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. Author manuscript; available in PMC: 2023 Feb 27.
Published in final edited form as: Bioessays. 2022 Oct 17;44(12):e2200181. doi: 10.1002/bies.202200181

Importin α/β and the tug of war to keep TDP-43 in solution: quo vadis?

Steven G Doll 1, Gino Cingolani 1,*
PMCID: PMC9969346  NIHMSID: NIHMS1876553  PMID: 36253101

SUMMARY

The transactivation response-DNA binding protein of 43 kDa (TDP-43) is an aggregation-prone nucleic acid-binding protein linked to the etiology of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). These conditions feature the accumulation of insoluble TDP-43 aggregates in the neuronal cytoplasm that lead to cell death. The dynamics between cytoplasmic and nuclear TDP-43 are altered in the disease state where TDP-43 mislocalizes to the cytoplasm, disrupting Nuclear Pore Complexes (NPCs), and ultimately forming large fibrils stabilized by the C-terminal prion-like domain. Here, we review three emerging and poorly understood aspects of TDP-43 biology linked to its aggregation. First, how post-translational modifications in the proximity of TDP-43 N-terminal domain (NTD) promote aggregation. Second, how TDP-43 engages FG-nucleoporins in the NPC, disrupting the pore permeability and function. Third, how the importin α/β heterodimer prevents TDP-43 aggregation, serving both as a nuclear import transporter and a cytoplasmic chaperone.

Keywords: Importins, TDP-43, protein aggregation, FG-nucleoporins, NTD, neurodegeneration

Introduction: TDP-43 domains are like knots on a rope

TDP-43 was identified nearly 20 years ago as a component of cytoplasmic and internuclear inclusion bodies in motor neurons taken from patients who had succumbed to ALS and FTLD [1]. The protein has since been implicated in 95% of cases of sporadic ALS [1,2]. TDP-43 binds and stabilizes nascent mRNA in the nucleus [3] and mature mRNA in the cytoplasm [4]. It is imported into the nucleus by virtue of a bi-partite nuclear localization sequence (NLS) located after an N-terminal domain (NTD) [5–7] (Figure 1A). The mRNA binding activity is conferred by two RNA recognition motifs (RRMs) that occur in tandem (RRM1 and RRM2), separated by a linker sequence [8] and followed by a C-terminal domain (CTD) containing an unstructured glycine-rich region [9]. A low-resolution model of the full-length TDP-43 in the presence of sarcosine determined using solution small angle X-ray scattering (SAXS) [10] revealed that the protein has a high degree of unfolding but is not completely disordered (Figure 1B). Three-dimensional structures of individual TDP-43 domains have been reported, namely, NTD [10–12], RRM1, and RRM2 [13–15]. In the full-length TDP-43, these domains are loosely connected by lengths of random coiled sequence, like knots on a rope. The CTD is intrinsically disordered at low concentrations but can form a stable amyloid-like filament at higher concentrations, typically when TDP-43 accumulates in the cytoplasm [16] (Figure 1B). Notably, the isolated CTD (263–414) acquires a fibrillary structure at one order of magnitude lower concentration than the full-length protein [17], suggesting the tug of war to keep TDP-43 in solution depends on the interplay between the N-terminal folded domains and its prion-like CTD.

Figure 1. TDP-43 topology and domain structure.

Figure 1.

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(A) Schematic diagram of full-length (FL) TDP-43 showing the NLS sequence, with key basic residues in red. (B) Cartoon representation of the TDP-43 domains, informed by the SAXS structure of the detergent-solubilized FL-TDP-43 [10] and published structures of each domain, namely, PDBs: 6T4B (NTD), 7N9H (NLS), RRM1 (4IUF), RRM2 (3D2W), 7PY2 (CTD). TDP-43 is shown as a dimer, with one protomer color-coded as in panel A and the second protomer in gray. The CTD is shown in the double-spiral-shaped fold described by Arseni et al. (PDB: 7PY2). (C) Zoom-in of the dimeric NTD with two protomers engaged in a head-over-tail dimerization interface (in magenta and red, respectively). (D) Magnified view of TDP-43 NLS (PDB: 7N9H).

TDP-43 NTD Dimerization and Aggregation

Dimerization of TDP-43 allows the protein to regulate the splicing of pre-mRNA [18–20]. This form of TDP-43 self-association was initially mapped biochemically to the NTD [21]. The first crystal structure of the TDP-43 NTD dimer indicated a “head-over-tail” stacking mechanism, whereby the N-terminus of one NTD interfaced with the C-terminus of the other NTD [11] (Figure 1C). This would be corroborated by an NMR structure of the NTD dimer the following year [22]. More recently, the crystal structure of an NTD oligomer has shown the extent to which NTD head-over-tail stacking leads to the formation of higher-order assembly [10]. Prior to the publication of this NTD oligomer structure, it was shown that aggregated TDP-43 that included the NTD was able to pull down the wild-type protein with higher affinity than TDP-43 lacking the NTD in a cellular model, suggesting an involvement of the NTD in the aggregation of the full-length protein [23]. However, the role of the NTD in TDP-43 aggregation remains contested. Jiang et al. demonstrated that alanine mutagenesis of Leu71 and Val72 within the NTD reduced TDP-43 aggregation [24]. However, the residues targeted by these authors do not contribute to the dimerization interface seen in the structures of the dimer [11,22] and oligomeric NTD [10]. This group may have discovered a secondary effect upon TDP-43 self-association, possibly through disruption of NTD folding. Thus, TDP-43 is naturally dimeric (Figure 1B), which was not appreciated in the SAXS analysis of the full-length protein, likely due to the presence of sarcosine [10] that disrupts NTD dimerization.

The interplay between TDP-43 dimerization and aggregation was investigated using equilibrium molecular dynamics (MD) simulations [25]. The NTD can sample several conformations en route to the fully folded state, and it has been hypothesized that these partially folded forms of the domain mediate aggregation rather than proper dimerization. Interestingly, the earliest structure of the NTD featured the monomeric form after exposure to denaturing conditions [12]. In this case, the NTD had an elongated fold relative to when dimerized, and it was not clear how self-association would be possible [11,12]. When the dimerization interface was disrupted through alanine scanning mutagenesis, the NTD could not oligomerize [11]. Accordingly, TDP-43 NTD stabilized in the monomeric form by Sulfobetane 3–10 formed oligomers with reduced kinetics relative to untreated TDP-43 NTD [26]. Indeed, the organization of the NTD dimerization interface is required for aggregation, as evidenced by an earlier study on the folding pathways of the NTD [27]. Thus, under physiological conditions, TDP-43 dimerizes through its NTD, and this facilitates aggregation.

Unique properties of TDP-43 bipartite NLS

The sequence of the TDP-43 NLS contains two patches of basic residues that allow binding of both the major and minor NLS sites on Imp α1 (Figure 1A). Doll et al.[7] have shown that the TDP-43 NLS does adhere to the bipartite binding mechanism, with the N-terminal 82-KRK-84 sequence located at the minor site in the structure, while the C-terminal 95-KVKR-98 sequence is at the major site. This is similar to that of other bi-partite NLSs, including that of nucleoplasmin [28,29]. Structural alignment of NLS sequences bound to Imp α1 reveals a conserved lysine, which protrudes into a surface groove of the major site, and a conserved arginine that protrudes into a second groove at the minor site. Indeed, these residues are topologically conserved across all bipartite NLSs, including TDP43, and used as major binding determinants. At a particular NLS binding site, major or minor, five residues are usually responsible for binding, termed the P1-P5 and P1’-P5’ at the major and minor NLS-binding sites, respectively. The conserved lysine and arginine occupy the P2 and P2’ positions, respectively, with other basic residues at flanking positions. The TDP-43 NLS deviates from other bipartite NLSs in that most of the bonding occurs at the minor site rather than the major site [7], similar to the yeast membrane protein Heh2 [30] (Figure 1D). Correspondingly, the TDP-43 NLS requires the P2’ arginine, but not the P2 lysine, to bind the Imp α1/β heterodimer in vitro [7]. Reliance on the P2’ site has been observed in previous work on TDP-43 nuclear import in cell-based assays [6,31,32]. These studies made use of TDP-43 constructs with poly-alanine mutations at the basic residues of the minor site 82-KRK-84 and major site 95-KVKR-98 sequences. In all cell lines used, over-expression of the ΔNLS construct in which the minor site sequence was disrupted resulted in depletion of nuclear TDP-43 and formation of cytoplasmic TDP-43 inclusions. Disruption of the major site also caused nuclear depletion, but this did not appear as substantial as when the minor site was affected [6,31,32]. Thus, TDP-43 harbors a bipartite NLS that contacts a heterodimer of Imp α1/β primarily via the minor NLS site basic residues.

TDP-43 disrupts the Nuclear Pore Complex

A heterotrimeric complex of TDP-43, Imp α1 (adaptor), and Imp β (receptor) moves through the NPC by virtue of the interaction between Imp β and the phenylalanine/glycine (FG) rich nucleoporins (nups) that comprise the channel [33,34]. However, TDP-43 may not simply translocate through the pore as other NLS-bearing cargoes. The selective permeability of the NPC is thought to be mediated by FG-nucleoporins that form a sieve-like matrix [35] through which nuclear import and export receptors can navigate [36]; such an FG-matrix is amenable to interaction with the disordered CTD of TDP-43. Using full-length TDP-43 and a fragment corresponding to the CTD of the protein, each fused to biotin ligase, Chou et al. demonstrated that CTD aggregates specifically were enriched for nucleoporins in a cellular model: interestingly, Nup214 was overrepresented in these aggregates [37]. Nup214 is a component of the cytoplasm facing side of the NPC and includes a disordered CTD with a number of FG-repeats [38]. Indeed, the overall topology of Nup214 resembles that of TDP-43 CTD, despite the significant difference in size between the two proteins. The CTD of TDP-43 is similarly rich in FG-like repeats [38] (Figure 2A) and may consequently interact with Imp β in addition to Imp α1 during nuclear import. This would be somewhat similar to the nuclear import of FUS, in which weak and transient contacts are made between the glycine-rich FUS NTD and Kap β2 [39]. As TDP-43 can sequester FG-nups, so can FG-nups be incorporated into RNA granules [40], providing further support for the relationship between FG composition, disorder, and phase separation. It is likely the case that aggregated TDP-43 is able to interact with nucleoporins, disrupting the NPC [37]. As the relationship between TDP-43 aggregation and dysfunctional nucleocytoplasmic transport has been well established [41], this serves as yet another mechanism by which TDP-43 becomes trapped in the cytoplasm. And as TDP-43 CTD aggregates included an abundance of Nup214 [37], it follows that the cytoplasm-facing nups are most susceptible to being drawn into TDP-43 aggregates. Thus TDP-43 is an aggregation-protein protein with FG-nup-like features that can interact with FG-nups, disrupting the NPC and interfering with nuclear protein import and RNA export.

Figure 2. The FG Repeats of the TDP-43 CTD.

Figure 2.

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(A) Amino acid sequence of TDP-43 with the CTD colored in red. Underlined is the CTD region visualized by Arseni et al. (PDB: 7PY2). All FG- and FG-like repeats are highlighted in yellow. (B) Structure of the aggregated TDP-43 CTD, residues 282–360 (PDB: 7PY2). All FG-like repeats are shown as yellow spheres.

Back to the Cytoplasm: Passive Diffusion of TDP-43

A putative nuclear export signal (NES) was identified within the C-terminal portion of the RRM2 domain [6]. Interestingly, most of the leucine and isoleucine residues of the NES needed to engage the nuclear export receptor CRM1 (XPO1, Exportin 1) are occluded by the folding of the RRM2 N-terminus [15]. This observation was corroborated by an NMR ensemble of structures for the RRM1 and RRM2 [42]. Yet the RRM2 domain can sample non-native conformations in which the NES is solvent exposed [43]. However, efforts to affect TDP-43 nuclear export through selective inhibitors of nuclear export (SINEs) indicated that TDP-43 does not require CRM1 to move from the nucleus to the cytoplasm in a cellular model [32,44,45]. Accordingly, SINE compounds did not appear effective in ameliorating motor deficiencies in a murine model of TDP-43 proteinopathy [44]. Two other groups would independently confirm that the TDP-43 NES is not required for export [32,45]. Ederle et al. found that TDP-43 could reach the cytoplasm even when the transcription-export complex was disrupted [45]. This led to a hypothesis of passive diffusion through the NPC [32], which is supported by the finding that TDP-43 CTD is enriched in FG-like repeats, potentially implicated in heterotypic interactions with NPC FG-nups (Figure 2A,B). Indeed, augmenting the mass of TDP-43 through the addition of fusion proteins drastically reduced the diffusion of TDP-43 from the nucleus to the cytoplasm, lending support to the export diffusion model. Pinarbasi et al. further investigated the role of the TDP-43 CTD in this diffusion by comparing the localization of wild-type TDP-43 and a TDP-43 ΔCTD construct [32]. Loss of the CTD, while reducing the size of the protein, nonetheless appeared to reduce the amount of TDP-43 that could pass through the NPC. Thus, the cell brings TDP-43 into the nucleus through the classical nuclear import pathway, and the protein may diffuse back into the cytoplasm. Under normal conditions, a combination of small size, sufficient disorder, and glycine richness mediates the diffusion of TDP-43.

Post-Translational Modifications

TDP-43 is subjected to many post-translational modifications (PTMs) before, during, and after aggregation. However, the kinetics of PTMs occurrence is poorly understood and challenging to study in neurons. Here we will only discuss those modifications that pertain to the NLS and the effects upon nucleocytoplasmic transport. There are several excellent reviews that describe the PTMs that run the entire length of the protein [46–48]. Ubiquitination and phosphorylation comprise the majority of PTMs [49], as expected for an aggregation-prone protein. Using a hyper-active form of casein kinase 1-δ, Kametani et al. [50] found that Thr88, Ser91, and Ser92 within the linker sequence of the TDP-43 NLS were subject to phosphorylation in vitro (Figure 3A,B). This group would later confirm the phosphorylation of Ser92 in a cellular model using the same hyperactive kinase [51]. Interestingly, mass spectrometry of TDP-43 fragments taken from affected tissue of two ALS patients’ brains did not find phosphorylation within the TDP-43 NLS [52]. However, this group did not detect any fragments that corresponded to the very N-terminus (residues 1–56) of TDP-43 and observed no phosphorylation until the RRM2 domain. Given that the TDP-43 extracted from each patient had experienced substantial N-terminal cleavage, it seems possible that any phosphorylated NTD/NLS had been cleaved during disease progression, preventing its identification. Kametani et al. did find that Lys79 was ubiquitinated (although not necessarily poly-ubiquitinated) and Lys82 acetylated [52] (Figure 3B). Lys82 occupies the P1’ position of the minor binding site residues immediately upstream of the P2’ position. This lysine engages in two hydrogen bonds with Imp α1 [7]. Indeed, substitution of Thr88, Ser91, and Ser92 with glutamic acid (to mimic phosphorylation) had the overall effect of reducing the affinity of the TDP-43 NLS for the classical nuclear import system in vitro [7]. Garcia Morato et al. found that Lys79 and Lys84 could be acetylated in a non-neuronal cell model [53]. Addition of this modification to Lys84 increased the cytoplasmic concentration of full-length TDP-43 [53]. Lys84 occupies the P3’ position immediately downstream of the P2’ position. The PTMs within the P1’-P5’ residues (Lys82 and Lys84 acetylation) likely affect the affinity of the NLS for Imp α1 at least as much as PTMs near the P1’-P5’ residues (Thr88, Ser91, and Ser92 phosphorylation) [7]. While it may appear that phosphorylation of TDP-43 would contribute to its aggregation, a recent study has determined a countervailing effect [54]. In this work, full-length TDP-43 was in vitro phosphorylated by casein kinase 1-δ [54], the same kinase that can phosphorylate the NLS in a cell extract [50]. TDP-43 modified in this way had impaired phase separation relative to unmodified TDP-43 [54]. However, this group did not consider the effect of phosphorylation at the N-terminus of the protein and how phosphorylation might disrupt the mechanism of TDP-43 sub-cellular localization. While CTD phosphorylation may impede TDP-43 aggregation, it may only delay the inevitable when the protein is unable to localize to the nucleus due to phosphorylation within the NLS. Thus, post-translational modifications in the neighborhood of TDP-43 NLS bias the kinetics of aggregation by altering its transport properties, while post-translational events in the CTD stabilize the double-spiral-shaped fold [16].

Figure 3. TDP-43 minor NLS site is a hot spot.

Figure 3.

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(A) A model of ΔIBB-Imp α1 bound to TDP-43 NTD-NLS-RRM1 [7]. (B) Zoom-in view of the TDP-43 NLS that illustrates all PTMs (left) and point mutations (right) that promote loss of importins-binding and aggregation.

Importins binding to the TDP-43 NLS as the foundation of disaggregation

TDP-43 fibrils formed in vitro can be returned to the soluble phase by adding Imp α1/β [55]. This finding has been reproduced by Hutten et al., [56] and demonstrates the potential of the Imp α1/β heterodimer to remove TDP-43 aggregates. Interestingly, the non-classical nuclear import receptor Kap β2 has been shown to have a disaggregating effect on phase-separated FUS droplets [39,55,57,58]. Disease-associated mutations within the FUS NLS attenuate the ability of Kap β2 to re-solubilize FUS, indicating that it is specifically NLS binding to the import receptor that interferes with FUS self-association [39,55,57,58]. Doll et al. found that TDP-43-NLS association with Imp α1 prevented TDP-43 NTD from self-association [7]. These authors investigated the ability of TDP-43 NTD to form dimers, tetramers, and oligomers in vitro as a function of concentration. When the TDP-43 NTD attached to the NLS was co-expressed and co-purified with ΔIBB-Imp α1 alone or the Imp α1/β heterodimer, the NTD remained monomeric [7]. This result, in conjunction with the structure of Imp α1 in a complex with a TDP-43 NTD-NLS-RRM1 fragment, led us to a model where the adaptor subunit behaves as a ‘molecular prybar’, which physically separates the TDP-43 NTDs impairing TDP-43 self-association (Figure 4). Through this mechanism, the Imp α1/β may be able to solubilize TDP-43 fibrils, as described previously [55,56]. The Imp β subunit, in this case, stabilizes the structure of Imp α1 and allows the NLS to bind with higher affinity. It is likely that Imp α1 alone, lacking the autoinhibitory IBB-domain [59], would be able to dissociate TDP-43 aggregates, albeit with reduced efficacy. Similarly, MD evidence suggested that post-translational modifications in the neighborhood of the minor NLS P2’ site can weaken binding affinity, reducing the chaperone-like activity of Imp β and promoting aggregation.

Figure 4. Role of importins in TDP-43 aggregation pathway.

Figure 4.

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(A) Monomeric TDP-43 forms a dimer at low concentrations that regulates pre-mRNA splicing within the nucleus. The dimerization interface allows for ‘head-over-tail’ stacking of TDP-43 monomers. (B) Imp α1, and potentially other isoforms of importin α [56,61], part of the Imp α/β heterodimer functions like a prybar binding the NLS with high affinity and disrupting NTD dimerization and possibly higher order assemblies. (C) Under the pressure of PTMs, mutations, TDP-43 phase separates, CTDs come together to generate a double spiral-shaped fiber (PDB: 7PY2) that leads over time to (D) amyloid-like filaments (reproduced from [62] with permission).

Conclusions and Perspectives

Nearly two decades after the discovery of TDP-43, most ALS and FTLD cases remain of unknown etiology but are linked by a common denominator, TDP-43 proteinopathy. The realization that an imbalance in TDP-43 nucleocytoplasmic trafficking leads to cytoplasmic aggregates and damages the NPC, interfering with nuclear protein import and RNA export, has opened new doors to studying TDP-43 proteinopathy and offers intriguing therapeutic opportunities. But there are many unresolved questions in the field. Our recent work on TDP-43 NLS recognition by Imp α/β1 [7] identified NLS residues in the proximity of the minor binding site as a hot spot for PTMs (Figure 3B), including putative phosphorylation sites at Thr88, Ser91, and Ser92; acetylation at Lys82; and ubiquitination at Lys79. We also established that the adaptor Imp α1 sterically interferes with NTD dimerization, preventing the formation of NTD oligomers (Figure 4A,B). These results, combined with previous findings in the literature, shed light on new aspects of TPD-43 biology. First, we provide a reading frame to decipher the chaperone-like function of Imp α1/β that exerts disaggregase activity toward TDP-43 in vitro [55,56]. We propose that the Imp α1/β can engage the TDP-43 at two contact points. On one side, the nanomolar affinity of the adaptor Imp α1 is sufficient to recruit the NLS and prevent NTD dimerization. As TDP-43 FG repeats promote CTD fibrillization [60] (Figure 2B), Imp β binding to these dipeptides could potentially attenuate this form of self-association. Together, Imp α1 and β preserve TDP-43 trafficking in healthy cells, averting TDP-43 mislocalization and NPC damage. Second, the TDP-43 cycle of active import into the nucleus and passive diffusion into the cytoplasm is disrupted during the lifespan of the motor neuron in ALS and cortical neuron in FTLD by PTMs and mutations. Some of these PTMs may reduce the affinity of the NLS minor binding site residues for Imp α1 (Figure 3B), rendering the classical nuclear import pathway ineffective at dissipating aggregated TDP-43 and maintaining the healthy subcellular distribution of the protein. With reduced Imp α1/β chaperone activity, TDP-43 preferentially binds to itself, forming dimers, tetramers, oligomers, and eventually phase-separated aggregates (Figure 4C,D). Third, TDP-43 aggregate can damage the NPC by interacting with FG-nups. We propose these interactions are promoted by TDP-43 own FG-like repeats that may engage in cohesive interactions with FGs in nups. Just as Imp β1 and other β-karyopherins bind FG-nups to promote movement through the NPC under healthy conditions, we propose that these proteins function as anti-aggregation chaperones preventing NPC disruption and pathogenic accumulation of TDP-43 aggregates.

ACKNOWLEDGMENTS

We apologize to all authors whose work could not be cited due to space constraints. This work was supported by NIH grants R01 GM122844, R35 GM140733, and R21 NS128396 to G.C.

Abbreviations:

ALS

Amyotrophic Lateral Sclerosis

CTD

C-terminal domain

FTLD

Frontotemporal Lobar Degeneration

NES

nuclear export signal

NLS

nuclear localization sequence

NPC

nuclear pore complex

NTD

N-terminal domain

PTM

post-translational modification

RRM

RNA recognition motif

SAXS

small angle X-ray scattering

Footnotes

CONFLICT OF INTEREST

The authors declare no competing financial interests.

DATA AVAILABILITY STATEMENT

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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