Abstract
Trans-activation response DNA binding protein of 43kDa (TDP-43)
regulates a great variety of cellular processes in the nucleus and
cytosol. In addition, a defined subset of neurodegenerative diseases is
characterized by nuclear depletion of TDP-43 as well as cytosolic
mislocalization and aggregation. To perform its diverse functions TDP-43
can associate with different ribonucleoprotein complexes. Combined with
transcriptomics, MS interactome studies have unveiled associations
between TDP-43 and the spliceosome machinery, polysomes and RNA
granules. Moreover, the highly dynamic, low-valency interactions
regulated by its low-complexity domain calls for innovative proximity
labeling methodologies. In addition to protein partners, the analysis of
posttranslational modifications showed that they may play a role in the
nucleocytoplasmic shuttling, RNA binding, liquid-liquid phase separation
and protein aggregation of TDP-43. Here we review the various TDP-43
ribonucleoprotein complexes characterized so far, how they contribute to
the diverse functions of TDP-43, and roles of post-translational
modifications. Further understanding of the fluid dynamic properties of
TDP-43 in ribonucleoprotein complexes, RNA granules, and self-assemblies
will advance the understanding of RNA processing in cells and perhaps
help to develop novel therapeutic approaches for TDPopathies.
INTRODUCTION
The trans-activation response DNA binding protein of 43kDa (TDP-43) is a
nucleic acid binding protein with many diverse functions. Although
originally cloned as a protein that binds to the trans-activation
response DNA element within the human immunodeficiency virus long
terminal repeat [1], very few DNA-binding functions have been
described for TDP-43. For example, TDP-43 binds to the SP-10insulator, acting as a repressor for this acrosomal gene product in
somatic tissues [2]. More recently, TDP-43 was reported to be
recruited to sites of DNA damage where it participates in non-homologous
end joining DNA repair [3, 4]. Moreover, TDP-43 confers DNA
protection and genome stability by alleviating the co-transcriptional
formation of R-loops that promote transcription/replication conflicts
[5, 6].
Beyond DNA binding, far more studies established the RNA binding roles
of TDP-43 (Figure 1). TDP-43 was initially discovered as a splicing
factor mediating exon 9 exclusion of the cystic fibrosis transmembrane
conductance regulator transcript [7]. By binding to mostly intronic
GU-rich sequences, TDP-43 regulates splicing of canonical as well as
cryptic exons [8-10]. By alternative splicing of cryptic exons
TDP-43 regulates the use of alternative poly-adenylation sites, thus
affecting the expression of its own transcript [11] or that of
stathmin-2 [12]. In fact, TDP-43 binds to thousands of transcripts,
not only pre-mRNAs (coding and non-coding) but also microRNA (miRNA)
precursors [13, 14]. In addition to the regulation of alternative
splicing, TDP-43 also mediates mRNA stability, as exemplified for the
transcripts of neurofilament L [15] and the histone deacetylase
(HDAC) 6 [16]. TDP-43 can form a complex with the fragile X mental
retardation protein (FMRP) and the Staufen homolog STAU1, promoting
stabilization of the HDAC1 transcript [17]. Moreover, TDP-43 can
enhance mRNA translation via association with ribosomes [18].
Finally, TDP-43 participates in RNA transport processes in axons
[19] and during the assembly of stress granules (SGs) [20].
The manyfold diverse functions of TDP-43 in all these steps of RNA
processing are not only fascinating from a basic cell biology point of
view, but also bear great disease relevance. Neuropathological TDP-43
inclusions [21, 22] constitute defining lesions of specific subtypes
of the neurodegenerative dementia frontotemporal lobar degeneration
(FTLD) and amyotrophic lateral sclerosis (ALS), a motoneuron
degeneration [23]. Moreover, mutations in the TDP-43 gene are linked
to ALS [24-27]. During pathogenesis, TDP-43 must partition from its
physiological complex sites to phase-separate into liquid droplets and
further solidify into pathological aggregates. The challenge is to
identify all these physiological complex compositions and the assemblies
formed in disease course. Another essential issue is the regulation of
TDP-43 targeting into the respective protein complexes, possibly
involving post-translational modifications. Proteomic investigations
continue to provide insight into this important topic.
PROTEOMIC METHODOLOGIES TO STUDY TDP-43 COMPLEXES As the majority of proteins do not act as single element but rather
carry out distinct cellular functions in form of cell-type and
context-specific protein complexes, the systematic dissection of
protein-protein interaction networks gained importance in the past
decades. In particular, the availability of fast and sensitive mass
spectrometers fueled this process. The classical way to analyze
protein complexes is the co-purification of interacting proteins with
a bait protein, either by the recombinant expression of tagged
proteins or the immunoprecipitation using primary antibodies against
the target protein. The precipitated proteins are then analyzed by MS.
Originally tandem-affinity purification has been used to get highly
pure protein complexes allowing to identify specific interactomes.
Initially used for the investigation of protein complexes in yeast,
protocols have been developed for the analysis of interatomic networks
in mammalian cells [28-31]. A tandem-affinity purification MS
approach using an extended HIS-tag system in combination with a
custom-made antibody has recently been applied to TDP-43 [32],
revealing interactions related to protein stabilization and protein
folding upon oxidative stress stimulation. In addition, pull-downs
with a recombinant protein or protein domain have widely been used to
study protein-protein interactions [33, 34]. However, one major
drawback of these approaches is its bias for rather stable protein
interactions, given stringent washing conditions. As an alternative
strategy to cope with unspecific background quantitative MS, either
label-free of label-based, has been established to identify specific
interactions. SILAC has widely been used for this purpose.
Immunoprecipitation combined with SILAC and a knock-down control
emerged as a powerful tool to investigate protein complexes at their
native expression levels [35, 36]. Affinity based methods have
also been applied to TDP-43, in particular to the TDP-25 fragment (see
below), to assign interatomic networks [37]. Another study
uncovered TDP-43 and FUS binding to several factors important to DNA
repair mechanisms by affinity proteomics [38]. Furthermore, an
ALS-linked mutation in TDP-43 has recently been shown to alter protein
interactions involved in the motor neuron response to oxidative stress
[39]. One of the most comprehensive studies so far to elucidate
TDP-43 complexes for WT and two disease-causing variants (A315T and
M337V) was an immunoprecipitation-based approach following the
recombinant expression of FLAG-tagged TDP-43 bait proteins. This study
revealed to clusters of distinct interaction networks, a
nuclear/splicing cluster and a cytoplasmic/translation cluster while
no alterations by the disease-causing variants were observed, most
likely as the identification of co-precipitated proteins was relying
on a qualitative, identification-based approach, not considering
quantitative changes of the interactome [40]. Finally, a study,
combining tandem-affinity purification with SILAC-based quantitative
proteomics not only revealed expected interactions with heterogeneous
nuclear ribonucleoproteins (hnRNPs) but also identified components of
the Drosha complexes in the TDP-43 interactome, which is consistent
with roles for TDP-43 in both mRNA processing and miRNA biogenesis
[41].
While these techniques certainly represent powerful tools to
investigate large interatomic networks, they do not necessarily
identify direct protein interactions. One method developed three
decades ago is the yeast two hybrid system which emerged, thanks to
the mating system, to a powerful screening tool of a large set of bait
proteins [42]. By this approach, a large protein interaction
networks associated with neurodegenerative diseases has been described
[43]. TDP-43 was one of the 500 bait proteins tested in this
study. A more focused yeast two hybrid screen identified enzymes
involved in the regulation of TDP-43 ubiquitinylation [44].
Despite of the significant achievements possible due to the systematic
application of this methods, there is still an unmet need for the
identification of transient interactions at physiological conditions
and within intact cells. This is especially important for TDP-43 with
its low-complexity domain that engages in fluid dynamic protein
assemblies rather than rigid stable complexes. Recent developments of
proximity-based methods allowing the direct or indirect proximity
biotinylation of proteins in a considerable small distance to the bait
protein might help to close this gap [45]. Proximity-dependent
biotin identification involves bait protein fusion to enzymes based on
bacterial BirA biotin ligases that have been subject of continuous
optimization, now allowing a very efficient biotinylation at low
biotin concentrations and in a short time. Alternatively, proximity
labeling can be achieved by oxidative activation of biotin-phenol
using ascorbate peroxidase. Both methods offer a great opportunity to
gain more insight into the spatial organization of protein complexes
orchestrated by TDP-43, in particular following specific interactions
associated with RNA and/or liquid-liquid phase separation (LLPS).
Consequently, a recent study has applied biotin proximity labeling to
identify novel interaction partners of detergent-insoluble TDP-43
aggregates. The identified proximity-proteome was enriched for
components of the nuclear pore complex and nucleocytoplasmic transport
machinery which strongly implicate TDP-43-mediated nucleocytoplasmic
transport defects as a common disease mechanism in ALS/FTLD [46].
The achievements of the interatomic studies and their impact on the
current understanding of the TDP-43 (patho-)physiology is discussed in
greater detail in the next section.
TDP-43 INTERACTOMES
While unbiased approaches searching for TDP-43 partners have provided a
wealth of information, we can now use these data to focus on TDP-43
functions and attempt to describe in detail the nature of TDP-43
interactions in relation to the function and complex where it is found
(Table 1). In the following sections this review will try to identify
specific roles of TDP-43 in the different ribonucleoprotein complexes
where it is found and describe the nature of its interactions there.
Transcription
Although most research focused RNA-related effects (elaborated below),
TDP-43 can bind to DNA, with a preference for TG-rich sequences
[47]. Reducing promoter binding of TDP-43 by RNA interference caused
a global dysregulation of nascent transcripts, with up to a third of the
detected transcripts going up and around 5% going down [48]. From
the repressed transcripts, it is interesting to note that there is an
increase in Alu retrotransposons, suggesting that TDP-43 acts as a
transcriptional repressor of these elements. However, TDP-43 does not
interact directly with Alu DNA sequences, therefore the interaction
between TDP-43 and Alu elements transcription must occur in a different
way.
Another example of the role of TDP-43 in transcription is its repression
of the Acrv1 gene in murine testis, where it can bind to the
TG-rich Acvr1 promoter region and act as a transcriptional
repressor in spermatids [49, 50]. Another specific example of
transcriptional repression involves the ESCRT component vacuolar protein
sorting associated protein VPS4B, which is involved in the trafficking
of recycling endosomes in neurons. When TDP-43 was silenced in
iPSC-derived neurons, VPS4B RNA and protein increase and the endosome
recycling in neurites was impaired [51]. These examples, together
with the larger scale studies, show that the role of TDP-43 in gene
regulation extends to RNA transcription, where TDP-43 can directly bind
to TG-rich promoter regions and influence the expression of several
hundreds of targets. While still underrepresented in the field, this is
an aspect of TDP-43 biology that most probably will be expanded in
future studies.
miRNA Processing
MiRNAs are small, non-coding RNAs with an average size of 22
nucleotides, which can regulate gene expression by affecting mRNA
stability and protein expression. They can be encoded in intergenic
regions of the genome or in introns of related or unrelated genes
[52]. Most of the miRNA transcripts are produced as pri-miRNA, which
is processed in the nucleus by the Drosha complex into pre-miRNA. After
that, pre-miRNA is exported via the exportin 5 system to the cytoplasm
[53], where it is further processed by the Dicer complex, producing
mature miRNAs. This mature miRNA can then altered gene expression or its
targets [53]. Interestingly, TDP-43 is involved in both the nuclear
and cytoplasmic portions of the miRNA maturation process.
The clearest evidence that shows an involvement of TDP-43 in the miRNA
maturation process is the direct interaction it has with Drosha, Dicer
and argonaute-2 [14, 40]. TDP-43 can bind to both Drosha and Dicer
via its C-terminal domain, and the Drosha interaction seemed to be
affected by TDP-43 phosphorylation at S409/410 [14, 54]. A closer
inspection of TDP-43 role in both complexes show that it can directly
bind to some pri-miRNA via the consensus poly(UG) sequence, such as
pri-miRNA-574, pri-miRNA-578 and increase the binding affinity of the
Drosha complex to those sequences [14]. This data is supported by
the fact that silencing TDP-43 triggers a nuclear accumulation of
unprocessed pri-miRNA let-7b, pri-R-181c, pri-miRNA-574 and
pri-miRNA-578 [13, 14, 55], suggesting that a lack of TDP-43 reduces
the pri-miRNA cleavage efficiency of the Drosha complex. Because of
this, it is not surprising that a reduction in TDP-43 levels can alter
the levels of several miRNAs [55, 56].
Interestingly, some of the gene targets of these miRNAs have been found
to be altered in TDP-43 depletion models, which offers a mechanism
linking both datasets [13, 56]. To link these results to disease,
the effect of TDP-43 on miRNA biogenesis has been investigated in
differentiating neurons and in ALS and FTD-TDP-43 patients. A knockdown
of TDP-43 in differentiating neurons causes reduction in Drosha levels,
however Drosha mRNA levels do not change and the exact mechanism for
this change is not clear [56]. In addition, several miRNAs have been
reported to be altered in patients with TDP-43 pathology, but the
disease relevance of this finding remains unknown [57]. Finally, it
is worth noting that the results of the studies seem to be very
cell-type specific and suggest a highly complex and dynamic regulation
of miRNAs by TDP-43.
RNA Splicing
The splicing of thousands of RNAs have been linked to the presence of
TDP-43, both in terms of exon splicing and in the suppression of cryptic
exons [8-10]. The role of TDP-43 in splicing it is further supported
by its interactions with several spliceosome and splicing-related
proteins. For instance, TDP-43 has been found associated with
proline/glutamine-rich and serine/arginine-rich splicing factors and
many hnRNPs [40]. Indeed, TDP-43 functions in conjunction with
hnRNPs to promote splicing [58-60].
More directly, TDP-43 interacts with the small nuclear ribonucleoprotein
of 70kDa (snRNP70) [61], a component of the U1 snRNP complex that
stabilizes the interaction between the snRNA U1 and the 5’ splice site
of a processed pre-mRNA [62]. TDP-43 was also detected by MS among
the proteins co-immunoprecipitated with FLAG-tagged C9ORF78, a natively
unfolded protein that acts within the U5 snRNP [63]. As the C9ORF78
interaction partner snRNP200/BRR2 helicase was enriched in TDP-43
immunoprecipitates [40], integration of TDP-43 into the 3’ splice
site helicase activity could in part explain the alternative splicing
functions of TDP-43, in addition to a potential role for pre-mRNA 5’
splice initiation at the U1 snRNP complex.
“Like Smith antigen” (LSm)6 was identified as a TDP-43 partner in a
yeast two-hybrid study [44]. LSm6 together with LSm5 and LSm7
organizes the formation of LSm heteroheptameric rings [64]. In the
nucleus, the LSm2-8 complex is engaged in the assembly of the
pre-catalytic spliceosomal B complex by binding to the 3’ end of U6
snRNA, and is part of the structural reorganizations that occur in the
subsequent spliceosome activation step involving the aforementioned
helicase snRNP200/BRR2 that unwinds the U4/U6 snRNAs [65].
Interestingly, there is a second LSm complex in the cytoplasm (LSm1-7)
that is part of the mRNA degradation machinery [66-68]. Likewise,
there are dual functions of the LSm associated “protein associated with
topoisomerase II” (Pat)1b in the nucleus and in the cytoplasm [69].
Because TDP-43 can also shuttle between the nucleus and the cytoplasm,
such interactions are particularly noteworthy. Moreover, Pat1 can
enhance LLPS of mRNA decay factors in processing bodies [70, 71],
membraneless organelles that are sites of mRNA degradation, storage and
repression. Taken together, TDP-43 with its intrinsically disordered
domains might be involved in structurally dynamic assemblies that
mediate RNA splicing in the nucleus as well as mRNA regulation in RNA
granules (see also below).
Coupling of RNA splicing and decay underlies the mechanism of TDP-43
autoregulation through a negative feedback loop [9, 72, 73]. TDP-43
autoregulation has been confirmed in vivo both in mouse models
[74] and in an FTLD patient with a mutation in TDP-43 3’UTR,
resulting in higher TDP-43 levels [75]. TDP-43 binds to a conserved
region in the 3’UTR of its own transcript. The binding to this sequence
promotes TDP-43 oligomerization and assembly into dynamic
ribonucleoprotein granules [76]. Two autoregulation mechanisms
involving alternative splicing have been described, one dependent on
nonsense-mediated RNA decay and the other one exosome-dependent. In the
nonsense-mediated decay model, TDP-43 binding favors alternative
polyadenylation signals pA2 and pA4, leading to RNA degradation
[73]. The second proposed mechanism involves inclusion of a cryptic
exon 7. This alternatively spliced mRNA is highly unstable, and it is
degraded via the exosome system [72, 77]. By a similar mechanism
targeting a cryptic exon TDP-43 regulates expression of the autophagy
gene product ATG4B [8, 78].
In addition to its own transcript, TDP-43 binds to 3’UTR regions of many
more mRNAs [10, 79, 80]. For example, TDP-43 binding to the 3’UTR
stabilizes the neurofilament L mRNA [15], which can be affected by
TDP-43 mislocalization and relieved by autophagy induction [81].
Likewise, TDP-43 binds to the 3’UTR of the transcript encoding
GTPase-activating protein binding protein (G3BP) to promote its
expression [82, 83]. Moreover, binding of TDP-43 within the coding
region of HDAC6 mRNA stabilized its protein expression [16, 84]. On
the other hand, overexpression of TDP-43 destabilized the mRNA coding
for the FTLD-linked gene product progranulin and hence reduced its
protein levels [80]. As this interaction was detected in the
cytosol, TDP-43 affects mRNA levels not only at the level of alternative
pre-mRNA splicing in the nucleus, but also controls mRNA stability in
the cytosol.
mRNA Transport
After splicing, capping and polyadenylation, mature RNA is transported
to the cytoplasm for further translation at the ribosome. Several
RNA-binding proteins are known to play a role in this process, being
TDP-43 one of them. While the mRNA transport takes place in every cell,
in large cells with complex morphology such as neurons it is a critical
process that allows for local translation of proteins that are needed at
neurites or distal segment of the axons. It is in this context where
TDP-43 has been found to interact with several other RNA-binding
proteins and play an important role.
The previously mentioned serine/arginine-rich splicing factors [40]
could couple splicing with nuclear export of mRNA [85]. Once in the
cytosol of neurons, TDP-43 colocalizes with mRNA granules in axons
[86], where it can interact with FMRP and STAU1 [17, 40, 87]. A
closer look at these interactions showed that TDP-43 can mediate both
anterograde and retrograde transport depending on the protein partner.
TDP-43 is engaged in anterograde transport when interacting with FMRP,
while interaction with STAU1 mediates retrograde transport [87].
Interestingly, pathological mutants M337V and A315T impaired RNA granule
transport along axons [19]. Since both of these mutations are
located in the C-terminal domain, linked with protein-protein
interaction, it is possible that the defect in mRNA transport comes not
from a change in mRNA binding affinity, as much as a change in protein
binding affinity to other partners, such as FMRP and STAU1.
Specifically, substitutions at W334 in TDP-43 C-terminal low-complexity
domain impact both the number of mRNA granules found in axons and mRNA
anterograde transport [88].
When involved in axonal transport, TDP-43 forms liquid droplets that,
interestingly, change their biophysical properties as they circulate
along the axon [89]. As TDP-43 granules move from proximal to distal
sections of the axons the liquid phase becomes more fluid and dynamic.
This transition is affected by pathological mutations (M337V and G298S).
The reasons behind this transition and its significance in TDP-43
pathophysiology remains to be uncovered.
Stress Granules
In addition to processing bodies and transport granules (see above),
cytosolic mRNA can also be packaged into SGs [90], along with
prominent recruitment of TDP-43 [20]. SGs are membrane-less
organelles that form upon different cell stressors, such as oxidative
stress or heat shock. They are formed by an inner stable core and a
fluid shell, and they are composed by a mixture of RNAs, RNA-binding
proteins, ribosomes and scaffold proteins [91]. Their canonical
function is to store stalled ribosomes during stress and continue
translation after the stress has disappeared, however this hypothesis
has been questioned in recent years. While translational repression
elements are enriched in SGs [92], they are not entirely essential
for translational repression as single molecule imaging has revealed
active translation within SGs [93]. It is at present unclear what
the ultimate function of these organelles are, or even if they are a
mere epiphenomenon triggered by stress without a specific function
[94].
Over 200 proteins have been found to be enriched in SGs from stressed
cells, and several of those, such as G3BP and T cell restricted
intracellular antigen-1 (TIA-1), have been identified as members of the
SG core [91]. Interestingly, TDP-43 has been found to interact with
both of them in an RNA-independent manner [95] and it is recruited
to SGs upon cell stress [20, 83]. However, the role of TDP-43 in SGs
is less clear. Knockdown of TDP-43 causes a reduction in G3BP mRNA and
protein levels, while on the other hand the same depletion can increase
TIA-1 amounts in cells [83, 96]. These modulations of SG core
components do not prevent their formation, but they do change the
engagement and disengagement dynamics. A closer look at the interaction
between G3BP and TDP-43 shows that upon artificial G3BP SG formation via
an optogenetic system, TDP-43 and TIA-1 are recruited to the initial
G3BP1 granules [97]. These results support the idea that G3BP is an
early core element of SGs and that TDP-43 is perhaps a recruiter of RNA
targets to SGs. The impact in SGs dynamics caused by TDP-43 knockdown
could be due to the modulation of SG members’ mRNAs, and not via direct
action of TDP-43 at the SGs. In SG-inducing cellular stress conditions
TDP-43 still forms insoluble aggregates when SG formation proper is
blocked [98, 99]. Thus, although TDP-43 clearly participates in SGs,
the (patho)physiological significance remains obscure.
Protein Translation
Beyond translational stalling in SGs TDP-43 may generally affect protein
translation via a cytosolic translation interactome cluster [40].
Particularly when the predominantly nuclear TDP-43 is mislocalized in
the cytoplasm it can cause a decrease in global protein synthesis
[100, 101]. Overexpression of TDP-43 did not seem to cause changes
in total translation both rather affected translation of select
neurodegeneration-relevant mRNAs identified by ribosome profiling
[18]. TDP-43 binding to such mRNAs caused altered translation in
overexpression or cytoplasmic mislocalization models [102-104].
Association of TDP-43 with polysomes [18, 101] may not only occur by
binding to RNA but also proteins, such as the receptor for activated C
kinase 1 (RACK1) [40, 44, 101], a multifunctional scaffold protein
present in ribosomes. Also, TDP-43 mediated alternative splicing of the
exon junction complex protein ribosomal S6 kinase 1 Aly/REF-like target
(also known as polymerase delta interacting protein 3) has been shown to
affect global translational yield [105]. Moreover, TDP-43 is
involved in the axonal transport of ribosomal proteins and reduced
levels of TDP-43 can cause a reduction in local translation in the axons
[103, 106]. Thus, in addition to its functions in RNA processing,
TDP-43 may also affect the (patho)physiological proteome via protein
translation regulation [107].
Non-Coding RNAs
In addition to the RNA species mentioned above, TDP-43 also interacts
with long non-coding RNAs (lncRNAs) [10]. The level of metastasis
associated lung adenocarcinoma transcript 1 (MALAT1) (aka noncoding
nuclear-enriched abundant transcript 2, NEAT2) is regulated by TDP-43,
with overexpression of TDP-43 causing an increase in MALAT1 RNA levels,
and TDP-43 knockdown causing the opposite [108]. A detail
examination of the MALAT1 elements identified a short interspersed
nuclear element to be involved in the localization of the molecule,
whose deletion would cause mislocalization to the cytoplasm and
sequester TDP-43 into liquid droplets [109].
In the context of NEAT1 interaction, it has been reported that TDP-43
can colocalize with it in paraspeckles [110]. Specifically, the
isoform NEAT1_2 seems to act as a scaffold and recruits TDP-43 into
these nuclear bodies, triggering TDP-43 LLPS [111, 112].
Interestingly, the disease-linked mutation D169G interfered with the
NEAT1-mediated TDP-43 LLPS [112]. On the other hand, silencing of
TDP-43 increased levels of the stress-induced long isoform NEAT1_2 and
stimulated paraspeckle assembly [113, 114]. Conversely, upon
accumulation of TDP-43 the major long isoform NEAT1_1 was found
up-regulated and appeared to counteract pathological effects of TDP-43
[115]. TDP-43 interplay with NEAT1 therefore seems to be an
important regulatory mechanism influencing cellular viability via
paraspeckles, nuclear domains mediating RNA processing and metabolism
[116].
MALAT1 and NEAT1 are among the strongest TDP-43 binding RNAs [10].
In addition, a lncRNA called growth-arrested DNA damage-inducible gene 7
was found to compete with TDP-43 binding to the mRNA encoding
cyclin-dependent kinase 6, thereby interfering with its mRNA decay and
thus controlling cell cycle progression in Chinese hamster ovary cells
[117]. Binding of TDP-43 to neuroLNC in this case selectively
stabilized mRNAs encoding synaptic vesicle proteins to ensure
presynaptic function and neuronal excitability [118]. Last but not
least, SILAC profiling found TDP-43 enriched in chromatin with the
X-inactive specific transcript [119] where it participates in a
condensate required for gene silencing [120]. The relevance of
specific TDP-43 interacting hnRNPs in this process [121] remains to
be elucidated. It emerges that TDP-43 regulates cellular proteomes not
only by interactions with and partitioning of protein-coding mRNAs and
miRNA processing, but also via lncRNAs.
Nucleocytoplasmic Shuttling
The manyfold functions of TDP-43 in the nucleus and cytoplasm obviously
require nucleocytoplasmic shuttling, which is mediated by active nuclear
import through a bipartite nuclear localization sequence (NLS) between
K82-K98 and probably passive, exportin-independent nuclear export
[122, 123]. Importin-α1 recognizes the TDP-43 NLS and forms a
heteromeric importin-α1/β complex with TDP-43, and this interaction can
be modulated by three phosphorylations at the NLS (T88, S91 and S92)
[124]. In addition, the key NLS residue K84 can be ubiquitinated as
well as acetylated, with both modifications altering TDP-43
nucleocytoplasmic shuttling [125, 126] (see below). In FTLD and ALS
this balance is altered and cytoplasmic mislocalization of TDP-43 is
considered a clear marker of its proteinopathy [21, 22]. Because of
this, it has been the focus of a considerable amount of research.
The deletion mutant ΔNLS-TDP-43 mislocalizes to the cytoplasm, where it
can become less soluble [127]. In addition, under arsenite stress
TDP-43 shifts into the cytoplasm where it may become less soluble and
phase-separates into stress granules and eventually turns into protein
inclusions similar to those found in patients [128]. In addition,
cytoplasmic aggregates of C-terminal truncated TDP-43 can recruit
nuclear import elements such as importin-α and nuclear pore components
such as the nuclear pore glycoprotein of 62kDa (NUP62), disrupting
general nuclear import and the nuclear lamina structure [46, 128].
Interestingly, NUP62 co-aggregated with cytoplasmic TDP-43 can recruit
karyopherin-β1, which in turn interacts with aggregated TDP-43
C-terminal fragment (CTF) to reduce aggregation [129]. This newly
reported interaction hints to a complex network of interactions between
TDP-43 and nuclear transport elements that is not limited to the
importin system and opens new ways to reduce TDP-43 aggregation.
POST-TRANSLATIONAL MODIFICATIONS OF TDP-43
It was evident already at the time of discovery that TDP-43 in disease
is altered by modifications including phosphorylation, ubiquitinylation,
and truncation leading to CTFs commonly referred to according to their
approximate molecular masses as TDP-35 and TDP-25 [21, 22].
Moreover, under oxidative stress TDP-43 cysteine residues C173-C175 can
form disulfide bonds [130] and in the concomitant presence of nitric
oxide these residues become S-nitrosylated [131], leading to
oxidation-mediated TDP-43 aggregation and neuropathology. Thus, the
investigation of TDP-43 post-translational modifications may provide
important clues for the regulation of (patho)physiological functions and
disease-promoting processing of TDP-43.
TDP-43 Phosphorylations
The most commonly detected pathological modification of TDP-43 is
phosphorylation of serines-409 and -410 [132, 133] (Figure 2A). It
is of note that potential phospho-acceptor threonine and serine residues
are spread among the entire sequence of TDP-43, immunoreactivity with
post-mortem disease tissue was only detected with antibodies selective
for serine residues in the C-terminus [134, 135]. Consistently,
phospho-sites were clustered in the TDP-43 C-terminus in a small LC-MS
analysis of insoluble material extracted from the brains of 2 ALS
patients [136]. Similar to other intracellular amyloidogenic
proteins [137, 138], the broadly acting casein kinases were capable
of phosphorylating TDP-43 in vitro . Casein kinase 1
(CK1 ) strongly phosphorylated TDP-43 at serines 379, 403/404
and 409/410 and appeared to promote TDP-43 aggregation in vitro[134]. The CK1ε homolog doubletime in Drosophila enhanced
TDP-43 toxicity in a fly model [139]. Expression of a constitutively
active form of CK1δ promoted TDP-43 pathology in SH-SY5Y neuroblastoma
cells [140]. Endoplasmic reticulum stress might be a trigger for
CK1-dependent TDP-43 phosphorylation and aggregation in motor
neuron-like cells [141]. Inhibition of CK1δ showed protective
effects in [A315T]TDP-43 transgenic mice and in lymphoblasts of ALS
patients [142]. However, Gruijs da Silva et al. recently
reported on the contrary that CK1δ phosphorylation as well as
phosphomimic mutagenesis did not affect TDP-43 functions but rather
suppressed TDP-43 aggregation, rendering TDP-43 condensates more
liquid-like and dynamic [37]. It remains to be further established
if CK1 inhibitors are a viable therapeutic option for the treatment of
ALS [143].
Screening for TDP-43 kinases in C. elegans , Liatchko et al. found
cell division cycle 7-related protein kinase (CDC7 ) to
phosphorylate TDP-43 S409/410 and promote toxicity in worms [144].
CDC7 inhibition decreased TDP-43 phosphorylation in a variety of models
and restored TDP-43 function in human patient lymphoblasts [145,
146]. Moreover, the tau-tubulin kinases 1 and 2 (TTBK1 /2)
were identified as putative disease promoting pS409/410 TDP-43 kinases
[147]. Unlike other putative TDP-43 kinases, TTBK1 is expressed
predominantly in the CNS and thus localized to
neurodegeneration-relevant areas [148]. TTBK1 was confirmed to
phosphorylate TDP-43 at disease-relevant sites S409/410 and S403/404in vitro and in arsenite-stressed cells and to induce
pathological TDP-43 effects including cytoplasmic mislocalization
[149]. As more effective TTBK1 inhibitors are being developed
[150], it will become interesting to explore the therapeutic
potential and relative contribution of TTBK1 and 2 for FTLD-TDP and
FTLD-TAU [151].
TDP-43 Ubiquitin Modifications
The attachment of ubiquitin moieties in post-mortem brain
tissue of TDPopathy patients was evident already from the first study
[21]. Formation of diverse ubiquitin chains on lysine residues of
target proteins are most important post-translational modifications that
regulate a large variety of cellular fates, including protein
trafficking and turnover [152]. Specifically, the attachment of
poly-ubiquitin chains linked via lysine-48 targets proteins to
proteasomal breakdown while ubiquitin binding motifs couple
ubiquitinylated cargo to the autophagy machinery. Thus, initial studies
dealt with the question of proteasomal and autophagic breakdown of
TDP-43. Cells treated with proteasome inhibitor showed accumulation of
poly-ubiquitinylated, insoluble TDP-43 [127, 153]. The proteasome
appears to act synergistically with the autophagy machinery in the
catabolism of aggregating TDP-43 [154, 155].
MS analyses assessing the characteristic DiGly-shifts indicated several
lysine residues within TDP-43 as putative ubiquitin anchor sites [126,
156]. Site-directed mutagenesis revealed not a single lysine residue
to account for TDP-43 ubiquitinylation effects, indicating considerable
redundancy of the TDP-43 lysine-ubiquitin system. The RNA binding region
harbored one cluster of ubiquitinylated TDP-43 lysine residues (K102,
K114, K145, K181) [156], which were also detected in global
ubiquitylome surveys [157, 158]. Explicit investigation of DiGly
motifs in pulled-down TDP-43 once more confirmed ubiquitinylation at
K181, which appeared constitutive in all conditions [126].
Ubiquitinylations at K102 and K114 were detectable when the 6HIS-tag was
attached to the TDP-43 C-Terminus [126]. Modifications at K145 were
not detected in this study, perhaps due to incomplete coverage.
Interestingly, a second cluster of putative TDP-43 ubiquitinylation
sites was detected after proteasome inhibition at the NLS residue K84
and K95 [126], as well as K160 that had been previously found in
proteome-wide screens of ubiquitinylation sites of proteasome-inhibited
cells [157, 158]. Site-directed mutagenesis confirmed K95 as a
potential proteasome-targeting ubiquitinylated residue [126] and was
a major residue mediating TDP-43 mislocalization upon proteasome
inhibition by administration of poly-GA protein [159] that can be
produced from pathogenic repeat expansions of the FTLD/ALS geneC9ORF72 .
Taken together, TDP-43 can be ubiquitinylated at several lysine
residues, and not a single site confers proteasome or autophagy
targeting except for possibly K95 under special conditions. Moreover,
any influences of non-classical ubiquitinylations on RNA binding,
subcellular localization, LLPS and aggregation remain to be further
explored. It is noteworthy that ubiquitinylated TDP-43 tends to be
shifted into mislocalized insoluble aggregates, for example after
expression of the ubiquitin ligase parkin [160], UBE2E
ubiquitin-conjugating enzymes [44], the cullin-2 substrate binding
component VHL [161] or FTLD/ALS-linked mutant
SCFcyclin F [162]. Conversely, the ubiquitin
ligases Znf179 [163] and Praja1 [164] enhanced
ubiquitin-dependent clearance of TDP-43. Clearly ubiquitinylations of
TDP-43 are highly complex and the outcomes likely depend on cellular
context and ubiquitin code written be distinct ubiquitin ligases. As for
de-ubiquitinating enzymes, only the rather general UBPY/USP8 has been
described so far, counteracting ubiquitinylated TDP-43 pathology in cell
and fly models [44].
TDP-43 is also a target for the small ubiquitin-related modifier
(SUMO ) [165, 166]. A SUMOylation consensus site comprises
K136 within the RNA binding domain. Inhibition of SUMOylation with
anacardic acid treatment of K136R substitution reduced TDP-43
aggregation and cytotoxicity [165]. Mutant [K136R]TDP-43 showed
reduced binding and splicing activity towards several target mRNAs
[166]. However, it should be noted that K136 is also subject to
lysine acetylation (see below), so it has to be carefully determined if
SUMOylation or acetylation account for effects observed by K136
mutagenesis.
TDP-43 Lysine Acetylations
In addition to ubiquitin modifications, protein lysine residues can also
be acetylated. TDP-43 acetylation at K145 and K192 was first described
by Cohen et al. [167]. It was found that stress-induced K145
modification directly alters RNA binding activities and aggregation
propensity of TDP-43. Histone deacetylase 6 removed this potentially
pathogenic modification. Similar effects were found for TDP-43 K136
acetylation, in this case sirtuin-1 acted as a relieving deacetylase
[125]. Importantly, in this study amber suppression expansion of the
genetic code was established for the first time to introduce the
authentic modified amino acid into defined sites. Although site-directed
mutagenesis is a powerful tool to stimulate small post-translational
modifications of amino acids such as serine/threonine phosphorylation
and lysine acetylation, respectively, the aspartate/glutamate and
glutamine substitutions are not identical to the phosphorylated or
acetylated residues. Although molecular dynamics simulations suggested
little impact of the K136R substitution on the local structure within
the RNA-binding domain [166], subtle clashes with bound RNA cannot
be ruled out [125]. Nevertheless, amber suppression confirmed the
results of site-directed mutagenesis, showing that K136 acetylation
reduced RNA binding and splicing activity. Importantly, the K145 or K136
acetylation-mediated disengagement of TDP-43 from native RNA-binding
protein complexes led to LLPS and subsequent pathological aggregation of
TDP-43 in the nucleus, or when combined with nuclear import deficiency,
formation of modified TDP-43 inclusions in the cytosol, the most common
hallmark of TDPopathies [125, 168]. Demixing of RNA binding
deficient TDP-43 into liquid droplets and conversion into gel/solid is
controlled by chaperone proteins, such as HSP70 in the nucleus [169]
or HSPB1 in the cytosol [170], likely as part of an HSF1-induced
chaperone response system [168].
Moreover, K84 was identified as another acetylated residue in TDP-43. As
this is a key determinant of the nuclear import sequence [126, 127],
K84 acetylation affected the nucleocytoplasmic distribution of TDP-43
[125]. Taken together, it is noteworthy that similar to the lysine
ubiquitin modifications also the acetylated lysine residues are
primarily located in the nuclear import sequence and the RNA binding
domain. These essential aspects of TDP-43 (patho)biology are at least in
part regulated by lysine modifications, with acetylations in the
RNA-binding region apparently having the most straightforward pathogenic
potential [171].
TDP-43 Truncations
The presence of TDP-43 CTFs is a conspicuous feature discovered in FTLD
and ALS patient brain [21, 22] but not as prominent in spinal cord
[39, 172]. There is considerable CTF heterogeneity in patient
isolates [136, 173, 174], making it hard to pinpoint a defined
protease cleavage mechanism. Nonaka et al. found putative cleavage sites
between M218-D219 and E246-D247 in a MS analysis of insoluble TDP-43
from FTLD brain and reported that such transfected CTF-GFP fusion
proteins caused aggregation and interfered with TDP-43 splicing activity
in neuroblastoma SH-SY5Y cells [175]. Caspases can cleave TDP-43 to
roughly appropriately sized CTFs [176-179]. Although apoptotic
caspase stimulation caused some CTF formation it did not appear to be an
indispensable factor for TDP-43 aggregation and cytotoxicity and might
even protect against full-length TDP-43 pathology [180, 181].
Calpain is another candidate protease for pathological TDP-43 cleavage
[182]. It has to be noted that CTFs may not only be formed by
post-translational proteolytic processing, but can also arise from
alternative splicing [183, 184].
Less is known about the N-terminal fragment counterparts, which appear
to be more short-lived [185]. They might be derived from TDP-43
cleavage by calpains [182] or by asparaginyl endopeptidase
[186], and they do have aggregation propensity [187, 188]. While
it is clear that truncated TDP-43 species exist, their production
mechanisms and (patho)physiological relevance needs to be further
established [189, 190].
CONCLUSIONS AND OUTLOOK
TDP-43 is a multifaceted protein with crucial roles RNA translation and
processing and, in addition, it is the main component of protein
aggregates in both ALS and FTD-TDP43. The knowledge about TDP-43
pathophysiology has exploded in the years after its identification as a
pathological marker of these diseases, but even now it is challenging to
establish the effect of TDP-43 dysfunction in the different cellular
loci where it is involved. However, new techniques such as proximity
labelling coupled to mass spectrometry are now able to identify partners
in highly dynamic contexts. The proliferation of such studies will
expand enormously our knowledge of membrane-less organelles and identify
how TDP-43 impacts the dynamics of RNA granules.
In addition to proximity labelling, the knowledge about protein
structures and molecular grammar of low valency interactions have been
expanded to a point where we can predict and to a certain extent
influence LLPS in cells. Future studies could establish how different
membrane-less organelles are organized, and how the interaction of their
components can alter the physical characteristics. The understanding of
this process could prove extremely valuable for the description of the
molecular causes of TDP-43 proteinopathies and could open the door to
new therapies and diagnostic tools.