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Phase to phase with TDP-43 Yulong Sun, and Avijit Chakrabartty Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01088 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 24, 2017

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Phase to phase with TDP-43 Funding Source Statement: Canadian Consortium of Neurodegeneration and Aging (CCNA), Canadian Institute of Health Research (CIHR), ALS Society of Canada (ALS Canada), Alzheimer Society of Canada (ASC) Yulong Sun1 and Avijit Chakrabartty1, 2* 1: Department of Medical Biophysics, University of Toronto. 2: Department of Biochemistry, University of Toronto. *To whom correspondence may be addressed: Dr. Avi Chakrabartty. University Health Network. Princess Margaret Cancer Research Tower. 101 College Street, Room 4-305. Toronto, Ontario, M5G1L7. Phone/Fax: (416)-581-7554. Email: [email protected]

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Biochemistry

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LIST OF ABBREVIATIONS Aβ, amyloid-beta; AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; bvFTLD, behavioral variant FTLD; CDK6, cyclin-dependent kinase 6; CFTR, cystic fibrosis transmembrane regulator; CTD, C-terminal domain; fALS, familial ALS; FTD, frontotemporal dementia; FTLD, frontotemporal lobar degeneration; FUS/TSL, Fused in Sarcoma/Translocated in Sarcoma; HIV-1, human immunodeficiency virus type 1; hnRNP, heterogeneous nuclear ribonucleoprotein; LLPS, liquid-liquid phase separation; lncRNA, long non-coding RNA; LTR, long terminal repeat; ncRNA, non-coding RNA; NES, nuclear export signal; NLS, nuclear localization signal; NMD, nonsense mediated decay; NTD, N-terminal domain; PLAAC, prionlike amino acid composition; PNFA, primary non-fluent aphasia; pRb, retinoblastoma protein; PrLD, prion-like domain; PrP, prion protein; RNP, ribonucleoprotein; RRM, RNA recognition motif; sALS, sporadic ALS; SAXS, small angle X-ray scattering; SD, semantic dementia; SG, stress granule; TAR, transactive response; TDP-43, TAR element DNA binding protein of 43 kDa; TTR, transthyretin


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ABSTRACT TDP-43 is a dimeric nuclear protein that plays a central role in RNA metabolism. In recent years, this protein has become a focal point of research in the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, as pathognomonic inclusions within affected neurons contain post-translationally modified TDP-43. A key question in TDP-43 research involves determining the mechanisms and triggers that cause TDP-43 to form pathological aggregates. This review gives a brief overview of the physiological and pathological roles of TDP-43 and focuses on the structural features of its protein domains and how they may contribute to normal protein function and to disease. A special emphasis is placed on the C-terminal prion-like region thought to be implicated in pathology, as it is where nearly all ALS/FTD-associated mutations reside. Recent structural studies on this domain revealed its crucial role in the formation of phase-separated liquid droplets through a partially populated α-helix. This new discovery provides further support for the theory that liquid droplets such as stress granules may be precursors to pathological aggregates, linking environmental effects such as stress to the potential etiology of the disease. The transition of TDP-43 between soluble, droplet, and aggregate phases and the implications of these transitions on pathological aggregation are summarized and discussed.


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INTRODUCTION Recent advances in modern medicine have led to dramatically longer lifespans in the human population. Ironically, through this increase in life expectancy, the imperfections in our evolutionary mechanisms that maintain the robustness of proteins appear to have been exposed. Molecular evolution has produced tremendously sturdy proteins that have little to no turnover in an organism’s lifetime such as proteins of the lens. However, even these proteins can accumulate modifications and aggregate in individuals of advanced age, leading to the disruption of optical properties of the lens and senile cataracts.1 As suggested by Christopher Dobson, the rapid increase in human life expectancy may have out-paced molecular evolution, giving rise to numerous diseases that are caused by protein misfolding and aggregation.2,3 Many of these diseases, such as senile cardiac amyloidosis caused by the deposition of the protein transthyretin (TTR) in heart tissue, and certain forms of cancer where the key regulatory protein p53 is known to aggregate, have only emerged in the past century.4–6 Neurons seem particularly vulnerable to late-onset protein misfolding diseases, possibly due to the lack of neuronal cellular turnover and age-dependent deficits in protein quality control. Indeed, the abnormal accumulation of protein in affected neurons has emerged as a common hallmark of neurodegenerative diseases. Identification and characterization of major protein components of these aggregates has often lead to transformative breakthroughs in uncovering the mechanisms of disease pathogenesis. Classic examples include the discovery of the prion protein (PrP) and formulation of the proteinonly hypothesis of prion disease and the identification and study of the amyloid-beta (Aβ) peptide in formulating the amyloid cascade model of Alzheimer’s disease (AD).7,8 This review will focus on the molecular mechanisms of misfolding and aggregation of transactive response (TAR) element DNA binding protein of 43 kDa (TDP-43), a key protein found in neuronal


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inclusions of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). ALS/FTD is a spectrum disorder ALS, also known as Lou Gehrig’s disease, is a devastating neurological disorder characterized by the progressive loss of lower motor neurons in the anterior horn of the spinal cord and upper motor neurons in the motor cortex, leading to initial paralysis of extremities followed by fatal paralysis of the diaphragm.9,10 ALS has a prevalence of 3.9 cases per 100,000 in the United States.11 The disease is incurable and progresses rapidly, resulting in an average life expectancy of 3-5 years after disease onset, usually occurring in mid-adult life.10,12 90% of ALS cases are sporadic (sALS) with unknown etiology, while 10% of cases have family history (fALS), although the two are clinically indistinguishable. Distinct from ALS, FTLD, presented clinically as frontotemporal dementia (FTD), belongs to a broad range of disorders leading to progressive cognitive, behavioral, and/or language deficits.13 It is the second most common form of dementia in people younger than 65 years of age after AD.14 Clinically, FTD can present as behavioral variant FTLD (bvFTLD) with predominantly behavioral changes, primary non-fluent aphasia (PNFA), affecting speech, or semantic dementia (SD), affecting comprehension.15 Approximately 30-50% of FTD cases show family history, with the majority of cases caused by mutations in three major genes: microtubule protein tau (MAPT), progranulin (GRN) and chromosome 9 open reading frame 72 (C9ORF72).16–21 To date, a large number of genes have been identified to be causative for ALS and FTD (reviewed in 22). Some of them are related to clinically pure ALS or FTD, but a large portion of genes are found in both diseases, suggesting a common disease mechanism. Additionally, despite distinction in disease presentation, co-occurrences of ALS and FTD have been widely reported.


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About 15% of FTD patients develop ALS and about 50% of ALS patients show some signs of cognitive impairment, meeting diagnostic criteria of FTD in about 5% cases, indicating significant clinical overlap.15,23 A major breakthrough linking the two diseases occurred in 2006, where TDP-43 was found to be ubiquitinated, hyperphosphorylated, and fragmented in neuronal inclusions of patients with sporadic and familial forms of ALS and FTD.24,25 Due to their clinical, genetic and pathological overlaps, it is now believed that the two diseases belong to a spectrum of disorders termed TDP-43 proteinopathies and that the disease phenotype arises from differences in the primary sites of neurodegeneration: motor neurons in ALS and cortical neurons in FTD. Despite the numerous genes involved in the disease spectrum, the fact that aggregates of TDP-43 have now been found in 97% of cases of ALS and 45% of cases of FTD suggests that it is directly linked to the disease mechanism.26 The study of TDP-43’s folding and aggregation is therefore invaluable to determining the cause of ALS/FTD. The identification of TDP-43 as a major component of ALS/FTD pathology catapulted the investigation of the protein’s structure, function (native and pathological), and biophysical characteristics. Since the discovery of its involvement in ALS/FTD ten years ago, over 1700 publications on TDP-43 have been produced. The biophysical features of this multi-domain dimeric protein and how the behavior of these domains may contribute to disease pathogenesis are discussed herein.

PHYSIOLOGICAL FUNCTIONS AND PATHOBIOLOGY OF TDP-43 Physiological functions of TDP-43 TDP-43 was first discovered as a ubiquitously expressed cellular factor that binds to the TAR element, an element in the long terminal repeat (LTR) region of human immunodeficiency virus type 1 (HIV-1) which is critical for the control of gene expression in the virus.27 The 43 kDa protein was named for its function as TAR DNA binding protein of 43 kDa upon its initial


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discovery. The role of the protein in human disease was first studied in the early 2000, where it was found to regulate splicing of the gene coding for the cystic fibrosis transmembrane regulator (CFTR) protein exon 9 by binding to a region near its 3’-splice site consisting of repeats of UG nucleotides.28 TDP-43 binds preferentially to TG-rich and UG-rich sequences (vide infra) and appears to be involved in a number of roles in splicing and transcription.28–33 To date, TDP-43 has been found to participate in a large number of nuclear and cytoplasmic functions as it is shuttled between the two cellular milieus.34 In brief, TDP-43 is known to be involved in premRNA processing and splicing, microRNA processing and regulation, control of long noncoding RNA (lncRNA) and non-coding RNA (ncRNA) expression, mRNA transport, mRNA stability through recruitment into stress granules (SGs) and mRNA translation (reviewed in 35). TDP-43 is also involved in various aspects of cell proliferation and apoptosis. It regulates the phosphorylation of retinoblastoma protein (pRb), a tumour suppressor dysfunctional in several major cancers, through the repression of cyclin-dependent kinase 6 (CDK6) expression.36 Mutant forms of TDP-43 are also more prone to cause neural apoptosis in chick embryos.37 Disruption to cell cycle and apoptotic proteins by TDP-43 mutations may implicate the protein in neuronal cancers. Due to its many functions, TDP-43 levels are tightly regulated through a negative feedback loop. TDP-43 binds to the 3’UTR of its own mRNA, leading to nonsense mediated decay (NMD)-independent mRNA degradation and reduction in TDP-43 production.38–40 Recent findings also suggest that RNA/DNA binding modulates TDP-43 solubility.41–43 In cells, TDP-43 localizes to the nucleus in both diffuse and speckled distributions.44 During stress response to heat shock or sodium arsenite, TDP-43 coalesces into SGs in the cytoplasm and modulates SG assembly and dynamics.45–49 Alterations to these SG processes have been suggested to play a key role in TDP-43 aggregation and pathology.50–52 The involvement of SGs also links


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environmental effects to protein aggregation, which may provide an explanation for the mostly sporadic nature of ALS (vide infra). Given these crucial RNA-related functions of TDP-43, it is not surprising that homozygous knockouts of TDP-43 are embryonically lethal in mouse models while heterozygous mice are not as affected, possibly due to the ability for TDP-43 to tightly control its expression levels through negative feedback.53,54 The gene encoding TDP-43 protein (TARDBP) is also highly conserved in human, mouse, Drosophila melanogaster, and Caenorhabditis elegans, with very low rates of divergence between the four eukaryotes, suggesting that TDP-43 likely has crucial roles as a gene regulator.55 TDP-43’s many functions have led to the suggestion that disruptions to TDP-43 expression level and function is at least partially responsible for neurotoxicity. Pathological functions of TDP-43 Aggregation of wild type TDP-43 primarily in the cytoplasm of neurons is a prominent feature in ALS/FTD, and research on the mechanistic relationship between aggregation and disease is ongoing. In pathognomonic, cytoplasmic aggregates, TDP-43 is aberrantly ubiquitinated, phosphorylated, acetylated, sumoylated, and cleaved into C-terminal fragments.24,25,56,57 The nature of how these post translational modifications relate to disease pathology is still under investigation. Unlike AD and prion disease, the aggregates in ALS/FTD neurons are amorphous and non-amyloid, and TDP-43 aggregates created in vitro and in vivo often share this property.43,58,59 Cytoplasmic aggregation is usually accompanied by depletion of native TDP-43 from the nucleus as well as sequestration of other RNA-binding proteins into these aggregates.60– 63

Whether depletion of TDP-43 and other RNA-binding proteins can cause RNA disruption and

the extent of this disruption on neurotoxicity still remains unclear. Current evidence suggests that the cytoplasmic aggregates themselves are toxic to cells and cause cell death through a toxic gain


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of function, although alternative theories of TDP-43 aggregates as cytoprotective structures do exist in Drosophila models.58,64–68 The current consensus is that the disease likely arises from a combination of loss of TDP-43 native function and gain of toxic function from aggregation.69 A number of factors can contribute to the aggregation of the protein, including cytoplasmic accumulation, changes in TDP-43 expression levels, aberrant cleavage and fragmentation, loss of native state binding partners, or the production of truncated isoforms through alternative splicing.39,43,70–72 Environmental factors such as stress have long been suspected as a contributing factor in ALS/FTD pathogenesis, and evidence for this suspicion has recently been growing. One mechanism mammalian cells use to cope with environmental stress is to transiently repress the translation of mRNAs for proteins not essential to survival by organizing these arrested mRNAs and their RNA binding proteins into small (≤ 5 µm) non-membrane bound cytoplasmic domains called SGs. The assembly of SGs can be induced by oxidative, genotoxic, osmotic and thermal stresses.73,74 SG assembly and disassembly are dynamic processes mediated by a number of proteins, including TDP-43.48,75,76 Knockdown of TDP-43 reduces the expression of levels of G3BP and increases TIA-1 levels, proteins known to affect SG assembly, causing SGs to form slower, take more time to reach the average size of normal SGs, and dissipates more quickly.49 The disruption to SG regulation and persistence is predicted to cause cytoplasmic inclusions similar to those observed in ALS/FTD neurons.47 How TDP-43 mutations impact the in vivo response to stress in motor neurons is disease relevant and remains to be explored. It has been suggested that a predisposing event that enriches a population of cytoplasmic, aggregation-prone TDP-43 (through mutation or other events) followed by chronic environmental stress and persistence of SGs can cause normally reversible, functional TDP-43 aggregation in SGs to form irreversible aggregates as seen in disease. However, it is still unclear whether SGs are direct


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precursors to TDP-43 aggregates, or whether they are formed independently and recruited to SGs afterwards.52 The assembly of these membraneless organelles, also known as ribonucleoprotein (RNP) granules, occurs through the physical process of liquid-liquid phase separation (LLPS).77 Current research into the formation of these RNP granules has made the SG precursor hypothesis increasingly popular, as it serves as a long missing link between environmental effects and ALS/FTD pathology. Recent structural studies of TDP-43 have shed light on the potentially detailed molecular mechanisms of how TDP-43 diverges from folding into reversible RNP granule assemblies versus pathological aggregates.

INSIGHTS INTO TDP-43 AGGREGATION FROM STRUCTURAL STUDIES Domain structure of TDP-43 TDP-43 is 414 amino acid residues in length, and is comprised of an N-terminal domain (NTD: 1-102) that includes a predicted nuclear localization signal (NLS: 82-98), two RNA recognition motifs (RRMs) composed of residues 106-177 (RRM1), and residues 192-259 (RRM2) which includes a nuclear export signal (NES) from residues 239-250, and a C-terminal domain (CTD: 274-414) (Figure 1A).34,78 In vitro biophysical characterization and crosslinking studies in cell culture and mouse brains all suggest that TDP-43 is intrinsically a dimeric protein and that dimer formation may be mediated by a number of regions across the entirety TDP-43, including the NTD, RRM2, and/or the CTD.43,44,79–81 The CTD is particularly disease relevant, as it is where nearly all ALS/FTD-associated mutations are found.37,82–123 This is a flexible region containing only a transient α-helical structure, and contains QN-rich residues implicated in aggregation (Figure 1D). The structural study of full length TDP-43 has been difficult due to its high aggregation propensity and difficulty of purification, as well as its flexible CTD.64 No crystal or 10 ACS Paragon Plus Environment

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NMR structures have been produced for the protein in its entirety but structures of the individual domains of TDP-43 have been solved. The ubiquitin-like fold of the NTD The N-terminus of TDP-43 contains a ubiquitin-like fold from residues 1-77 and a nonstructured region from residues 78-102, but depending on experimental conditions, NMR spectroscopy of residues 1-77 from two groups have reported conflicting results on the stability of this domain. While Qin et al. reports that it is at an equilibrium with an unstructured state, Mompean et al. reports a single stable structure at high resolution.124,125 In the latter condition, residues 1-77 appear to adopt a well-folded structure that consists of six β-strands and an α-helix arranged in β1β2α1β3β4β5β6 topology (Figure 1B, Figure 2A). The low resolution structure by Qin et al. reports a similar conformation except β4 and β5 were not observed and appeared as a single β strand. Strands β1β2β3 and β6 form one β sheet that is similar to ubiquitin, while a second smaller sheet composed of β4 and β5 appears to be a novel feature unique to TDP-43 NTD, resembling the structure of the C-terminal Dix domain of the scaffolding protein axin 1.125 The remaining residues (78-102) in the N-terminus is rich in positively charged amino acids. This region appears to bind non-specifically to DNA only at pH 4.0, likely due to charge-charge interactions.44,124,125 The relative position of the NTD to the rest of TDP-43 is unclear, but small angle X-ray scattering (SAXS) data suggests that it may dock closely to the tandem RRMs (Figure 2).126 The N-terminus is involved in aggregation and splicing The NTD of TDP-43 appears essential to TDP-43 normal function, but is also required for pathological aggregation. Cytosolic localization of ectopically expressed TDP-43 without a nuclear localization signal caused the formation of TDP-43 inclusions in HEK293T cells, but


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expression of the same construct without the first 10 N-terminal residues, or constructs containing mutations of residues 6-9 (RVTE) to glycine residues showed diffuse cytoplasmic localization, suggesting that the N-terminus is required for aggregation.79 Additionally, the same mutations resulted in loss of TDP-43 splicing activity when the mutants were expressed in conjunction with knockdown of endogenous TDP-43 in cell culture.79 This suggests that loss or mutation of these first 10 residues may adversely affect the ability for TDP-43 to form its native dimeric structure and its ability to recruit proteins needed in the splicing machinery. This is expected since residues 6-8 also form the first β-strand in the N-terminal fold, and residues 6-9 are involved in stabilizing the first β-sheet of the N-terminal domain (Figure 1B). Mutations of these residues would disrupt the structure and dimerization of NTD, which agrees with predictions made by computer simulations.79 In a cellular aggregation model of TDP-43 where exogenous TDP-43 protein containing additional 12 tandem repeats of its aggregation-prone QNrich sequence at its C-terminus was expressed, aggregates resembling pathological inclusions formed and sequestered endogenous TDP-43, causing loss of TDP-43 exon skipping function.127 However, when the same construct without the N-terminal 75 residues was expressed, aggregates formed but without sequestration of native TDP-43 or loss of splicing function.128 Furthermore, the N-terminal TDP-43 fragment containing residues 1-105 also appears to oligomerize into larger species in a concentration dependent manner and the constructs containing the NTD plus RRM domains shows improved DNA binding activity compared to the tandem RRMs alone.81 Taken together, these evidence suggest that the NTD of TDP-43, and specifically the β-sheet structural motif, contributes to both native TDP-43 function as well as aggregation. This region of the protein is required for the initial dimerization of TDP-43 and recruitment of other RNA binding proteins, an event required for RNA splicing and perhaps the formation of RNP granules


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such as SGs. The N-terminal region effectively increases the local concentration of the TDP-43 and other RNA binding proteins, which enhances RNA binding and splicing functions. On the other hand, this very mechanism of congregating proteins to close proximities may also serve as a prerequisite for aggregation of the protein or recruitment of native proteins into established aggregates.128 The only ALS/FTD-associated mutation in this region is A90V located at the predicted nuclear localization signal at residues 83-98.84,107 The location in the nuclear localization signal suggests a possible mechanism of pathology through disruption to nuclear localization and cytosolic accumulation. Tandem RRMs contain canonical folds but are uniquely arranged TDP-43 contains two RRMs in tandem, separated by a 15-amino acid linker. Initial co-crystal structures of individual RRM1 and RRM2 domains of TDP-43 bound to single-stranded DNA demonstrated that the structures of these RRMs and the molecular interactions involved in oligonucleotide binding are congruent with typical RRM domains.80,129,130 Structurally, they consist of a β-sheet sandwiched between two α-helices arranged in the β1α1β2β3α2β4β5 topology, where the β4 strand can also be referred to as β-hairpin.131 Two segments of 6 and 8 amino acids rich in aromatic residues on the β1 and β3 strands form the typical interacting surface on the β-sheet for nucleotide binding through direct stacking interactions with the ribonucleic bases, while a few amino acids on the loop regions between β1 and α1 (Loop1) as well as between β2 and β3 (Loop3) provide hydrogen bonding interactions (Figure 1B, 2B).80,132 Both RRMs share this canonical structure, except that RRM1 possesses a longer Loop3 region than RRM2, which is thought to contribute to RRM1’s higher affinity for targets due to the more numerous amino acid-DNA interactions generated from this longer loop region (Figure 2B).129


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The two RRMs are individually capable of binding to relatively short poly-UG RNA sequences. RRM1 binds 6 UG repeats (Kd = 65.2 nM) while RRM2 binds to 3 UG repeats (Kd = 379 nM), but both RRM domains are required for high affinity, synergistic binding to sequences with greater than 6 UG repeats (Kd = 14.2 nM).30,80 Indeed, the RNA binding targets of TDP-43 are very numerous, and not all possess short UG repeats. The 3’UTR sequence through which TDP43 modulates autoregulation contains 34 nucleotides and some targets of TDP-43 can extend up to 100 nucleotides in length.133,134 Recent NMR studies have produced a structure of tandem RRM domains interacting with a single RNA strand with the sequence 5′-GUGUGAAUGAAU3′, termed AUG12, revealing the role of both RRMs in binding to this target. The tandem domains reside side-by-side upon RNA binding and use both of their hydrophobic β-sheet regions and its loops to generate an extended groove to accommodate the RNA molecule (Figure 2B). However, unlike usual tandem RRM domains in which RNA binds to the grove in a 3’-to-5’ direction from RRM1 to RRM2, the TDP-43 tandem RRMs are arranged in reverse. Subsequently, the linker between the two RRMs that canonically spans only 2 β-strands now spans across a larger area, across 4 β-strands, which allows for this linker region to participate in more extensive interactions with RNA targets as well as the RRMs themselves and potentially other regions of the protein such as the N-terminus (Figure 2B).131 The structural study also reveals that a degenerate consensus sequence of (5′-GNGUGNNUGN-3′) is recognized by the tandem RRMs, unlike other RRMs that require a continuous stretch of 6-9 nucleotides for high affinity binding.132 It is possible that this extended inter-RRM linker region provides the basis for TDP-43’s ability to participate in a large number of RNA processing functions due to its ability to recognize a large number of specific sequences as well as potentially interacting with other RNA binding molecules.131


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Role of RRM domains in aggregation The role of RRM domains are most often associated with the native RNA processing functions of the protein. In vitro studies have shown, however, that binding of oligonucleotide targets such as 12 TG repeat DNA sequences to TDP-43 through its RRMs prevents TDP-43 aggregation, suggesting some involvement of RRMs in aggregation, directly or indirectly.42,43 Only two ALSassociated mutants have been identified in the RRM domains. The recently identified P112H mutation resides on the Loop1 region of RRM1 between β1 and α1, which may affect RNA binding interactions (Figure 1C). However due to the novelty of the study, the effect of this mutation on TDP-43 structure or function is not well-characterized.122 The other mutant caused by the mutation D169G, located at a short loop region between β4 and β5 of RRM1, shares the same overall structure as the wild type protein and actually has slightly higher binding affinity to oligonucleotide targets, suggesting that it is unlikely to disrupt normal binding functions. Furthermore, this mutant increased the thermal stability of RRM1 due to increased hydrophobic interactions from the D to G mutation. Interestingly, this mutant is more susceptible to caspase 3 cleavage between D208 and V209, which effectively separates α1 from β2 in RRM2, and potentially exposes one side of the β-sheet within the RRM to aberrant protein-protein or proteinDNA interactions, while causing cytoplasmic mislocalization though loss of the nuclear localization signal (Figure 2B). The effect of this cleavage reaction may contribute to aggregation or disruption to native protein function.130 The C-terminal region has a dynamic structure Perhaps the most rigorously studied region of the protein is the CTD (274-414). Since nearly all ALS-associated mutations are found in this region, it has been implicated as an important contributor to pathogenesis. 35 kDa and 25 kDa C-terminal fragments of TDP-43 can be


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generated by caspase 3 cleavage or alternative splicing and are found in pathological inclusions. The exact mechanism as to how these fragments are generated in disease is still a subject of debate.70,71,135,136 This low-complexity region is glycine-rich and resembles sequences of yeast prions.137. The CTD appears to adopt transient and dynamic secondary structures ranging from αhelix to β-strand conformations. The structural study of the CTD is difficult due to its flexible nature, but current studies have focused primarily on a segment of the CTD approximately between residues 318 to 369. This region is considered to be the amyloidogenic core of the protein, as it contains the QN-rich segment at residues 331-369 that is capable of forming amyloid-like β-sheet structures implicated in aggregation, although TDP-43 aggregates formed in vitro and found in patients do not stain positively with amyloid-specific dyes such as Congo Red and Thioflavin S.58,59,64,138–140 Expression of 12 tandem repeats (12×QN) within this region is sufficient to induce the formation of phosphorylated and ubiquitinated inclusions in a cell culture model.127 The residues 321-366 have also been implicated in protein-protein interactions and specifically binding to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and several other members of the hnRNP family.141,142 Structurally, the amyloidogenic core can be largely divided into two segments, one approximately between residues 320-343, which is capable of forming a transient helix-turn-helix structure and the remaining residues 341-366 which are predicted to form two antiparallel β-sheet structures in molecular dynamics simulations.138,140,143,144 Interestingly, the α-helical region can also undergo α-to-β secondary structure transitions as measured by CD spectroscopy.138 Recent studies have implied that the key α-helix formed cooperatively from residues 321-330 is required for in the formation of RNP granules such as SGs through LLPS, which may be a key mechanism of how TDP-43 performs its native and pathological functions.143,145


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THE C-TERMINAL DOMAIN: PRIONS, DROPLETS AND AGGREGATION The C-terminal domain resembles yeast prions The most studied region of TDP-43 is its CTD due to its direct involvement in aggregation and pathology. This region can be considered a prion-like domain (PrLD) due to its sequence similarity to yeast prions. Recent findings suggest that this PrLD is not entirely disordered, but can fold dynamically into α-helices or β-strands, which may govern both native protein function in reaction to environmental stress as well as pathological aggregation. The PrLD is not a unique property of TDP-43, and may trace its evolutionary history to early eukaryotes. Although identified as pathological, infectious agents in prion disease in humans, prions in yeast play a major role in yeast metabolism and may confer selective advantages.7,146 Classically, yeast protein Sup35 can misfold into its prion state Ψ+ via its N-terminal domains into the typical amyloid structures composed of cross β-sheets.146,147 Sup35 is a translation termination factor in yeast, but upon folding into its prion conformation, it loses this function and leads to readthrough of nonsense mutations. In the laboratory it was demonstrated that in yeast strains harboring a premature stop codon in their ADE1 gene, cells without the capacity to form Sup35 prion state (Ψ- strain) become auxotrophic for adenine, whereas Ψ+ cells can grow on media lacking adenine. The Ψ+ state can be propagated through template-directed misfolding during mitosis, and amyloid aggregates in the diploid cells are segregated in the four spores during meiosis, leading to non-Mendelian propagation of the Ψ+ state to yeast progeny and retention of this selective advantage in future generations. Another yeast prion Mot3 is a transcription factor that regulates mating, carbon metabolism and stress response under its native state, but the prion state [MOT3+] allows for facultative multicellular growth phenotypes.148 These examples show 17 ACS Paragon Plus Environment

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that under certain environmental conditions, the ability to form prions can confer a survival advantage. However, these prion phenotypes are not without disadvantages, since prion formation triggers the increased activity of HSP40/70 in yeast and Ψ+ strains have reduced growth rates compared to their Ψ- counterparts, implying that the presence of prions induces a certain degree of cellular stress.149 Yeast prions appear to be bet-hedging mechanisms that allow adaptive response to environmental stress albeit with the risk of gaining pathology.77,148,150,151 Human proteins containing PrLD form membraneless organelles In addition to TDP-43, PrLD are also found in other human proteins. 70% of human proteins predicted to contain PrLDs by the PLAAC (Prion-Like Amino Acid Composition) search algorithm have molecular functions related to RNA/DNA binding, transcription factor activity or mRNA processing.77 Several of these proteins are implicated in human neurodegenerative disease, such as ataxin 1 and ataxin 2 in spinocerebellar ataxias, and more significantly, hnRNPA1, hnRNPA2/B1, TDP-43 and the RNA binding protein FUS (FUS/TLS; Fused in Sarcoma/Translocated in Sarcoma), which are proteins whose mutations are known to cause ALS/FTD.116,120,152–154 These proteins all share the common feature of having a disordered PrLD and RRMsto mediate RNA binding. In the case of TDP-43, regions within the PrLD are often considered the amyloidogenic core of the protein, responsible for TDP-43 aggregation 64,138,139. Despite the association of these PrLD with disease, there would be no selective pressure to retain these domains if they only confer detrimental effects of protein misfolding in the form of neurodegenerative diseases, yet regions of the PrLD such as the α-helical segment of TDP-43 PrLD is well-conserved in vertebrates.143,155 Thus, it is likely that in humans, the PrLD of proteins have functions that may confer selective advantages with risk of pathology, similar to yeast prions.


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One possible functional advantage of PrLDs is their role in the formation of membraneless organelles or RNP granules. The lack of a lipid-rich barrier to enclose its constituents is advantageous because it allows environmental changes to rapidly alter the internal equilibrium of the organelle.156 The physical properties of RNP granules were initially studied in germline P granules in C. elegans embryos, where P granules showed classic liquid droplet properties such as spherical morphology, fusion, dissolution, and concentration-dependent condensation, strongly implicating LLPS as their mechanism of formation.157 Many other RNP granules have since been reported such as processing bodies (P-bodies) which are sites of mRNA decay, and SGs which are assemblies of translationally stalled ribosomal subunits and its associated mRNAs that form during cellular stress.158,159 Proteins such as hnRNPA1 and TDP-43 are known to be recruited to SGs, and TDP-43 also modulates SG formation and dynamics.160,161 Membraneless organelles such as SGs allow for transient and reversible aggregation of unneeded transcripts and allows for cell survival under stress conditions.158 Recently, RNA binding proteins containing PrLD, such as FUS, hnRNPA1 and TDP-43, have been reported to undergo LLPS through their PrLD, which is thought to be the underlying mechanism of the formation RNP granules or bodies.143,145,155,160,162–165 The structural changes that occur within these proteins during LLPS, however, are not well understood and remain an active area of research. While a NMR study of FUS droplets suggests that the PrLD remains entirely disordered, a study using mass spectrometry-based footprinting of the PrLD in hnRNPA2 droplets asserts that it adopts a structure rich in cross-β sheets.163,166 It also remains uncertain whether liquid droplets formed in vitro are reflective of the phase architecture of membraneless organelles. TDP-43 undergoes phase transitions through its PrLD


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The component of TDP-43 responsible for phase separation into liquid droplets, emerged very recently. Unlike FUS, which appears to have no apparent structure at its PrLD in the droplet state, NMR studies on a TDP-43 PrLD construct consisting of residues 267-414 revealed that it can assemble into liquid droplets through a cooperatively folded, partially populated α-helix, initiated by the addition of 150 mM NaCl or addition of yeast RNA extract.143 Although NMR spectroscopy of the entire CTD indicates that the region is almost entirely disordered, this αhelix formed by residues 321-330 appears to be populated 50% of the time, and interacts with helices from other TDP-43 CTD molecules during liquid droplet formation.143 This apparent RRM-independent RNA interaction may be mediated through a RRG motif on the C-terminus.167 ALS/FTD-associated mutations A321G, Q331K and M337V disrupted phase separation and encouraged conversion to aggregates.143 Although residues Q331 and M337 do not reside within the transiently formed α-helix, structural studies indicate that these residues belong to the helixloop-helix region formed by resides 319-341, a region perfectly conserved among vertebrates and rich in aliphatic residues.168 Additionally, in a cell culture system where the full length TDP43 construct was modified by replacing its RRMs with a GFP reporter, the expressed construct formed nuclear droplets with “bubbles” containing nuclear millieu.145 In agreement with the NMR studies, ALS-causing mutation M337V altered the dynamics of these droplets, whereas mutations N345K and A382T outside the α-helical region did not have as significant of an effect.145 This suggests that the α-helical segment of the CTD and its inter-molecular interactions are critical for the formation of liquid droplets. It is unclear how the N-terminus biophysically affects TDP-43 droplet formation, as LLPS through the C-terminal critical residues have occurred with or without an N-terminal component.143,145 It is possible that the environment within a test tube allows for much higher than physiological achievable concentrations of TDP-


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43, thus the N-terminus that would normally be required for oligomerization and consolidating TDP-43 would no longer be necessary. In addition to liquid droplets, FUS, TDP-43 and hnRNPA1/A2 can also form amyloid-like folds rich in β-structure.140,144,154,169 TDP-43 is predicted to form β-rich structures through its QN-rich region consisting of residues 341-366, while hnRNPA1 is intrinsically prone to forming irreversible amyloid fibrils.140,144,154 FUS, hnRNPA2, and TDP-43 PrLD are also capable of phase separating into a gel phase known as “hydrogels”.163,165,166,169,170 Unlike the liquid droplet phase, the structural basis of hydrogels appears to be distinctly amyloid-like.166,169 Electron microscopy and X-ray diffraction of FUS and hnRNPA2 hydrogels reveal that they are composed of cross-β, amyloid-like folds. But unlike typical, irreversible amyloid, these hydrogels are readily solubilized by SDS or mild heating.169 The relationship between hydrogels and liquid droplets is not well defined, but recent studies suggest that unlike the present model of SGs where they behave as purely liquid compartments, SGs may contain “cores” of denser material held together by strong interactions within the PrLD.171 These core particles can be purified by conventional centrifugation. They are surrounded by a liquid shell in SGs that allows for the free exchange of materials with other liquid compartments, while exchange of material between the core and the shell is an ATP-dependent process that involves ribonucleoprotein remodeling mechanisms.171 It remains uncertain whether these cores are formed first, followed by recruitment of the outer shell, or whether the SG is formed, followed by condensation of the liquid phase into more stable core structure. We speculate that these core structures may be held together by β-rich, amyloid-like interactions, similar to those found in hydrogels, which PrLDs are capable of forming. It is however uncertain whether TDP-43 forms β-rich core structures in SGs as they were not found in the centrifugation-based purification procedure, but it is becoming


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clear that like other proteins with PrLDs, TDP-43 is capable of forming both liquid droplets mediated by α-helical interactions and amyloid-like hydrogel structures through β-rich folds. While the liquid states appear to be a part of physiological SG function, the conversion of these structures into irreversible aggregates may be associated with pathology. LLPS as drivers for aggregation The formation and maintenance of liquid droplets appears to be a delicate process and a number of factors can cause their transition into aggregates. In fact, liquid droplets tend to become less dynamic and less reversible over time and appear to have an intrinsic propensity to aggregate.51,52 For instance, constructs containing the PrLD of several RNA binding proteins including FUS and hnRNPA1 all produce liquid droplets in vitro under low salt conditions. The PrLD of different proteins can be recruited into the same droplet, forming a heterogeneous mixture, similar to the environment within SGs. However, over timespans of 24 hours, these droplets lose their liquid-like properties and change from dynamic spheres into more stable, irregular or filamentous structures.172 ThT-positive fibrils eventually form on the surface of full length hnRNPA1 liquid droplets after 24 hours and time-dependent “maturation” of SGs that lead to stable and β-rich SG core structures or amyloid-like fibrillization are also observed.160,171 In the case of TDP-43, droplets formed by the PrLD construct only remain stable in the timescale of hours before conversion to aggregates, suggesting that these droplets are transient structures that inevitably enter an irreversible state over time unless otherwise maintained.143 This property of TDP-43 liquid droplets may be attributed to transient nature of the α-helix segment of the PrLD, which readily undergoes α-β transitions, and ALS/FTD mutations that disrupt LLPS encouraged this conversion process.138,143


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In SGs, the β-rich core structures may be a means of liquid droplet maintenance to sequester proteins entering the aggregating phase and convert them to the droplet state through active, ATP-dependent remodeling mechanisms. SGs normally only persist for durations of hours, and it is possible that prolonged or repeated environmental stresses can overwhelm the remodeling mechanisms and cause irreversible aggregates to form.52,173 The conversion of cellular liquid droplets such as SGs into aggregates is a plausible pathway through which TDP-43 can form pathological inclusions. It follows that any factors that enhances TDP-43’s propensity to enter the aggregate state such as disruption of the α-helix interactions, increase in the propensity to form β-folds, or alterations to RNA content of SGs would accelerate the process of dropletaggregate conversion. Notably, a significant number of ALS/FTD-associated mutations in FUS and hnRNPA1, as well as nearly all disease-associated mutations in TDP-43 occur in their PrLD, and these mutations cause defects in droplet formation leading to decreased reversibility and higher propensity to convert into fibrils.154,160,163,165,174 In FUS, ALS/FTD-associated mutations reduces the speed at which FUS traverses through the liquid droplet and causes formation of starburst-shaped aggregates after in vitro aging.164 In TDP-43, ALS/FTD mutations that occur at the α-helix segment cause disruptions to the intermolecular interactions of the PrLD transient αhelices and altered droplet formation and reversibility.143 Although the effect of mutations outside the α-helix segment on the properties of liquid droplets remains to be tested, we speculate that these mutations may affect LLPS formation through altered protein-protein interactions. Under physiological conditions, membraneless organelles are not purely protein droplets, but contain a collection of different proteins and their associated RNA targets, and the biophysical properties of these droplets such as viscosity and droplet fusion rates, can be altered depending on the identity of the RNA to which the proteins are bound.175 Thus mutations on the


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PrLD that can potentially cause aberrant recruitment of RNA or RNA/protein complexes can contribute to aggregate formation in this manner. The specific occurrence of ALS/FTD mutations on the PrLD suggests that the disease pathology is intimately linked to the formation and aggregation of SGs. Spreading and propagation of ALS/FTD An unanswered question of ALS/FTD is the mechanism of cell to cell spread of the disease. It is tempting to attribute the spreading agent to the aggregation prone, β-rich fold of the TDP-43 PrLD, as prions spread by template-directed misfolding in prion disease. In vitro aggregates of recombinant TDP-43 can seed the aggregation of endogenous TDP-43 when transduced into HEK293T cells and multiple TDP-43 CTD fragments containing residues 287-322 are capable of forming amyloid that possesses the same seeding properties as classic amyloid fibrils.139,176,177 Moreover, transduction of insoluble fractions from ALS/FTD patient brain lysates into SHSY5Y neuronal cells can lead to the formation of phosphorylated and ubiquitinated aggregates.67 In order for cell-to-cell transmission to occur, the aggregation-prone form of TDP-43 must be expelled from the affected neuron, whether through endosomal pathways or cell death, and cross the cell membrane into the extracellular milieu and be taken up by a subsequent neuron. One proposed mechanism of membrane entry is through the microvesicle/exosome pathway through axonal terminals, while others have proposed that aggregates can rupture unstructured macropinosomes through “membrane ruffling” to gain cell entry.178,179 Recent findings suggest that segments containing the α-helical region of the PrLD (311-343) can interact with DMPC/DHPC bicelles, suggesting the possibility of membrane disruption through membranehelix interactions.139,180 The formation of α-helices may also play a more direct role in cell-tocell spreading, since α-helices are also known to form large supramolecular structures through


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helix-helix interactions between varying numbers of helices such as 3-helix micelles which are used as nanocarrier polymers that can cross a multitude of biological barriers for the delivery of its contents.181 Further studies to identify the mechanism and agent of cell-to-cell spread can open new avenues of research for the treatment of ALS/FTD by potentially blocking the entry of these agents from the affected neuron to neighboring healthy neurons, or sequester these agents using conformation-specific antibodies before they can gain cellular entry.

CONCLUSIONS Recent structural findings in the TDP-43 CTD have produced unprecedented insights into the molecular mechanism of TDP-43 function and aggregation. In particular, the observation of TDP-43 forming liquid droplets through its PrLD has provided a strong line of evidence for the intimate link between SG formation and pathological aggregation. The molecular mechanisms of pathogenesis are starting to come into focus (summarized in Figure 3). The PrLD containing the amyloidogeneic core plays a central role in TDP-43 phase changes and there is a clear correlation between secondary structure of the PrLD and the propensity to form functional versus pathological states. The conversion between these conformations may be a key step in pathogenesis. TDP-43 PrLD appears to wobble at a cusp of the protein folding energy landscape, where any number of factors can tip the balance and trigger the conversion from its native function to its pathological one. This structural conversion could be template-directed in nature, and its occurrence in soluble cytosolic TDP-43 and subsequent formation of pathological aggregates cannot be ruled out. However, as a more exquisite alternative, the conversion may occur within liquid droplets such as SGs, where the increased protein density can accelerate the propagation of β-rich folds. SGs have an intrinsic propensity to aggregate, and mutations that render TDP-43 more prone to adopt a β-fold at the PrLD, that alter concentration through


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cytoplasmic mislocalization, or that can alter protein-protein interactions and in turn alter RNA composition, can all affect SG assembly and dynamics. Additionally, environmental factors such as persistent or chronic stress due to exposure to extreme temperatures, toxins or physical harm, can also accelerate this intrinsic property of SGs. The clearance of these β-rich structures may eventually become stagnant due to the inevitable age-dependent decline of the protein quality control mechanisms, leading to accumulated aggregates and neurotoxicity.


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ACKNOWLEDGEMENTS This study was supported in whole or in parts by the Canadian Consortium of Neurodegeneration and Aging (CCNA), the Canadian Institute of Health Research (CIHR), the ALS Society of Canada (ALS Canada) and the Alzheimer Society of Canada (ASC). The authors would like to thank Kevin C. Hadley for helpful discussions and critically reading the manuscript.


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FIGURE LEGENDS Figure 1: Domain arrangement and secondary structures of TDP-43. A) Schematic representation of the TDP-43 domains. Domain boundaries are numbered according to full length TDP-43. TDP-43 consists of a N-terminal domain (brown) which contains the nuclear localization signal (grey), tandem RNA recognition motifs (dark yellow) containing predicted nuclear export signal (grey) and the C-terminal domain (blue). Nearly all known ALS/FTD associated mutation occur in the CTD (green box).37,82–123 B) Secondary structure of the NTD. Residues 1-77 contain a ubiquitin-like fold consisting of six β-strands (orange) and one α-helix (green). The nuclear localization sequence is underlined. C) Secondary structure of the tandem RRM domains of TDP-43. Both RRMs share similar secondary structure consisting of five βstrands (dark yellow) and helices α1 (light blue) and α2 (dark blue). D) Secondary structure of Cterminal domain. Only sequences containing observed or simulated secondary structures are shown. These include the helix-turn-helix motif (dark blue) and predicted β-strands (underlined).

Figure 2: Combined molecular structure of TDP-43 bound to a single strand of AUG12 RNA. Separate PDB files were joined together for a conceptual representation of the TDP-43 molecule. Dotted lines represent gaps in the sequence containing unknown structure. The speculative position of the N-terminal domain relative to the tandem RRM domains is based on small angle X-ray scattering data.126 NLS and NES sequences are shown in purple boxes. The color scheme of the secondary structures is consistent with that of Figure 1. A) The N-terminal ubiquitin-like fold consists of residues 1-77, derived from the NMR structure 2N4P.125 β-strands 1, 2, 3, and 6 form sheet 1 (dark orange), while strands β4 and β5 form sheet 2 (light orange). The α1 helix is colored in green. B) Structure of tandem TDP-43 RRM domains bound to


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AUG12 (pink line drawing) generated from the NMR structure 4BS2.131 Color scheme of the secondary structures in each domain is consistent, with β-sheet consisting of strands 1-5 (yellow) sandwiched between helices α1 (cyan) and α2 (light blue). The (’) notation denotes the matching secondary structures in RRM2. Key loop regions loop 1 (pink), loop 3 (orange) and the loop joining the two RRMs (green) are indicated. These loop regions combined with the extensive βsheet surface of both RRMs creates the binding surface for RNA target AUG12 (5′GUGUGAAUGAAU-3′). C) The secondary structure of TPD-43 amyloidogenic region derived from NMR structure 2N3X.168 This region contains extensive loops of unstructured regions except for residues 320-343 which consists of a helix-turn-helix motif (dark blue).

Figure 3: Graphical representation of TDP-43 aggregation model. TDP-43 is represented by a round blue circle (N-terminus) and two orange squares (tandem RRMs) bound to RNA (red half-ladders), attached to a C-terminal helix (blue lines) in its native state as a dimer. In the stress granule, TDP-43 C-terminal helix interact to form liquid droplets containing other proteins (green circles) and RNA (green half-ladders). The structural transition of the prion-like domain of TDP-43 to β-rich folds is represented with pink antiparallel arrows. The fibril-like arrangement of the antiparallel arrows reflect amyloid-like folds associated with stable stress granule cores or irreversible aggregates in the cytoplasm. Post-translational modifications to these aggregates are represented by yellow circles labeled P for phosphorylation, or purple circles labeled U to represent ubiquitination. The pink-blue gradient on the left represents the conversion of α-helix to β-rich folds in the prion-like domain, reflecting the transition from native to pathological TDP-43 states. The schematic presents both stress granule dependent and independent TDP-43 aggregation pathways.


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Biochemistry

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Micelle Revealed by Small-Angle Neutron Scattering and Coarse-Grained MD Simulation. Biomacromolecules 17, 3262–3267.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 1 A

ALS/FTD mutations

A90V 1

102

NTD

K263E N267S G287S G290A S292N G294A/V G295C/R/S G298S M311V

A315T/E A321G/V Q331K S332N G335D M337V Q343R N345K G348C/R/V

N352S/T G357R/S M359V R361S/T P363A G368S Y374X G376D N378D/S

S379C/P A382P/T I383V G384R W385G S387indel N390S/D S393L

P112H D169G 106

L

177 192

RRM1

259 274

RRM2

E

414

CTD

B NTD β1 β2 α1 β3 1 MSEY TED ENDEP PS EDDGTV PGAC NPVSQC β4 β5 β6 51 MRGV EG APDAGWGN L YPKD NKRKMDETDA SSAVKVKRAV NLS 101 QK

C

RRM1 β1 α1 106 GLPWKT T α2 β4 β5 156 R DG RRM2 β1 192 242

β2 TFGEVL

FTE

L

α1

RCTED M β4 β5 LCGED K G NES

β3 KDLKTGHSK G

β2 QYGD

β3 IPKPFRA

α2 ADD

N

D CTD 318 IN

helix-turn helix SS

QNQS GPSGNNQNQG NMQREPNQA simulated β-strands

ACS Paragon Plus Environment

Biochemistry

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Figure 2


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Figure 3 α-helix

Nuclear TDP-43 Functional dimer; RNA/DNA binding

Stress; N-terminal oligomerization; α-helical interactions

Stress granule TDP-43 LLPS; Contains other RNA and RNPs

Cytoplasmic shuttling

PrLD secondary structure

fragmentation

α-helix to β-strand conversion

N-terminal oligomerization; C-terminal aggregation

Expulsion of infectious agent

β-strand

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Biochemistry

Disruption to SG dynamics Persistent SGs

Persistent or ”Aged” SGs

ATP-dependent remodeling

Irreversible aggregates

Stress granule “core”

Amorphous; Phosphorylated; Ubiquitinated

Hydrogel-like; β-rich

intracellular Spreading agent uncharacterized

extracellular


Biochemistry

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For ToC use only Į-helix

TDP-43 PrLD 2º structure /LTXLGGURSOHWV

1DWLYHQXFOHDU

ß-strand 3DWKRORJLFDO DJJUHJDWHV

ZLWKJHOOLNHFRUHV


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Phase to Phase with TDP-43 - ACS Publications - American Chemical

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