In healthy cells, TDP-43 is predominantly localized in the nucleus where it regulates multiple steps of gene expression including transcription [
69] and splicing [
70] and participates in DNA repair [
71,
72]. In addition, a small proportion of the protein is localized in the cytoplasm where it is involved in mRNA stabilization and transport [
73‐
76], translation [
77,
78], microRNA biogenesis [
79,
80] and stress granule assembly [
81‐
84]. In the context of ALS, TDP-43 becomes depleted from the nucleus and mislocalizes in the cytoplasm where it accumulates and forms insoluble aggregates [
19‐
21]. These changes in subcellular localization and solubility may critically alter the functions of TDP-43 (most probably via a combination of loss- and gain-of-function mechanisms), which eventually exerts deleterious effects on NMJs and MN survival. In the translucent zebrafish, optogenetic induction of cytoplasmic mislocalization and aggregation of wild-type TDP-43 is sufficient to trigger axonal defects and endplate denervation [
85], consistent with the hypothesis that pathogenic dysregulation of TDP-43 may underlie NMJ disruption. We focus our attention on perturbed TDP-43 functions of potential importance for the loss of NMJ integrity in ALS (Fig.
1).
Impaired RNA processing
In ALS, it is hypothesized that the loss of nuclear localization of TDP-43 may alter RNA processing that usually occurs in the nucleus, which may lead to dysfunction of cellular pathways critical for neuron health and NMJ integrity. In fact, mutations in
TARDBP have been shown to cause various RNA abnormalities such as changes in gene expression, mis-splicing and reduced transcript stability [
60,
86‐
89]. While TDP-43 normally functions as a splicing repressor regulating the inclusion of alternatively spliced exons [
41], widespread splicing alterations have repeatedly been described in TDP-43 downregulation and mutant models [
60,
87‐
94]. Pathologically altered TDP-43 can induce (1) the inclusion of normally excluded exons (cryptic exons) [
91‐
94], and (2) the exclusion of normally constitutively expressed exons (skiptic exons) [
88], suggesting both loss- and gain-of-function mechanisms with regard to TDP-43 splicing functions [
88]. Incorrect splicing can cause a frameshift, introduction of a stop codon and/or generation of an aberrant splicing product that yields a non-functional protein. Of note, splicing alterations have sometimes been observed without detectable aggregation or nuclear clearing [
60] and in the absence of neurodegeneration [
89], implying that impaired RNA processing may be an early event in ALS pathogenesis.
The first studies characterizing RNA targets of TDP-43 using cross-linking immunoprecipitation combined with high-throughput RNA sequencing revealed that TDP-43 binds to thousands of transcripts derived from genes implicated in RNA metabolism, neurodevelopment, neuronal survival and synaptic function [
90,
95‐
97]. Polymenidou and colleagues found that the most downregulated genes in TDP-43-depleted mouse brains encode proteins critical for synaptic formation and neurotransmission such as glutamate receptor subunits (
Gria2/3,
Grik2,
Grin1,
Grin2a/b), ion channels (
Cacna1,
Kcnma1) and synaptic vesicle proteins neurexin 1 to 3 (
Nrxn1/2/3) and neuroligin 1 (
Nlgn1) [
90]. Similarly, analysis of post-mortem cortical tissues of patients with TDP-43 pathology revealed significant downregulation of genes involved in synaptic functions, including synaptic vesicle proteins synaptobrevin 1 (
VAMP1), synaptotagmins (
SYT1,
SYT13) and synaptosomal-associated protein 25 (
SNAP25) [
98]. Recently, loss of TDP-43 was found to induce cryptic splicing of the critical synaptic gene
UNC13A in iPSC-derived motor and cortical neurons and post-mortem brain neuronal nuclei, resulting in depletion of the
UNC13A transcript and protein [
94,
99]. Furthermore, single nucleotide polymorphisms (SNPs) in
UNC13A (associated with increased ALS and frontotemporal dementia (FTD) risk through genome-wide association studies) were found to promote this incorrect splicing in patient brain tissues [
94].
Differential expression of synaptic transcripts was also observed in several cellular and animal TDP-43 models [
46,
60,
100]. Of particular interest, some studies revealed interactions between TDP-43 and transcripts encoding proteins with critical roles at the neuromuscular synapse [
52,
95]. TDP-43 binds the
AGRN transcript encoding agrin [
95], a key regulator of NMJ development and maintenance [
101].
AGRN was shown to undergo cryptic splicing upon TDP-43 depletion [
93,
94]. Lower levels of agrin are detected in the cerebrospinal fluid of ALS patients compared with non-ALS patients and healthy controls [
102]. Additionally, TDP-43 was found to directly interact with the
MAP1B transcript [
95], which encodes a protein responsible for stabilizing microtubules at presynaptic terminals during NMJ formation. Altered subcellular localization of
MAP1B transcripts has been described in spinal cord specimens of ALS patients [
103]. Levels of the
MAP1B ortholog
futsch have been repeatedly shown to be decreased with TBPH/TDP-43 loss-of-function in flies [
39,
46,
104]. Interestingly, mutations in
futsch phenocopy several pathogenic changes observed with TBPH/TDP-43 depletion [
105], supporting the idea that TDP-43 dysfunction may result in structural defects at the NMJ. Recently, a novel role of TDP-43 in regulating acetylcholinesterase (AChE) expression was described [
52]. AChE, classically known for hydrolyzing the neurotransmitter acetylcholine (ACh) in the synaptic cleft, has been demonstrated to be involved in NMJ development and NMJ stabilization at the adult synapse [
106‐
109]. TDP-43 knockdown in zebrafish is associated with decreases of AChE activity and expression, while overexpression of human AChE ameliorates NMJ pathology and locomotive deficits [
52]. Moreover, reduced transcript levels of
ACHE have been reported in ALS spinal cord tissue sections related to the site of symptom onset [
110], highlighting a potential contribution of AChE to disease pathogenesis.
Overall, these findings strengthen the hypothesis that dysregulated TDP-43 may lead to synaptic destabilization through altered gene expression. Given the thousands of RNA targets regulated by TDP-43, the challenge now is to identify the transcriptomic changes most relevant to the development and progression of ALS.
DNA damage
In addition to impaired RNA processing, TDP-43 dysfunction has been linked to defective DNA damage response (DDR) [
71,
72]. In healthy neurons, TDP-43 is involved in the detection and repair of double-stranded DNA breaks (DSBs) via non-homologous end joining (NEHJ) [
71,
72], a major DNA repair pathway as neurons are unable to divide or undergo homologous recombination. TDP-43 is rapidly recruited at DNA damage sites where it interacts with factors of DDR and NHEJ-mediated DSB repair, including the XRCC4–DNA ligase 4 complex [
71,
72,
111]. TDP-43 depletion in multiple neuronal cell models causes a significant accumulation of DSBs due to a reduction in NHEJ-mediated DSB repair efficiency [
71,
72]. In particular, TDP-43 is involved in the prevention and repair of transcription-associated DNA damage, specifically, the formation of R-loops [
112,
113]. These are three-stranded DNA:RNA hybrid structures which can lead to spontaneous DSBs when unresolved. In HeLa cells, silencing of TDP-43 leads to increased R-loop formation and R-loop-mediated DNA damage [
113].
Hence, it is hypothesized that the loss of TDP-43 nuclear functions in ALS may cause persistent DNA repair defects and genome instability. In fact, TDP-43 nuclear clearing correlates with DNA damage and activation of DDR in sporadic ALS spinal cord tissues [
71]. Similarly, transfection of TDP-43
A315T and TDP-43
Q331K in multiple cellular models lead to higher levels of the DSB marker γH2AX, indicating a loss of DNA repair function induced by ALS mutations [
72,
114]. Increased DNA damage was detected in spinal cord tissues from patients expressing TDP-43
Q331K [
114] as well as in the frontal cortex of patients with FTD-TDP-43 [
115]. Interestingly, fibroblasts obtained from two pre-symptomatic individuals with
TARDBP mutations encoding TDP-43
M337V also display increased levels of DNA damage and impaired NHEJ, implying that failure of DNA repair mechanisms by TDP-43 may occur early in the disease course [
72].
Focussing here on potential mechanisms of NMJ disruption, it could be hypothesized that persistent DNA damage can provoke MN death [
116], thereby triggering the retraction of motor terminals. An alternative hypothesis is that DNA damage in MNs may cause NMJ dismantling prior to neurodegeneration. Consistent with this idea, early accumulation of DNA damage was detected in the cortex of inducible hTDP-43ΔNLS mice preceding NMJ denervation, followed later by spinal MN loss [
72,
117,
118]. Although this study did not examine the presence of DNA damage in spinal cord tissues, another group established a link between early DNA damage and distal axonal defects [
119]. Naumann and colleagues performed a sequential characterization of mutant
FUS phenotypes in iPSC-derived MNs and reported early DNA damage, followed by defects in axonal trafficking of organelles, axonal degeneration, and finally death of MNs [
119]. Unfortunately, to our knowledge, no equivalent study has yet been performed in a TDP-43 model. Overall, these studies support a critical role of defective DNA repair mechanisms by dysfunctional TDP-43 in the pathogenesis of ALS. Further work is required to determine the downstream consequences of DNA damage and how they may relate to denervation.
Mitochondrial dysfunction
Mitochondria are the main producers of reactive oxygen species (ROS), which cause oxidative stress and lead to cell death through apoptosis at excessive amounts [
120]. Mitochondria also play a critical role in energy production, which is crucial for MNs due to their high metabolic demand to sustain their large size and long axons. Oxidative stress and metabolic imbalance can result from mitochondrial dysfunction, which is hypothesized to contribute to ALS pathogenesis. In fact, evidence of increased oxidative stress was found in the motor cortex [
121,
122] and spinal cord [
123] of sporadic ALS patients. Additionally, abnormal mitochondrial morphology was observed in ALS spinal cord specimens [
121].
Mitochondrial dysfunction has been repeatedly described in cellular models expressing human wild-type TDP-43 or ALS variants (Q331K, M337V, A382T, I383T), including increased levels of mitochondrial ROS [
124], activation of mitophagy [
125,
126], reduced basal respiration [
127] and transmembrane potential [
128], and deficiency in calcium uptake [
129]. While TDP-43 is normally detected in mitochondria, this localization is increased in ALS patient specimens [
130]. TDP-43 mitochondrial localization is also enhanced by TDP-43 variants [
131,
132], perhaps reflecting a gain of toxic function. Consistent with this idea, inhibition of TDP-43 mitochondrial localization mitigates neurodegeneration and NMJ loss in TDP-43
A315T mice [
132].
Mitochondria have also been detected within large TDP-43 aggregates in TDP-43 transgenic mice [
133,
134], leading to the hypothesis that aggregates may sequester this organelle. Furthermore, aggregates have been shown to dysregulate the expression of nucleus-encoded mitochondrial proteins via sequestration of mRNA, microRNAs and other RNA-binding proteins, resulting in enhanced oxidative stress [
135], fewer and dysfunctional mitochondria at NMJ pre-synapses, and denervation [
136].
Abnormalities in mitochondrial morphology and distribution are a prominent TDP-43 phenotype [
126,
127,
131,
134,
136‐
138]. Furthermore, abnormal mitochondria have been shown to accumulate in presynaptic terminals of ALS patients [
121], although this has been recapitulated inconsistently in TDP-43 transgenic mice. Two studies have described depletion of mitochondria at nerve terminals of NMJs in mice expressing human wild-type TDP-43 [
133] or hTDP-43
ΔNLS [
136]. In concordance with post-mortem studies, Magrané and colleagues noted an accumulation of mitochondria in distal axons and at NMJs of presymptomatic mice expressing TDP-43
A315T [
138]. Despite these conflicting results, both accumulation and depletion of mitochondria may have profound consequences at the NMJ, as the localization and integrity of mitochondria at nerve terminals is directly correlated with NMJ function [
136,
139,
140].
In summary, mitochondrial dysfunction is commonly linked to TDP-43 dysregulation. In ALS, aggregation and enhanced mitochondrial localization of TDP-43 along with abnormal distribution of mitochondria may induce the loss of MNs and NMJs.
Defective anterograde axonal transport and transport-translation coupling
In the cytoplasm, TDP-43 associates with RNA and other effector proteins to form transport ribonucleoproteins (RNPs) responsible for RNA transport along microtubules in both anterograde and retrograde trajectories [
73‐
76]. This enables control of protein expression in specific regions of the cell, a process that is particularly important for MNs as they are large cells with multiple cellular compartments (cell body, dendrites and axons) that have local translational needs. Altered axonal transport has been one of the earliest proposed mechanisms to explain NMJ disruption in ALS and constitutes a frequently identified phenotype in TDP-43 models [
74,
138,
141]. Furthermore, genetic defects and abnormalities in cytoskeletal components and motor complexes are commonly linked to ALS [
142‐
146] (reviewed in [
147]).
One hypothesis is that impairment in anterograde transport (from the cell body to neuronal processes) may prevent adequate maintenance of distal axons and presynaptic membranes, leading to denervation and neuronal cell death. ALS-associated mutations in
TARDBP (M337V, A315T and G298S) have been shown to decrease anterograde transport and enhance accumulation of transport RNPs in the cell body [
74]. As a result, delivery of transcripts to distal compartments is impaired, as shown by altered mRNA content in axonal processes of mutant MNs [
74]. Similarly, axon sequencing (axon-seq) analyses identified broad changes in the subcellular localization of mRNAs and microRNAs in the cell soma and axons of primary mouse MNs depleted of TDP-43 or expressing the TDP-43
A315T variant [
148,
149]. Thus, it is conceivable that alterations in the spatiotemporal localization of RNA species within MNs due to defective axonal transport may impact local protein synthesis at the presynaptic membrane, compromising the integrity of neuromuscular synapses.
TDP-43 is detected at presynaptic membranes of NMJs [
74,
150], suggesting that it may also directly contribute to local regulation of translation at this synapse. At least in dendrites, there is accumulating evidence that TDP-43 regulates local translation along with Fragile X mental retardation protein (FMRP) [
73,
77,
78,
151,
152]. TDP-43 acts as a translational repressor and stabilizes RNA until a stimulus (such as neuronal activity) signals a need for novel proteins at the synapse [
78]. Given that TDP-43 interacts with the D1 domain of FRMP via its C-terminal domain (where the vast majority of ALS mutations cluster) [
151], it has been proposed that this interaction could be perturbed in ALS, preventing MNs from adequately modulating transport-translation coupling of RNPs [
73]. Interestingly, loss-of-function mutations in the FMRP ortholog dFXR lead to morphological defects and alterations of neurotransmission at the NMJ in fruit flies [
153,
154].
Moreover, Nagano and colleagues recently showed that TDP-43 binds and transports along axons the mRNAs of ribosomal proteins (RPs) that are locally translated and assembled into ribosomes which, in turn, participate in local protein synthesis themselves [
155]. Using in situ hybridization, they showed that the RP mRNA signal is significantly decreased along axons of TDP-43-depleted mouse cortical neurons [
155], revealing a broader role of TDP-43 in modulation of protein synthesis. It is worthy noting that, in addition to RNPs, the delivery of other vital cargos which depends on anterograde transport to reach the pre-synaptic compartment (e.g., synaptic vesicles precursors, mitochondria and proteins [
139,
140,
156,
157]) may also become compromised in TDP-43-ALS.
Defective retrograde axonal transport
Another proposed mechanism for NMJ disruption in ALS is the abnormalities of retrograde transport that may prevent the delivery of factors supporting neuron survival back to the cell body, such as neurotrophin-containing signaling endosomes [
23]. Neurotrophins (such as brain-derived neurotrophic factor and nerve growth factor) are normally internalized through receptor-mediated endocytosis and retrogradely transported to cell bodies to modulate various aspects of the developing and adult neurons including cell survival, neurite outgrowth and synaptic function [
158]. The TDP-43
M337V variant was recently found to impair the retrograde axonal transport of neurotrophin-containing signaling endosomes in mice, preceding NMJ dismantling and motor symptoms [
58].
In addition to neurotrophins, other pathways that initiate at the NMJ are crucial for regulation of the formation and function of this synapse, including the bone morphogenetic protein (BMP) signaling pathway [
159,
160]. Mutations in essential components of this signaling cascade (i.e., BMP, BMP receptors and Smad transcription factors) induce changes in NMJ morphology and a decrease in neurotransmitter release [
159,
160]. In fruit flies, defects in endocytic traffic of BMP receptors have been described with both loss- and gain-of-function of TDP-43/TBPH, as demonstrated by a shift from Rab5
+ early endosomes to Rab11
+ recycling endosomes at motor terminals [
47]. These results were accompanied by a decrease in pMAD staining indicative of decreased BMP signaling at the NMJ, while rerouting BMP receptors via Rab11 inhibition partially restores BMP signaling, NMJ defects and motor deficits [
47]. There is also pathological evidence of dysfunctional BMP/TGF-β signaling in sporadic ALS spinal cord specimens, with MNs showing accumulation of pSmad in cytosolic TDP-43 aggregates [
161]. Taken together, TDP-43 dysfunction could prevent MNs from maintaining the integrity of NMJs by disrupting the long-range signal transduction required to respond appropriately to external stimuli.
Axonal degeneration
Axonal fragmentation is a prominent feature of neurodegeneration. According to the dying-back theory, degeneration originates distally at nerve terminals and progresses in a retrograde fashion to sequentially affect the axons and cell bodies, eventually leading to MN loss [
23,
25]. This phenomenon is reminiscent of Wallerian degeneration (also known as programmed axon death), a tightly regulated process of axonal fragmentation and neuronal death, distinct from apoptosis, which occurs following a nerve injury [
162,
163]. Sterile Alpha and TIR Motif-Containing 1 (
SARM1) has been identified as a key initiator of programmed axon death, as depletion of this gene confers long-term resistance to degeneration [
164‐
169]. The
SARM1 locus has been associated with an increased susceptibility to sporadic ALS [
170] and constitutively active SARM1 variants have been recently identified in ALS patients [
171,
172]. ALS, as well as other neurodegenerative diseases where axons may be affected before neuronal cells bodies (e.g., Parkinson’s disease, Alzheimer’s disease and Huntington’s disease [
173‐
177]), is increasingly believed to be Wallerian-like disorders in which a similar cell death program is triggered in the absence of a physical insult. Metabolic stress and disruption of axonal transport, two processes which have been repeatedly associated with ALS pathophysiology, are thought to be responsible for initiating this response [
146,
178‐
180]. In particular, studies have consistently reported both mitochondrial and axonal dysfunction in TDP-43 models [
58,
74,
125,
129,
131,
138,
141], raising a possible link between TDP-43 and programmed axon death. The role of TDP-43 in response to cellular injury reinforces this hypothesis, as in vivo axotomy or axon ligation triggers upregulation and transient accumulation of TDP-43 at the site of injury [
181‐
183]. Furthermore, TDP-43
G348C mice exhibit sustained cytoplasmic mislocalization of TDP-43 and impaired recovery after nerve crush injury, as shown by fewer regenerating axons and persistent motility impairments compared with control animals [
184].
More direct evidence implicating TDP-43 in the Wallerian pathway was demonstrated by genetic ablation of
SARM1 resulting in improvement of disease phenotypes in
TARDBP models [
66,
185]. In
C. elegans expressing TDP-43
A315T, loss-of-function mutation in the
SARM1 ortholog
tir-1 improves motility deficits and MN survival [
185]. Similarly,
SARM1 knockout mitigates axonal degeneration and MN loss in TDP-43
Q331K mice [
66]. Importantly, these findings were accompanied by a significant decrease in NMJ denervation [
66]. These results imply that the activation of the axonal death program is involved in disruption of NMJs, and preserving the motor terminal-muscle interaction and axonal integrity may be required for the survival of MN cell bodies [
66]. Recently, patient-associated SARM1 variants were shown to promote neurodegeneration in primary neurons and mice, due to a constitutive NAD
+ hydrolase activity [
171,
172]. In this regard, we speculate that TDP-43 dysregulation in ALS may confer an increased susceptibility to activation of the Wallerian pathway via SARM1, causing NAD
+ depletion (and consequently ATP depletion), axonal degeneration, NMJ denervation and MN loss. It is worthy of note, however, that
SARM1 deletion does not mitigate neurodegenerative phenotypes in the SOD1
G93A mouse model, suggesting distinct mechanisms in SOD1-ALS [
186,
187].
TDP-43 is also associated with other mediators of the Wallerian pathway, namely PHR1 (also known as PLEKHB1) [
188] and stathmin-2 (also known as SCG10) [
189,
190]. PHR1 promotes Wallerian degeneration, as its conditional knockout delays degeneration of severed axons and NMJ loss similar to
SARM1 depletion [
191]. Paradoxically, it is also involved in axon outgrowth and synaptic formation [
192‐
194]. PHR1 is essential for the development of NMJs: its constitutive knockout is lethal at birth due to incomplete innervation of the diaphragm, causing respiratory failure [
192,
194]. PHR1 is significantly downregulated in MNs of TDP-43
A315T mice in the early symptomatic phase of the disease, preceding NMJ morphological defects [
188]. Further investigation is required to determine the possible pathological role of PHR1 in TDP-43-mediated ALS.
Two studies have clearly shown that TDP-43 regulates expression of stathmin-2 (
STMN2) [
189,
190], an axon-maintenance factor that is rapidly depleted in distal axons upon injury [
195,
196]. It is considered an early marker of subsequent axonal degeneration, potentially acting upstream of
SARM1 [
195]. Stathmin-2 was shown to be significantly downregulated in spinal cord and cortical specimens from ALS patients as well as in iPSC-derived MNs depleted of TDP-43 [
189,
190]. Mechanistically, the decline of stathmin-2 level is due to altered TDP-43 splicing activity, causing the inclusion of a cryptic exon that results in a non-functional protein [
189,
190]. Stathmin-2 downregulation has also been observed in patient-derived neurons expressing TDP-43 variants (G298S, A382T, N390S), suggesting a loss of normal splicing function (i.e., cryptic exon repression) conferred by the mutations [
190]. Loss of stathmin-2 is associated with impaired axonal regeneration following in vitro axotomy [
189,
190], consistent with its role in maintaining the integrity of axons. Stathmin is also shown to be required for maintenance of NMJ stability. In fruit flies, neuron-specific knockdown of
stathmin, or expression of a loss-of-function mutant, causes a reduction of bouton number and axonal retractions at the NMJ [
197,
198]. Similarly,
Stathmin mutant or knockout mice develop a late-onset axonopathy and NMJ denervation, leading to muscle atrophy and severe motor impairments [
199,
200].
In summary, TDP-43 is functionally linked to factors involved in the Wallerian degeneration pathway, with dual roles in axonal outgrowth and NMJ maintenance. Disturbances in TDP-43 homeostasis in ALS may affect the expression levels of these factors, which in turn may contribute to defects at the NMJ, axonal degeneration and MN loss that characterize this disease.
Aggregation and RNA sequestration
TDP-43 aggregation is a core feature of ALS [
20]. These insoluble aggregates, detected in nearly all ALS cases, contain ubiquitinated and hyperphosphorylated full-length TDP-43 as well as truncated C-terminal fragments of the protein [
20]. When ALS-associated mutations are present in
TARDBP, TDP-43 has an increased propensity to aggregate and is capable of interacting with the wild-type protein, recruiting it into further aggregates [
61,
201]. The majority of mutations are found in exon 6 of
TARDBP encoding the protein’s glycine-rich C-terminal domain, which has been proposed to mediate solubility and oligomerization [
202]. This implies that aggregation may be an important contributor to disease phenotype.
It has been proposed that aggregates can sequester RNA from the translational machinery, thereby depleting MNs of critical proteins for NMJ maintenance. Indeed, an emerging property of pathologically altered TDP-43 is sequestration of mRNA into insoluble complexes [
136,
203]. Coyne and colleagues have shown that the TDP-43
G298S variant can sequester transcripts of the chaperone Hsc-70-4/HSPA8, resulting in decreased expression of the protein at the NMJ in transgenic
Drosophila and mice [
203]. These changes are accompanied by deficits in synaptic vesicle endocytosis, defects in NMJs and locomotion, and decreased lifespan [
203]. HSPA8 protein levels, but not transcript levels, are also reduced in human MNs differentiated from iPSCs expressing
C9ORF72 or
TARDBP mutations, confirming a post-transcriptional mechanism of expression inhibition [
203]. It is plausible that this process may take place within aggregates given their resemblance to RNA granules, which are known to contain mRNA in a translationally silent state (i.e., stalled translation initiation complexes) [
204]. Under physiological conditions, TDP-43 participates in the assembly of both transport RNPs [
73‐
76] and stress granules [
81‐
84], highlighting its role in modulating mRNA availability in time and space. In ALS, a gain-of-function of TDP-43 could result in mRNA trapping within insoluble aggregates rather than being stabilized temporarily within granules. In support of this idea, Altman and colleagues showed that “aggregate-like” TDP-43 RNP condensates drive suppression of local protein synthesis in sciatic MN axons and presynaptic terminals of inducible hTDP-43ΔNLS mice [
117,
118,
136]. Specifically, they demonstrated that mRNAs of nucleus-encoded mitochondrial genes
Cox4il and
ATP5A1 are directly bound by TDP-43 and sequestrated within axonal condensates, resulting in decreased levels of the proteins [
136]. Ceasing hTDP-43ΔNLS expression induces clearance of axonal and synaptic condensates and consequently restores local protein synthesis as well as the number of innervated NMJs and contracting muscle fibers. However, the precise mechanisms through which TDP-43 condensates inhibit protein synthesis remain to be investigated. In addition to mRNAs, Zuo and colleagues showed that H
2O
2-induced TDP-43 aggregates also sequester specific microRNAs in mouse neuroblastoma-derived N2a cells, leading to upregulation of their corresponding targets [
135]. RNA immunoprecipitation experiments showed that TDP-43
M337V enhances the capture of the microRNAs compared with the wild-type protein, supporting a gain-of-function mechanism. Furthermore, TDP-43 co-aggregates with other RNA-binding proteins (i.e., hnRNP M, hnRNP H1 and RMB14), raising the possibility that RNA sequestration within aggregates may not be limited to direct TDP-43 targets. Taken together, these studies support the hypothesis that TDP-43 aggregates may negatively impact NMJs by interfering with the expression of essential proteins for NMJ maintenance and function.