The Self-interaction of Native TDP-43 C-terminus Inhibits Its Degradation and Contributes to Early Proteinopathies
I-Fan Wang1,2,*, Hsiang-Yu Chang1,2,3, Shin-Chen Hou3, Gunn-Guang Liou4, Tzong-Der Way3, C-K James Shen1
1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115
2Garage Brain Science, Taichung, Taiwan 413
3Department of Biological Science and Technology, College of Life Sciences, China Medical University, Taichung, Taiwan 404
4Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan 350
*Corresponding Author: I-Fan Wang
Author Contributions: I.-F. Wang conceived the research, designed and performed the experiments, wrote the manuscript and coordinated collaboration. H.-Y. Chang performed bioinformatic analysis, conducted the experiments and the data analysis and assisted in writing the manuscript. S.-C. Hou and T.-D. Way assisted in EGCG related experiments and provided reagents. G.-G. Liou carried out the EM. C.-K. James Shen helped in revision of the manuscript.
Key words: TDP-43 C-terminus, Pathological degradation, Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS), Aggregation-prone domain, Epigallocatechin gallate (EGCG)
ABSTRACT
The degraded, misfolded C-terminus of TDP-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Cleavage of TDP-43, rather than its phosphorylation or ubiquitination, is the central event of early pathogenesis.
However, the precise mechanism of pathological cleavage of TDP-43 remains unknown.
We found a novel type of protein-interaction in which native TDP-43 C-terminus protein physically interacts with itself or with cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of TDP-43, including subcellular localization and exon skipping of CFTR. Significantly, mutants with a failure to engage in prion-like interactions were cleaved into a ~24 kDa C-terminus fragment of TDP-43. The estimated cleavage site of the degraded TDP-43 fragment corresponds to the pathological-cleavage site identified in patients with TDP-43 proteinopathies. Consistently, constraining the prion-like interactions of these mutants using EGCG attuned pathological-like degradation. Briefly, the native function of the TDP-43 C-terminus acts as a guardian of pathogenesis, which is directly associated with loss of function. Moreover, our insights into native aggregation-prone domains further explain the reciprocal effects of TDP-43 proteinopathies and other misfolding diseases in their early stage of pathogenesis.
INTRODUCTION
The aggregation of misfolded, phosphorylated, ubiquitnated C-terminal fragments of
TAR DNA-binding protein 43 (TDP-43) is a common characteristic in frontotemporal
lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral
sclerosis (ALS)1-8. Among mammals, zebrafish and flies, the protein sequence,
biochemical properties and biological functions of TDP-43 are well conserved. In the cell
nucleus, TDP-43 forms visible 50- to 250-nm granules that serve as a scaffold to link
other functionally related sub-compartmentsand participates in transcriptional repression
as well as alternative splicing9-12. TDP-43 specifically binds to UG repeat sequences in
RNA, altering pre-mRNA splicing of cystic fibrosis transmembrane conductance
regulator (CFTR) and may associate with long ncRNA to regulate nuclear speckle or
paraspeckle11, 13. TDP-43 mutants lacking a C-terminal glycine-rich region fail to excise
exon 9 in the CFTR gene9, but how TDP-43 C-terminus governs exon 9 skipping of
CFTR, as well as its role in pathogenesis of TDP-43 proteinopathies is still unknown.
Given that the TDP-43 C-terminus binds directly to hnRNP A1 and hnRNP A2/B1 in
pull-down assays14, the loss of association between TDP-43 and the hnRNPs has been
proposed as a possible mechanism behind TDP-43 proteinopathies, causing aberrant
pre-mRNA splicing or mislocalization of TDP-43. However, this hypothesis does not
adequately account for pathological degradation of TDP-43 protein itself, which is a
major feature of TDP-43 proteinopathies in the early stage1.
Recently, we have reported a database search for homologs of the mTDP-43
C-terminus across multiple species and found that the mTDP-43 C-terminal domain (a.a.
274-414) shares 24.2% sequence identity with the N-terminal yeast prion domain of
Sup35 (a.a. 1-125; SupN)15. This analysis led us to propose that the TDP-43 C-terminus
might have prion-related properties. Based on the atypical Q/N rich-domain of TDP-43
involved in the self-assembly of misfolded C-terminal fragments and the sequestration of
TDP-43 into polyglutamine aggregates, TDP-43 has been further proposed to act as a
prion16. However, in contrast to most known prions, misfolded pathological fragments of
TDP-43 or full-length TDP-43 aggregates in vitro do not react with the amyloid-specific
dye Congo Red1,17, indicating that the TDP-43 Q/N-rich domain may not be a
prionogenic domain. We were, thus, interested in understanding whether the intriguing
sequence similarity in Q/N residues between the TDP-43 C-terminus and known prion
domains is critical to the cellular functions of TDP-43 C-terminus, rather than misfolded
related. However, to date, no cellular roles have been clearly elucidated for a prion
domain. To gain insight into its cellular role, we developed assays to explore the native
functions of TDP-43 C-terminus in cells.
RESULTS
Highly Dynamic Assembly of TDP-43 Aggregates in Normal Cells via Q/N-rich
C-terminus
Based on structure and intensity, two groups of TDP-43 clusters appeared in the
nucleus: visible granules of 50-250 nm in diameter (arrow, Fig. 1a) and irregular puncta
(arrowhead, Fig. 1a), as shown by endogenous TDP-43 and GFP-tagged, full-length
mTDP-43 (TDP-43-FL) (Fig. 1a). The dynamics of TDP-43 clusters in living cells were
further visualized by time-lapse microscopy. In GFP-TDP-43-FL transfected cells, the
small irregular puncta were rapidly burst, reorganized, or disassembled, and were
restricted to a local area (Fig. 1b). High magnifications of irregular puncta of
GFP-TDP-43-FL are shown in Fig. 1b (arrowheads, lower panel). Individual
GFP-TDP-43 small puncta histories suggest that they have similar motility characteristics.
In contrast, most of the visual granules were more stable and traveled within a limited
localization. A few granules were stationary (white arrowheads, Fig. 1c). Also observed,
but rare, were fission and fusion events (yellow arrowheads and red arrowheads,
respectively, Fig. 1c). Occasionally two independent granules of GFP-TDP-43-FL from
two different areas collided and then separated (blue arrowheads), possibly exchanging
material between granules (Fig. 1c). With gentle 0.5% Triton X-100 pre-extraction of
cells to remove diffuse nucleoplasmic TDP-43 proteins, TDP-43 clusters in nuclei exhibit
clear shapes by immunostaining (Fig. 1d, middle)18. An example of a nucleus is shown in
Fig. 1d. No signal was detected in cells without permeabilization (Fig. 1d, right). Western
blot further confirmed that a fraction of mTDP-43 FL proteins was retained in the
detergent-insoluble fraction (Fig. 1e, left lane). These results suggest that these insoluble
supermolecules of TDP-43 protein, behave as highly dynamic functional aggregates in
the nucleus.
An atypical Q/N-rich domain of TDP-43 is capable of spontaneously forming a
filamentous structure and retains TDP-43 proteins in the insoluble fraction in mammalian
cells in the same way as other aggregation-prone domains (Supplementary S1). To
validate whether the aggregation propensity of TDP-43 C-terminus contribute to form
functional TDP-43 aggregates in physiology statues as well, we constructed a TDP-43
C-terminal deletion mutant, TDP-43-PLD∆. In the absence of the PLD, TDP-43-PLD∆
diffused in the nucleus (Fig. 1f). We termed the region as prion-like domain (PLD)19. It
appears that, in addition to forming misfolding aggregates, the Q/N-rich domain is
essential for the assembly of reversal functional TDP-43 aggregates in cells.
Functional Substitutions of GQN-rich C-terminus of Mammalian TDP-43 by a Yeast
Prion Domain of Sup35
To clarify the intriguing sequence similarity in Q/N residues between the TDP-43
C-terminus and yeast prion domain of Sup35 (SupN), we replaced the PLD of
GFP-tagged mTDP-43 with yeast prion domains (TDPSupN) (Fig. 2a). As the sequence
homology or the polyQ/N context may affect the conformation in cells, an additional
replacement by the N-rich yeast prion domain, UreN (aa. 1-84) was also generated. UreN
shares only 12% similarity with the PLD. The distribution of the Q and N residues of
these three Q/N-rich domains was illustrated in Fig. 2b. Interestingly, chimeric TDPSupN
proteins restored two groups of clustering aggregates: visible granules of 50-250 nm in
diameter (arrow, Fig. 2c) and irregular puncta (arrowhead, Fig. 2c) in the nuclei, as
shown by the GFP-tagged, full-length mTDP-43 (mTDP-43-FL). In contrast, mTDPUreN
failed to recapitulate full-length TDP-43 localization, although mTDPUreN formed large
foci in the cytosol (Fig. 2c). Live-cell imaging showed that the clustered mTDPSupN
proteins had identical behavior to mTDP-43-FL. Fluorescence recovery after
photobleaching (FRAP) analysis of GFP-mTDP-43-FL and GFP-mTDPSupN movement
showed that fluorescence recovery of these proteins was rapid, but significantly slower
than that of mTDP-43-PLD∆ (Fig. 2d). The half-recovery time was calculated to be 8.23s,
7.28s and 1.34s, for GFP-mTDP-43-FL, GFP-mTDPSupN, and GFP-mTDP-43-PLD∆,
respectively. The recording images obtained from two bleached areas of a single
GFP-mTDPSupN expressing cell showed fluorescence recovery (Fig. 2e). We noticed
that the fluorescence recovery in visible granules was faster than that in irregular puncta,
which may imply differential kinetics in the redistribution of protein into the two types of
functional aggregates (Movie 1). Significantly, in vivo splicing assays revealed that
mTDPSupN effectively promoted exon 9 skipping of CFTR in the same way as
TDP-43-FL (Fig. 2f and Supplementary S2). Cotransfection with α-globin promoter and
mTDPSupN in 293T cells did not alter mTDP-43 transcription repression
(Supplementary S3). As expected, the C-terminus of TDP-43 was not involved in
TDP-mediated transcriptional repression12. In summary, mTDPSupN localized to the
nucleus, where it effectively promoted exon 9 skipping in the same manner as
endogenous TDP-43.
As GFP-TDP-43-FL and GFP-TDPSupN proteins dynamically clustered in the
nucleus, we examined whether self-interaction of TDP-43 occur via prion-like domain.
Using co-immunoprecipitation with FLAG antibodies, we found that endogenous 43-kD
and 95-kD TDP proteins co-precipitated with FLAG-tagged TDP-43, suggesting TDP-43
self-interaction (Fig. 2g). 95-kD TDP-43 protein was likely constituted by a 43-kD
TDP-43 and a 52-kD ubiquitinated TDP-43 (Supplementary S4). This self-interaction can
be recapitulated by GFP-tagged FL and mTDPSupN (Fig. 2h). Notably, the
coprecipitation efficiency of endogenous TDP-43 with mTDPSupN was similar to that
with TDP-43 FL, and more effective than with TDP-43-PLD∆ (Fig. 2h). These results
suggested that the cellular role of the Q/N-rich domain is to promote protein self- or
cross-interactions and act as a protein-protein interaction domain in the cell.
Comparisons among the cellular and misfolded properties of the known Q/N-rich
domain were shown in Table 120-31. The cellular-folded Q/N-rich domain shared key
properties characteristic of the misfolded proteins, including insolubility and a tendency
to form aggregates via intrinsic element- and self-interactions. However, the interaction
of these prion-like supramolecules was dynamic in the cell in response to stimuli. We
hypothesized that these interactions occur because of the structural plasticity of the
aggregation-prone domain, which allows temporal folding of parallel β-sheets in the cell.
We thus described this interaction of cellular-folded Q/N-rich domain as “prion-like
interaction”. Notably, although natively folded Sup35N and natively folded TDP-43
C-terminus adopt substitutable structure in cells, only Sup35N aggregate into an
in-register β-sheet amyloid structure when misfolded, suggesting that a protein that has
prion-like properties does not have to be a prion.
A Single Proline Substitution at Aggregation-prone-domain of TDP-43 Induced
Pathological Degradation of TDP-43
Substitution of the structural breaking amino acid proline has been demonstrated to destroy β-sheets32. The simplified model was illustrated in Fig. 3a. As glycine- rich region localizes at adjacent the 1st Q/N rich region, two glycine residues G296 and G300 were selected to examine the effect of glycine on prion like nature of TDP-43 C-terminus.
We thus substituted proline at amino acids G296, G300, Q302 or Q340 of TDP-43 PLD (Fig. 3b). Notably, only TDP-43 Q302P proteins were partially degraded into ~24kD pathological fragments (asterisk, Fig. 3b). In vivo splicing assay showed that TDP-43 Q302P attenuated exon 9 skipping efficiency by 23.5% (Supplementary S5). Four days post-transfection with Q302P, a portion of the transfected cells showed cytoplasmic accumulation of TDP-43 Q302P fusion proteins (arrow, Fig. 3c) and formed cytosolic
aggregates (arrowhead, Fig. 3c). The ratio of cells with cytoplasmic accumulated fusion proteins in TDP-43-FL and TDP-43 Q302P- expressing transfectants was shown in Fig.
3d. Indeed, a subset of the TDP-43 Q302P-expressing cells had a lower fluorescence half-recovery time, which was similar to that of TDP-43-PLD∆ (Fig. 3e). A lower viability of TDP-43 Q302P-expressing cells was further confirmed by MTT assay (Supplementary S6).
Furthermore, substitution of proline at amino acid 25 of Sup35 has been suggested
to abolish the propagation and infectivity of all three [PSI] variants33. To confirm that
impairing the cellular properties of “prion domain” leads to pathological degradation of
TDP-43 proteins, we generated a mTDPSupN mutant, GFP-mTDPSupN*, which
contained a G25P substitution in SupN, and assessed its localization and ability to skip
exon 9 of CFTR. As expected, immunoblot analysis showed that the majority of
GFP-mTDPSupN* proteins shifted to the soluble fraction and were degraded into
~48-kD fragments (Fig. 3f). The estimated cleavage site of the degraded mTDPSupN*
fragment corresponded to the cleavage site of a ~24 kD fragment that has been detected
in patients with TDP-43 proteinopathies1. Tubulin and actin immunoblot were used as
controls for non-specific protein degradation (lower panel, Fig. 3f). The degraded
mTDPSupN* fragment did not translocate into the insoluble fraction, possibly because a
G25P substitution in SupN* completely abolished the intrinsic propensity of
mTDPSupN* to aggregate. GFP-mTDPSupN* was evenly distributed in the nucleus and
cytosol (Fig. 3g) and failed to promote alternative splicing activity that confirmed loss of functional prion-like domain of TDP-43 (Fig. 3h).
Epigallocatechin Gallate (EGCG) Induced Oligomerization of TDP-43 that Attuned
Its Protein Degradation
If the disruption of prion-like interactions of the Sup35 or TDP-43 C-terminus leads
to a shift into the soluble fraction and protein degradation (Fig. 3), constraining prion-like
domains should cause oligomerlization/functional aggregates and then impede the
cleavage of TDP-43 proteins. EGCG has been suggested to redirect an array of
amyloidogenic molecules, including synuclin, huntingtin, amyloid-β, TTR and yeast
prion [PSI], into nontoxic oligomers. A recent study of crystallization of a V30M
TTR-EGCG complex further indicated that EGCG directly bound and stabilized TTR
tetramer in vitro and induced TTR oligomerization in cells34. The pharmaceutical
mechanism of EGCG curing of yeast prions [PSI] could be via stabilization of the native
prion domain Sup35 as well35. To address this, EGCG was applied to block the rapidly
degraded mutant proteins of TDPSupN* proteins (Fig. 4a). TDPSupN* mutants were
treated with mock or 20 µM EGCG. An increase in full-length TDPSupN* in cells treated
with 20 µM EGCG for 3 days relative to the level in mock-treated cells was observed
(Fig. 4a). We further speculated that EGCG may function in preservation of the native
folded TDP-43 C-terminus as well (Fig. 2c, f, h). Indeed, EGCG induced oligomerization
of TDP-43 in a dosage-dependent manner (Fig. 4b). In GFP-Q302P-TDP-43 expressing
cells with rapid degradation, the degradation of GFP-Q302P-TDP-43 proteins was
reduced by EGCG and corresponded with the appearance of TDP-43 aggregates in nuclei
(Fig. 4c and d). To confirm that EGCG-induced aggregation was mediated via TDP-43
C-terminus, GFP-TDP-43-FL or GFP-TDP-43-PLD∆ expressing cells were treated with
20 µM EGCG. Upon stimulation of EGCG, GFP-TDP-43-FL proteins aggregated in
nuclei (arrow), but GFP-TDP-43-PLD∆ proteins did not (Fig. 4e). MTT assay further
confirmed that these EGCG-induced TDP-43 aggregates were non-toxic (Fig 4f). In
confluence-cultured cells, with EGCG 20 or 50 µM, the normal protein degradation of
GFP-TDP-43-FL expressing cells also attuned (Fig. 4g left). Consistent with the increase
of TDP-43 aggregates upon EGCG, the insoluble protein level of GFP-TDP-43-FL
increased simultaneously (Fig. 4g right). The identical effect of EGCG on endogenous
TDP-43 proteins was observed in confluence-cultured cells (Supplementary S7). Taken
together with the results of Figure 3, we concluded that the impairment of interactions
between the cellular-folded prion domains is a prerequisite for its protein degradation
under both normal and pathological conditions.
DISCUSSION
Increasing evidence indicates that aggregation-prone domains play crucial roles in
various cellular functions, such as granular assembly and nuclear export (Table 1). In this
study we further found that the Q/N-driving propensity of TDP-43 is involved in
alternative splicing and protein stability. Recently an increasing number of prionogenic
domain-containing proteins have been identified, among them many, such as Mot3p and
Lsm4p,contain a Q/N-rich domain and are involved in transcription or RNA processing36.
Additionally, a self-perpetuating Q/N-rich domain of apCPEB was suggested to
participate in the long-term facilitation of Aplysia31. A set of proteins which control the
stability of mRNAs as well as their translational silencing are co-localized in cytoplasmic
foci called processing bodies or P bodies37. Interestingly, several P-body components
contain a potential prionogenic domain. In yeast, the prion-like properties of this motif
may be related to granule assembly and can be restored by replacement with yeast prion
RNQ1. Lsm4p is one of these well characterized proteins27. Ultrastructural studies have
demonstrated that P-bodies are condensed, fibrillar aggregates as that of the prion [PSI]
particles in yeast38,39. In the same way as shown here in the assembly of TDP-43
functional aggregates, assembly of P-bodies is dynamic and, in neurons, is regulated by
neuronal activity41. In consistent with this kind of physiological aggregates, a new type of
off-pathologenesis pathway contains a non-toxicity ~ 500 kD oligomer of amyloidogenic
molecules has been found in cells42. We suggested that the higher bands of TDP-43
shown in Fig. 2h-g, would be assembled prion-like oligomers of TDP-43, as the
overexpressing TDP-43 Q302P interfered the generation of endogenous higher bands of
TDP-43 upon oxidative stress (Unpublished data).
Based on the results of the current study we propose a model, outlined in Fig. 5, in
which the ambivalent propensity of the TDP-43 C-terminus allows to either form
structurally flexible aggregates under physiological processes (Fig. 5, conformer B and
conformer B’), or rigid pathological inclusions (Fig. 5, conformer C). The restorative
formation of nuclear clustering and alternative splicing ability when TDP-43 C-terminus
is replaced by yeast sup35N strongly suggests that a prion-like property is involved in
subcellular localization and exon 9 skipping of CFTR (Fig. 4). Interestingly,
ALS-inherited G348C and R361S mutants attenuated exon 9 skipping efficiency by
12.5% and 28.9%, respectively, whereas G296P, a disease-unrelated mutant, did not
(Supplementary S8). These results implied that ALS-inherited mutations showed
attenuation of the prion-like activity of TDP-43. We prefer that the impairment of
interactions between prion-like domains occurs prior to caspase cleavage, since an
oligomer stabilizer, EGCG can reduce protein degradation of TDP-43 mutants defected in
prion-like interaction (Fig. 4a, c, d). The failure of the PLD to adopt proper cellular
folding, due to a loss of prion-like interactions or impairment by ALS-linked mutations at
the early stages of pathogenesis, may trigger pathological fragment cleavage, convert the
PLD to a misfolded structure, and ultimately result in disease inclusions. We also found
that the TDP-43 N-terminus stabilized the cellular-folded conformation and altered the
insoluble propensity of TDP-43 C-terminus, suggesting that the TDP-43 N-terminus
(1-105) may act as a chaperon-like domain to autoinhibit the disorder tendency of the
PLD through long-range intramolecular interaction (Supplementary S9). A recent study
showed that physical interaction of mutant SOD1 proteins with the TDP-43 N-terminus
consistently resulted in an increase in TDP-43 proteins in the detergent-insoluble
fraction43. The comparison of TDP-43 variants is summarized in table S1.
Additionally, the co-immunoprecipitation of endogenous TDP with mTDPSupN
suggested that prion-like properties mediate the interactions between heterogenous
Q/N-rich domains (Fig. 2). Indeed, we have found that proteins containing polyQ
domains are often functionally linked to other poly-(amino acid) proteins by database
analysis (http://string.embl.de/newstring_cgi/) of the functional interactomes of Q/N-rich
proteins. For example, functional interactome analysis of ATXN2 reveals ten functional
interactors. Seven of the ten interactors, FOX1, TBP, ATN1, CACNA1A, ATXN1,
ATXN3, ATXN7 and DDX6 contain a polyQ-rich element. In addition to the mTDPSupN
used in this study, TDP-43 has recently been shown to interact with DDX6 and
ATXN244,45,46. These three Q/N-rich proteins could physically interact each other and
localize to stress granules or P-bodies under certain conditions45. More recently, Elden et
al. demonstrated that treatment with RNase abolished co-immunoprecipitatation of
ATXN2 with TDP-43-YFP, suggesting that the interaction of ATXN2 and TDP-43 occurs
in an RNA dependent manner44. With regard to the RNA dependent regulation of TDP-43
activity, we found that RNA binding deficient mutants of TDP-43 proteins would form
visible granular aggregates and gained a ~20-fold TDP-43 visiable granules in nucleus
(Supplementary S10a)44. The relocalization of TDP-43 proteins from irregular puncta to
visible granules, where TDP-43 did not bind to RNAs, suggests that prion-like activity
may be regulated by RNA binding then affecting TDP-43 functions. We propose that the
Q/N-rich element of TDP-43 can be exposed when RNAs bind to TDP-43. The
allosterism can allow natively folded TDP-43 to interact with natively folded ATXN2 or
DDX6 in a prion-like manner (Fig. 5, conformer B). In the presence of RNAse, a
significant shifting of TDP-43 from monomer to dimer was observed by western blotting
analysis (Supplementary S10b). This result was consistent to observations in
supplementary S9a and c, and supported in absent of RNA, TDP-43 proteins formed the
other type of functional aggregates, proposed as conformer B’ of Fig. 5.
The cross- interactions between “cellular folded Q/N-rich domains” provide an
explanation for the reciprocal effects of prone-to-aggregation protein in common protein
misfolding disease. The biochemical and biophysical roles of the cellular folded Q/N-rich
domain are likely to retain the protein in the insoluble fraction by self- or
cross-interaction (Fig. 2 and Table 1). This function would allow the protein to form
dynamic scaffolds in a stable conformation and have a lower mobility, which is suited for
restricting the protein to a microenvironment optimal for performing particular
bio-reactions, such as alternative splicing and sub-compartment assembly.
As the aggregation-prone domain has structural plasticity and can spontaneously
self-assemble into ordered pathological or functional aggregates, it is reasonable to
speculate that these two pathways may act antagonistically, as shown in Fig. 5. Ideally,
compounds that stabilize functional aggregates of aggregation-prone domains may be
investigated as therapies for protein misfolding. This is particularly important for TDP-43
proteinopathies, because cleavage of TDP-43 proteins occurs prior to inclusion formation
during disease progression. Loss of normal full-length TDP-43 proteins perturbs
pre-mRNA splicing and ultimately leads to cell death. Growing evidence shows that
polyphenol compounds, such as EGCG, redirect an array of amyloidogenic molecules and
effectively cured huntingtin inclusions by oral administration in a Drosophlia
melanogaster model47,48. In our studies, EGCG induced oligomerlization of natively
folded TDP-43 and thus inhibited pathological-like degradation, suggested targeting to
stabilization of natively folded prion domains is a promise therapeutic approach for
FTLD-U and ALS. With an eye to therapy design for command of cellular- folded
aggregation- prone domain, future work should focus on the resolution of an accurate
cellular structure of the TDP-43 C-terminus and the identification of factors affecting
prion-like interactions under physiological and pathological processes. A potential
mechanism could be the interplay of post-translation modifications on the prion-like
domain.
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40. We are grateful to Dr. C.-H. Wong at Academia Sinica for discussion and
proofreading, to Dr. Carmay Lim at Academia Sinica for providing structural
insights, to Tsung-Yu Tsai for her assistance in writing this article and to Dr. F.
Baralle at the International Centre for Genetic Engineering and Biotechnology in
Italy for providing us with the hCF-TG13T5 minigene plasmid. We also thank the
staff of the Imaging Core of IMB, Academia Sinica. This research was supported by
Academia Sinica, National Health Research Institutes and National Science Council
grants to C.-K. James Shen (NSC 99-2321-B-001-033-) and G.-G.
Liou (MG-099-PP-08) and Garage Brain Science, Com (ND-01-001). I.-F. Wang
dedicates this article to her late sister.
LEGENDS
Figure 1. Dynamics of Detergent-Insoluble TDP-43 Aggregates in Normal Cells (a)
GFP-mTDP-43-FL, as endogenous TDP-43, formed dispersed visible granules
(arrow) and irregularly shaped puncta (arrowhead) in the cell nucleus. Scale bars:
10 µm. (b) Visualization of supramolecular GFP-mTDP-43-FL proteins by
time-lapse microscopy. Supramolecular GFP-mTDP-43-FL proteins can be
classified into two groups on the basis of their structure and dynamics. Time-lapse
fluorescent image of GFP-mTDP-43-FL expressing cells were taken every 1 min
for 30 min. Arrowheads in high-magnification images, upper panels, are shown to
demonstrate the dynamics of irregularly shaped puncta. Scale bars: 10 µm. (c)
Tracking of visible granules of GFP-mTDP-43-FL. White arrowheads indicate
stationary granules; yellow arrowheads indicate fission; red arrowheads indicate
fusion; blue arrowheads indicate the collision and separation of two distinct
granules. (d) Pre-extraction of nucleoplasmic proteins revealed clear TDP-43
aggregates in 293T cells. In the regular protocol (fixation/permeabilization; F/P),
transfectants were fixed with 3.75% paraformadehyde and permeabilized with
0.5% Triton X-100. In the pre-extraction protocol (P/F), transfectants were
extracted with 0.5% Triton X-100, and then fixed with 3.75% paraformaldehyde.
Cells without permeabilization (F) were used as a negative immunostaining
control. These fixed 293T cells were then immunostained with antibodies against
TDP-43. Images of cells were acquired by confocal microscopy. (e) Validation of
the solubility of GFP- mTDP-43-FL by western blot. The protein lysate harvested
from GFP-mTDP-43-FL-expressing 293T cells was used for immunoblotting with
anti-GFP antibody. (f) The disordered C-terminus of mTDP-43 (prion-like domain;
PLD) is required for induced granular assembly. In contrast to full-length TDP-43
(mTDP-43-FL), mTDP-43-PLD∆ appears evenly distributed in nuclei without TDP
granules.
Figure 2. Functional Substitutions of the GQN-rich, C-terminus of Mammalian
TDP-43 by a Prion Domain SupN. (a) Scheme of GFP-tagged chimerical
mTDP-yeast prion domain fusion construct. (b) Illustration of Q/N distribution in
the PLD, yeast prion domain Sup35 (1-125) (SupN) and UreN (1-84). (c)
Localization of GFP-tagged mTDPSupN and mTDPUreN. Replacement of the
PLD with SupN restored nuclear clustering in the manner of GFP-mTDP-43-FL.
The construct mTDPSupN formed dispersed visible granules (arrow) and
irregularly shaped puncta (arrowhead) in the cell nucleus. mTDPUreN formed
large foci in cytosol. Scale bars: 10 µm. (d) Visualization of TDP-43-FL proteins
and TDPSupN proteins by time-lapse microscopy. FRAP analysis of
GFP-TDP-43-FL, GFP-TDPSupN and GFP-TDP-43-PLD∆ movement. (e)
Selected images of TDPSupN expressing cells demonstrate fluorescence
recovery of GFP-tagged TDP-43 proteins. (f) Further examination of the
alternative splicing ability of GFP-tagged mTDPSupN by in vivo splicing assay.
The chimera mTDPSupN promoted exon-9 exclusion in a manner similar to
TDP-43-FL. Exon-9 inclusion (+) and exon-9 exclusion (-) bands are indicated. All
data are presented as means with SD (n = 3). M: marker; *: aberrant splicing
product. (g) An examination of TDP-43 self-interaction in vivo by cross-IP. The
protein lysate harvested from Flag- or Flag-TDP-43-expressing 293T cells was
used for immunoprecipitation with anti-Flag antibody and was further examined by
immunoblotting with anti-TDP antibody. Endogenous 43 kD TDP and 95 kD TDP
were co-precipitated with Flag-TDP-43. (h) Co-precipitation efficiency of
mTDP-43 variants with 95 kD TDP and 43kD TDP. The co-purification of TDP-43
with associated proteins was performed by cross-IP analysis with anti-GFP
antibody from the lysate of overexpressing GFP-tagged mTDP-43 variants FL,
PLD∆, or TDPSupN. The immunoprecipitates were further examined by
immunoblotting with an anti-TDP antibody. SupN complemented PLD in
interaction with endogenous TDP.
Figure 3. A proline Substitution in the Prion Domain of mTDPSupN or TDP-43
C-terminus Abolishes the Prion-like Interaction and Induces Pathological
Degradation of TDP-43 Proteins. (a) As illustrated, Proline substitution could
abolish the clustering of PLD if the substitute resides in a b-sheet (top); Proline
substitution has no effect on the clustering if the substitute resides in a loop region
(bottom). (b) Immunoblot analysis of G296P, G300P, Q302P and Q342P
mTDP-43 mutants. Linear illustration of the mutations substituted with proline is
shown in the upper panel. Anti-GFP antibodies were used for the immunobloting.
Note that TDP-43 Q302P substitution resulted in a pathological fragment
indicated by an asterisk. (c) At 4 days post-transfection, cytosplasmic
accumulation (arrow) and aggregation (arrowhead) was significantly increased in
the TDP-43 Q302P-expressing cells. Scale bar: 20 µm. (d) Statistical analysis of
cells with TDP-43 proteins accumulated in the cytosol for TDP-43-FL and TDP-43
Q302P transfectants. (e) Comparisons of half-recovery time of fluorescence
between GFP-TDP-43-FL proteins, GFP-TDP-43-PLD∆ proteins and
GFP-TDP-43 Q302P proteins by FRAP. In a small portion of TDP-43
Q302P-expressing cells, GFP-TDP-43 Q302P proteins have a lower half-recovery
time similar to the GFP-TDP-43-PLD∆ proteins. (f) A G25P substitution in the
prion domain of mTDPSupN abolishes the prion-like interaction and induces pathological degradation of mTDPSupN proteins. A biochemical analysis of mTDPSupN and mTDPSupN* proteins was examined using anti-GFP antibodies.
The construct mTDPSupN*, but not mTDPSupN, was cleaved into a ~48 kD fragment (*) and a 32 kD fragment. Tubulin and actin immunoblots were used as controls for non-specific protein degradation. (g) Localization of GFP-tagged mTDPSupN*. In contrast to GFP-tagged mTDPSupN, GFP-tagged mTDPSupN*
appeared diffuse in the cytosol and the nucleus of 293T cells. Scale bar: 10 µm.
(h) Deficiency of CFTR exon-9 skipping by GFP-tagged mTDPSupN*. Exon-9 inclusion (+) and exon-9 exclusion (-) bands are indicated. M: marker; *: aberrant splicing product. All data are presented as means with SD (n = 3).
Figure 4. The Effects of Epigallocatechin Gallate (EGCG) on TDP-43. (a) Stimulation with 20 µM EGCG increased the full length of TDPSupN*. The
TDPSupN*-expressing cells were treated with or without EGCG for 3 days and
were then harvested for analysis using anti-GFP antibodies. *Full length. All data
are presented as means with SD (n = 3). (b) EGCG induced oligomerization of
TDP-43. A biochemical analysis of endogenous proteins with or without EGCG
using anti-TDP-43 antibodies. The quantification of the oligomeric TDP-43
proteins is shown in the right panel. All data are presented as means with SD (n =
3). *P < 0.05 by ANOVA. (c) EGCG attuned the protein degradation of
GFP-Q302P-TDP-43. (d) Upon EGCG addition, GFP-Q302P-TDP-43 aggregates
(arrow) appeared by using anti-GFP antibodies. Scale bar: 10 µm. (e) Failure of
induced PLD truncated mTDP-43 mutant to form aggregates by EGCG. Scale bar:
10 µm. (f) MTT assay of viability of TDP-43-FL-expressing cells given EGCG. All
data are presented as means with SD (n = 5). (g) A biochemical analysis of
GFP-TDP-43-FL proteins with or without EGCG using anti-GFP antibodies.
Figure 5. Working Model for the Intrinsic Propensities of TDP-43 C-terminus in
Normal Cellular Functions and Disease Progression. As part of the normal
physiological processes of the cell, the intrinsic propensity of the TDP-43
C-terminus is to confer assembly of nuclear bodies, protein stability, and promote
exon9 skipping of CFTR by adopting flexible prion-like folding; however, if loss of
TDP-43 N-terminus by pathological cleavage, ALS-linked mutations or other
unknown cellular factors could cause disruption of cellular prion-like folding of the
TDP-43 C-terminus, TDP-43 inclusions will form. EGCG induced oligomerization
of TDP-43 and reduced its degradation.
Table 1. Properties of Cellular-folded and Misfolded Q/N-rich Domain.
