is expressed only in the brain (180). Finally, TENR, likely another

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10 C. A. Droppelmann et al.

catalytically inactive ADAR, is only expressed in the testis (181). Among the catalytically active ADARs, ADAR1 does not have a clear biological role. Con-versely, ADAR2 is a genuine RNA-editing enzyme that has a clearly defi ned key target. ADAR2 is expressed in many tissues; however, the mRNAs it specifi cally edits are expressed within the CNS (182). One of the critical sites that ADAR2 edits is the Q/R site of ionotropic AMPA glutamate receptor subunit 2 (GluR2) mRNA (183).

It has been shown that the expression of ADAR2 is signifi cantly down-regulated in the motor neurons of ALS patients. Moreover, ADAR2 activity is decreased in ALS motor neurons (184). Reduced GluR2 Q/R site editing by ADAR2 appears to be specifi c to sporadic ALS among other neurodegen-erative diseases (185,186). Interestingly, ADAR2-negative motor neurons always show phosphorylated TDP-43 pathology in ALS (187), suggesting a molec-ular link between reduced ADAR2 activity and TDP-43 pathology. Similar results have been observed in aged mouse motor neurons, but decreased ADAR2 levels occurred without a signifi cant reduction in the editing effi ciency of the ADAR2-mediated A-to-I positions (188). Although ADAR2 seems to be rele-vant for ALS, none of the ADARs appears to play a role in its pathogenesis (188). To date, it is unclear whether the reduced ADAR2 expression is a cause or a consequence of TDP-43 pathology. It has also been shown that ADAR2 mRNA is a target for TDP-43 (189) and ADAR2 gene expression is reduced in the brains of TDP-43 knockdown mice (172). These studies suggest a regulatory role for TDP-43 in the expression of ADAR2, which may induce the down-regulation of ADAR2 in ALS motor neurons that lack nuclear TDP-43. However, it has been found that the abnormal processing of TDP-43 is not an upstream event of the down-regulation of ADAR2 in ALS motor neurons (190). While of interest, the functional relationship between ADAR2 and TDP-43 remains to be clarifi ed.

Besides its relation with TDP-43 pathology, the ADAR editing pathway also interacts with the microRNA pathway (191 – 193) adding more com-plexity and interrelation among these processes.

Final remarks and hypothesis

Despite the overwhelming evidence suggesting that ALS is a disease of aberrant RNA metabolism, important aspects of its pathogenesis remain poorly understood. However, new studies regarding the molecular and mechanistic aspects of ALS led us to create a hypothesis describing how this disease progresses and ultimately leads to motor neuron death in affected individuals (Figure 4). External factors such as environmental stress could alter post-translational modifi cations in RNA-binding proteins. These alterations, together with aggregation-prone mutations in RNA-binding proteins, could lead to

the alteration of the formation and function of SGs, concluding in the formation of protein aggregates. The resulting aggregates could dramatically alter RNA metabolism, affecting mRNA stability, tran-scription regulation, transport of RNA granules, splicing, miRNA biogenesis and RNA editing. In this light, it is intriguing that exosomes (extracellular vesicles of endocytic origin) carry functional mRNAs, proteins and miRNAs (194,195). Through exocyto-sis and the subsequent endocytosis by adjacent motor neurons, such translationally competent vesi-cles become interesting candidates to explain ALS disease spreading among motor neurons.

One of the consequences of altered RNA metab-olism could be the formation of RNA-unrelated pro-tein aggregates such as cytoskeleton protein aggregates, like those described for neurofi lament (196). Ultimately, impaired neuronal function as consequence of aberrant RNA processing could lead to motor neuron death either through impaired axonal transport or through a loss or gain of function of these aggregates.

Acknowledgements

We are grateful to Kevin Cheung for his critical reading of the manuscript and Wencheng Yang for his assistance with the immunohistochemistry images. MJS research is supported by the Canadian

Figure 4. Hypothesis. Alterations in post-translational modifi cations, together with aggregation-prone mutations in RNA-binding proteins, lead to an alteration in stress granule assembly, inducing the formation of protein aggregates. Consequently, RNA metabolism is dramatically affected, leading to motor neuron death.

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RNA metabolism in ALS 11

Institutes for Health Research (CIHR), the ALS Society of Canada and the Michael Halls Endowment.

Declaration of interest: The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of the paper.

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