Understanding age-related pathologic changes in TDP-43 functions and the consequence on RNA splicing and signalling in health and disease

TAR DNA binding protein-43 (TDP-43) is a key component in RNA splicing which plays a crucial role in the aging process . In neurodegenerative diseases such as amyotrophic lateral sclerosis, frontotemporal dementia and limbic-predominant age-related TDP-43 encephalopathy, TDP-43 can be mutated, mislocalised out of the nucleus of neurons and glial cells and form cytoplasmic inclusions. These TDP-43 alterations can lead to its RNA splicing dysregulation and contribute to mis-splicing of various types of RNA, such as mRNA, microRNA, and circular RNA. These changes can result in the generation of an altered transcriptome and proteome within cells, ultimately changing the diversity and quantity of gene products. In this review, we summarise the findings of novel atypical RNAs resulting from TDP-43 dysfunction and their potential as biomarkers or targets for therapeutic development.


An overview of RNA splicing
Genes that are transcribed as mRNA consist of exons and introns.mRNA is first transcribed into precursor mRNA (pre-mRNA).This pre-mRNA becomes mature mRNA through the removal of introns and religation of exons in a process known as splicing (Marasco and Kornblihtt, 2023).Once matured, this mRNA transcript is then recognised by the ribosome and is translated into protein.Historically, introns were considered "junk" DNA.However nowadays, it is clear that both introns and exons play an essential role in the expression of eukaryotic genes.
Constitutive splicing typically occurs when exons are selected and ligated into mature mRNA in the same order that they occur in the gene.However, splicing can also occur in different ways, which are categorised as alternative splicing.In 1977, alternative splicing was first observed in adenovirus transcripts (Berget et al., 1977;Chow et al., 1977), whereby different exons are selected for inclusion or exclusion in the final transcript depending on the circumstances of the gene expressed, rather than sequentially as with constitutive splicing.
The spliceosome is a large macromolecular complex responsible for splicing pre-mRNA into mature mRNA in eukaryotic cells through a series of catalytic reactions.It is comprised of five small nuclear RNAs and numerous interacting proteins which form RNA-protein complexes known as small nuclear ribonucleic proteins (snRNPs).Numerous human diseases, particularly those affecting the nervous system, have been associated with mutations in splice site recognition sequences or in proteins involved in pre-mRNA processing (Hutton et al., 1998;Lin et al., 1998;Pellizzoni et al., 1998).One such example is Transactive Response DNA binding Protein of 43 kDa (TDP-43) which is a known key component in RNA splicing due to its ability to bind both introns and exons (Gu et al., 2017;Sephton et al., 2011).

Spliceosome machinery
The spliceosome typically functions to exclude introns from pre-mRNA.Effective splicing requires three short conserved sequences within the introns: the 5' splice sites (5'SS) (consensus: GU), branching point (BP) (Consensus: A), and 3' splice site (3'SS) (consensus: YAG, where Y is C or U).The spliceosomal cycle is executed by following 4 steps: assembly, activation, splicing and disassembly (Rodrigues et al., 2023) (Fig. 1).A detailed overview of the molecules involved throughout the distinct steps of splicing was presented in early 2023 by Rodrigues and colleagues, who curated an atlas of the biogenesis of spliceosome machinery during RNA splicing (Rodrigues et al., 2023).In this section, a summary of the mechanism of splicing is detailed below, highlighting the five main small nuclear ribonucleoprotein complexes (snRNP) (U1, U2, U3, U4 and U5 snRNPs).

Assembly
The cascade is initiated through the formation of the so-called E complex at the 3' splicing and BP site.This formation occurs when U2 auxiliary factor (U2AF) and splicing factor 1 (SF1) bind to the 3' splicing site and BP site respectively.Conversely, U1 snRNP attaches to the 5'SS through base-pairing between the 5'SS and the 5' end of U1 snRNA (Bai et al., 2018;Kondo et al., 2015) and the attachment is stabilised with the binding of TDP-43 to the U1-70 K of the snRNA U1 complex (Bishof et al., 2018).Once established, the DEAD-box ATPase Prp5 removes SF1 from the BP site and recruits U2 snRNP to the location, giving rise to the A complex (Liang and Cheng, 2015;Zhang et al., 2020).With U2 snRNP securely integrated, the BP adenosine base is flipped out to interact with SF3B1 within the U2 snRNP complex.

Activation
The first fully assembled spliceosome with all five snRNPs simultaneously present, also known as pre-B complex, forms through somewhat unstable binding of the tri-snRNP (U4/U6/U5) with U2 snRNP to complex A, with U6 and U5 not yet recognising the pre-mRNA (Agafonov et al., 2016).Following the binding of the tri-snRNP with U2 snRNP, conformational changes occur in the pre-mRNA intron, facilitating some rearrangement within the U2 snRNP.This leads to the creation of the U2/U6 duplex and the replacement of U1 with U6, orchestrated by the DDX23 helicase (Zhan et al., 2018) which generates the B complex.
Subsequently, several proteins are recruited including the RES complex which enable the dissociation of U4 from U6 snRNP.Following these events, splicing factors like NTC and NTR proteins are brought in to shape a mature B act complex (Zhang et al., 2018).However, due to the spatial separation between the BP site and the 5'SS splicing active site, the branching reaction cannot be catalysed until various factors (DHX16, the SF3A, SF3B, and RES complexes) have been released.This allows for the recruitment of other specific splicing factors (DHX38, CWC25, YJU2, SFY2, ISY1, PPWD1, and PPIG) for the creation of a 5' exon and an intron lariat-3' exon intermediate, which forms the B* complex.

Splicing
Prior to 3' exon ligation, which would form the C* complex, exonligation factors (Prp18 and SLU7) along with DHX8 facilitate the connection of the 3'SS with the 5'OH of the exon 5' on the active site (Fica et al., 2017).Following exon ligation, the 5' exon and 3' exon are joined to create the mRNA and the P complex.Within this complex, the connected exons remain bound to the spliceosome.

Fig. 1.
Simplified spliceosomal cycle illustrating the main steps from the spliceosome assembly to its disassembly and the involvement of TDP-43.Spliceosome assembly is initiated in the E complex by the binding of U1 snRNP to the 5' SS and SF1 to the branch-point A in the pre-mRNA (in red boldface).SF1 recruits U2 snRNP and is displaced from the branch site forming the A complex.Additional U4/U6/U5 tri-snRNPs join to form the B complex and the departure of U1 and U4 with the recruitment of NTC/NTR proteins forms the B act complex.Once the U2 snRNP is destabilised from the rest of the machinery in the B* complex, cleavage of 5' SS occurs in the C complex followed by the spliceosome undergoing conformational change into C* complex.Exons are ligated in the P complex and the intron lariat is removed from the complex for degradation.
F. Cheng et al.

Disassembly
Following the release of the bound exons from the spliceosome by DHX8, a series of protein dissociations is triggered, culminating in the formation of the ILS complex.As the mRNA is liberated from the spliceosome, the other complexes attached to the intron lariats also undergo dissociation by the ATPase/helicase prp43 with help from the NTrelated protein 1 and 2 (Fourmann et al., 2017;Tsai et al., 2005).This event permits the eventual release and degradation of the intron lariat, enabling the recycling of the snRNAs and associated factors for future cycles of splicing.

Structure of TDP-43
TDP-43 was initially discovered through a genomic screening aimed at identifying new transcriptional inhibitors that could attach to the TAR-DNA component of the HIV-1 virus, where it operates as a transcriptional repressor (Ou et al., 1995).Decades later, TDP-43 was associated with neurodegenerative diseases by Neumann et al. when the protein was identified as a major protein component in insoluble inclusions of patients with Frontotemporal Lobal Degeneration with Ubiquitin-positive inclusions (FTLD-U) and Amyotrophic Lateral Sclerosis (ALS) patients (Arai et al., 2006;Neumann et al., 2006).TDP-43 is a ubiquitously expressed RNA-binding protein encoded by the TARDBP gene and it is primarily located in the nucleus.
TDP-43 is composed of a N-Terminal Domain (NTD) with a Nuclear Localisation Sequence (NLS), two RNA-Recognition Motifs (RRM1 and RRM2) and a glycine-rich sequence at the C-terminus that mediates protein-protein interactions (François-Moutal et al., 2019) (Fig. 2).The NTD has also been shown to promote self-oligomerisation in a concentration-dependent manner (Chang et al., 2012).Through the NTD, TDP-43 is able to form reversible polymers, which was demonstrated to be required for RNA splicing activity (Afroz et al., 2017;Jiang et al., 2017;Mompeán et al., 2017;Zhang et al., 2013).The NLS in TDP-43, located from amino acid 82-98, enables the protein to shuttle into the nucleus.Accordingly, mutating or deleting the NLS leads to cytoplasmic localisation of TDP-43 (Barmada et al., 2010).
Aging is a significant factor linked to the development of both genetic and sporadic forms of ALS/FTD.Recently, there is a growing awareness that RNA processing, particularly pre-mRNA splicing, plays a crucial role in the aging process (Harries et al., 2011).Angarola and Anczuków presented a list of age-related changes in splicing factor levels detected across multiple studies of differential expression of genes or proteins from young versus old human or mouse tissues.They found that TARDBP mRNA decreases with age in mouse brain, liver and heart and increases in human blood and muscle (Angarola and Anczuków, 2021).
In addition to ALS/FTD, which ALS presents a spectrum disease with a lifetime risk of 1:350 (Ryan et al., 2019), LATE presents another TDP-43 proteinopathy that impacts adults of advanced age with a 1:3 lifetime risk (Nelson, 2021).LATE neuropathological change (LATE-NC) is defined by the presence of mislocalised and phosphorylated TDP-43, leading to the formation of neuronal cytoplasmic inclusions.These inclusions appear in the amygdala and subsequently advance to the hippocampus and middle frontal gyrus of the neocortex (Nag et al., 2017;Nelson et al., 2022Nelson et al., , 2019)).Primarily impacting limbic structures, LATE frequently coexists with AD and individuals with AD who exhibit abnormal TDP-43 pathology tend to experience more severe dementia than those without TDP-43 inclusions (Tomé et al., 2023).Ayuso and colleagues used the term "AD-TDP" to refer to the large group of patients with both ADNC and LATE-NC at autopsy (Ayuso et al., 2023) and these individuals demonstrate a faster decline in global cognition and episodic memory compared to individuals with either pure LATE-NC or ADNC (Kapasi et al., 2020).The severe dementia observed in cases of AD with occurrent LATE-NC may be associated with increased levels of hippocampal p-tau pretangles and neurofibrillary tangles (Koper et al., 2022).However, additional research is essential to investigate the intricate connections and interactions among TDP-43, amyloid beta and Tau.Understanding these relationships is crucial for determining whether targeting these interactions could be a viable approach for therapeutic intervention in both AD and LATE.
In this review, we will explore the recent findings of RNA missplicing in TDP-43 proteinopathies (Table 1) and the potential use of these mis-spliced RNA regions as a diagnostic tool for neurodegenerative diseases with TDP-43 pathologies.

TDP-43 plays a role in RNA splicing
In 2010, Freibaum and team utilised a proteomics approach to identify proteins that interact with TDP-43 (Freibaum et al., 2010).They described two distinct protein interaction networks, one consisting of nuclear proteins involved in RNA splicing and metabolism and the other composed of cytoplasmic proteins that regulate mRNA translation.TDP-43 was also discovered to bind to U1-70 K, a component of the U1 snRNP complex responsible for enhancing the stability of the interaction between snRNA U1 and the 5'SS of a processed pre-mRNA (Bishof et al., 2018).
No binding alterations with TDP-43 was observed by Freibaum and colleagues, upon comparing the TDP-43 WT interactome to diseasecausing mutations A315T and M337V.However, this study focused on visualising the immunoprecipitation profile by Sypro-Ruby staining of 1D SDS-PAGE gels and analysing the TDP-43 WT interactome only by mass spectrometry.Several years later, Feneberg and colleagues investigated the human TDP-43 WT interactome in differentiated mouse motor neuron lysates and found that it was enriched with proteins involved in transcription, translation and poly(A)-RNA binding (Feneberg et al., 2020).When compared with mutant TDP-43 M337V interactome using a proteomic approach, they reported a loss of binding with splicing factors, such as Pabpc and Eif4a1, which suggests a potential contribution from TDP-43 splicing dysregulation, mRNA export and translation to ALS pathogenesis.TDP-43 K263E possesses a mutation within the RRM2 domain that was identified in one patient diagnosed with sporadic FTD (Kovacs et al., 2009).It was shown in cellular studies that this variant disrupted the capacity of TDP-43 to bind RNA, similarly to TDP-43 K181E (Chen et al., 2019).It also caused RNA processing machinery impairments, such as intron splicing and 3' polyadenylation regulation, in iPSC-derived neurons, similar to TDP-43 knock-down (KD) (Imaizumi et al., 2022).However, in contrast to neurons, the expression of TDP-43 K263E in iPSCs and neural progenitor cells did not recapitulate TDP-43's RNA binding deficiency.Therefore, these studies identify RNA processing deficiencies caused by mutant TDP-43 and suggest that mutations in the RRM domains in TDP-43 can have a cell type-specific dysfunction effect.

Effect of TDP-43 on its RNA targets
A key nuclear function of TDP-43 is to regulate alternative splicing.So far, seven basic types of alternative splicing have been identified including exon skipping, alternative 5' splicing, alternative 3' splicing, mutually exclusive exon, intron retention, alternative promoter, and alternative polyadenylation (Fig. 3).Upon loss of nuclear TDP-43, researchers have reported several mis-splicing consequences as a result.
One of the early studies generated a list of RNA targets of TDP-43 using a TDP-43 RNA immunoprecipitation (RIP) approach on cultured rat cortical neurons and the eluted peptides were subjected to deep RNAsequencing (Sephton et al., 2011).The major groups of TDP-43 were identified as exonic targets consisting of transcripts for proteins involved in RNA metabolism, synaptic function and nervous system development.In the same year, another research group utilised ultraviolet Cross-Linking ImmunoPrecipitation sequencing (CLIP-seq) and splicing-sensitive microarrays on mice brains.They reported that the RNAs dependent on TDP-43 and downregulated the most when TDP-43 is depleted in adult mouse brain are associated with pre-mRNAs possessing long introns with multiple TDP-43 binding sites.These encoded proteins were linked to synaptic activity (Polymenidou et al., 2011).With advancements in proteomics, functional genomics, and high throughput sequencing, different TDP-43 RNA target libraries have been developed (Hallegger et al., 2021;Herzog et al., 2020;Rengifo-Gonzalez et al., 2021) to contribute to the understanding of the splicing function of TDP-43 and identifying biomarkers associated with TDP-43 proteinopathies.One of the targets of TDP-43 is HNRNPA1 which was demonstrated that endogenous TDP-43 can bind to it to modulate its alternative splicing.SiRNA-mediated depletion of TDP-43 was found to increase exon 7B inclusion in HNRNPA1 transcripts thus indicating that TDP-43 represses the use of the 5' splice site of exon 7B in this transcript (Deshaies et al., 2018).
From those repertoires, researchers specifically studied the splicing dysregulation of mutant TDP-43 on its targeted RNA.In 2001, TDP-43 was reported to promote Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) exon 9 skipping (Buratti et al., 2001).Using this information, researchers employed a TDP-43-dependent CFTR splicing reporter, in which a full-length GFP gene is fused to a mCherry gene that is interrupted by the exon 9 of CFTR, to access the impact of PTM on TDP-43's splicing regulation, such as phosphorylation.The  phosphorylation at Ser48 on the NTD of TDP-43 was reported to be affecting its polymerisation and splicing activity (Wang et al., 2018).
Wang and colleagues showed phosphomimetic variant S48E had reduced CFTR exon 9 splicing ability.Similarly, other self-assembly deficient variants such as Y4R and E17R, also showed splicing dysregulation, strongly suggesting the requirement for NTD oligomerisation of TDP-43 in splicing regulatory function.SUMOylation, a PTM that occurs on lysine residues, plays a crucial role in regulating a wide array of cellular processes.By modifying the structure, stability, solubility, localization, and interactions with protein partners of the target proteins, SUMOylation has been observed to influence multiple cellular processes (Celen and Sahin, 2020).Since SUMOylation has already been studied in association to neurodegenerative pathologies and TDP-43 being susceptible for SUMOylation (Maurel et al., 2020), minigene splicing assays were performed on an artificial SUMOylation-resistant TDP-43 K136R to demonstrate the less effective skipping activity on targeted transcripts (Maraschi et al., 2021).
Another PTM that affects TDP-43 splicing function is o-GlcNAcylation, which was shown to be involved in regulating mRNA splicing.O-GlcNAcylation is an intracellular O-glycosylation that attaches the monosaccharide N-acetylglycosamine to Ser/Thr residues via an Olinked glycosidic bond.Double O-GlcNAcylated sites mutated TDP-43 T233A and TDP-43 T199A (TDP-43 2TA ) were introduced to TDP-43 KD cells, which was previously shown to produce unspliced CFTR exon 9 transcripts using the cell-based nuclear CFTR splicing assay.The ratio of spliced to unspliced CFTR exon 9 transcripts observed in TDP-43 KD cells expressing TDP-43 2TA was reduced compared to TDP-43 KD cells expressing TDP-43 WT, meaning that the splicing effectiveness was lost (Zhao et al., 2021).A positive RNA-binding-deficient 4FL (F147/149/229/231 L) mutant also shows loss of splicing activity.
Lastly, Necarsulmer and colleagues used TDP-43 K145Q/K145Q (TDP-43 KQ/KQ ) primary neuron model to access whether acetylation on TDP-43 affects its splicing function (Necarsulmer et al., 2023) as previously reported in human embryonic kidney-derived QBI-293 cell line (Cohen et al., 2015).They discovered that artificial acetyl-mimic TDP-43 leads to impaired splicing by the reduced exclusion of CFTR exon 9 and therefore adjusting TDP-43 acetylation may offer a promising therapeutic strategy for precisely regulating TDP-43 activity in cases of TDP-43 proteinopathies.The artificial acetylation mimic variant of TDP-43 K145Q , led to stress-induced nuclear TDP-43 loci and dysfunction in primary mouse and human-induced pluripotent stem cell-derived cortical neurons.RNA sequencing of the neocortex and hippocampus tissue from 18-month-old TDP-43 WT and TDP-43 KQ/KQ acetylation mimic mice showed splicing deficits in the presence of acetylated TDP-43, with SORT1 being the most significantly altered transcript in both brain regions.The exclusion of an exon toward the 3′ end of the SORT1 transcript was reduced by 55.9% in the TDP-43 KQ/KQ cortex and by 57.0% in the hippocampus (Necarsulmer et al., 2023).Another acetyl-mimic mutation K136Q was tested in HEK293E cells and found that TDP-43 K136Q showed reduced RNA binding and splicing capability (Garcia Morato et al., 2022).
TDP-43 familial ALS mutations were also found to affect TDP-43 splicing regulation.Mice with the TDP-43 M337V/M337V mutation have also been reported to exhibit splicing abnormalities in TDP-43 target mRNAs such as KCINP2, SORT1, and SEMA3F and despite being homozygous, these mice did not display significant TDP-43 pathology (Watanabe et al., 2020).This finding might indicate that the splicing regulation of mutant TDP-43 is lost prior to its mislocalisation to the cytoplasm.TDP-43 A315T mice were also reported to display abnormal splicing of ZMYND11, an RNA target of TDP-43, prior to the onset of motor symptoms (Narayanan et al., 2023) however this study has not looked into the localisation of TDP-43.
Cao and Scotter reported that RNA targets of TDP-43 vary across different studies, and the authors systematically compared publicly available RNA-sequencing databases from six TDP-43-depleted model systems and a human ALS/FTD neuronal nuclei dataset.They explored shared transcriptional patterns associated with TDP-43 dysfunction and reported that the top three upregulated and downregulated differentially expressed genes in TDP-43 KD models and ALS/FTD TDP-43 negative neuronal nuclei were KIAA1324, CFP and ITGA4 and TRHDE, PFKP and MASP2 respectively (Cao and Scotter, 2022).Additionally, they investigated shared Differential Exon Usage (DEU) events and found that only two genes, STMN2 and POLDIP3, exhibited a decrease in DEU event that was common between the ALS/FTD TDP-43-negative neuronal nuclei dataset and a rodent dataset.Moreover, these two genes displayed sequence homology in the corresponding regions between humans and mice.This discovery facilitates more precise investigation into TDP-43 splicing dysregulation within mouse models and provide valuable insights into the specific genes or biomarkers that should be prioritised when employing targeted therapies to rectify the splicing deficiency.TDP-43 splicing has been linked to the presence of abnormal cytoplasmic intron retention (IRT) in a human stem cell model of ALS, derived from ALS patients with VCP mutations R155C and R191Q (Tyzack et al., 2021).These mutations display TDP-43 mislocalisation from the nucleus to the cytoplasm.According to the study, a higher level of IRT corresponds to a greater sequestration of functional RNA-binding proteins (RBP), including TDP-43.The researchers suggested that cytoplasmically retained introns might function as RNA regulators, influencing the homeostatic control of RBP localisation during both development and disease.This delicate balance, if disturbed, could lead to a dysfunction of these RBPs, potentially affecting TDP-43 further and the splicing machinery in the nucleus.
Alternative polyadenylation (APA) represents another splicing alternative regulated by TDP-43.Approximately 70% of all human pre-mRNAs feature multiple polyadenylation (PA) site, and differential PA sites utilisation results in transcripts with unique 3' untranslated regions (3'UTR) with distinct binding motifs for miRNAs and RBPs (Gu et al., 2009;Hoque et al., 2013;Ramakrishnan and Janga, 2019).TDP-43 regulates the PA sites through its interaction with target RNAs near the PA signals (Rot et al., 2017).Recently, studies have focused on the differential utilisation of PA sites in both cell culture models and post-mortem tissues from ALS/FTD patients exhibiting TDP-43 pathology.Researchers revealed that changes in APA, whether shifting from proximal to distal or the reverse, can occasionally result in increased RNA stability (eg.MARK3, ELK1, SIX3, TLX1 and ELP1), consequently leading to the upregulation of specific protein levels (Arnold et al., 2024;Bryce-Smith et al., 2024;Zeng et al., 2024).Conversely, an increased usage of NEFL and TMEM106M distal PA has been observed in TDP-43 KD cortical neurons differentiated from human stem cells (iNeurons), resulting in a decrease of their corresponding protein levels (Arnold et al., 2024).Identifying genes susceptible to TDP-43 APA should become a major area of investigation in future studies.This is crucial because several of these genes could serve as targets for the development of biomarkers and the design of innovative therapies.

Effect of TDP-43 on cryptic exon suppression
Intron sequences that are not typically included in the mature mRNA transcripts are referred as cryptic exons (CE).CEs are also considered alternative splice site regions that lead to the inclusion of non-conserved exons upon TDP-43 dysregulation.The inclusion of CEs can lead to alterations such as frameshifts, premature stop codons, and other  modifications within the resulting mRNA.In 2015, Ling and team discovered the inclusion of CEs, in particular in ATG4B and RANBP1 mRNA transcripts, as a distinctive characteristic of TDP-43 KD (Ling et al., 2015).They observed that TDP-43 bound to UG-repeat-rich regions nearby or overlapping cryptic exons, thereby suppressing the inclusion of these cryptic exons, and preserving the regions as intronic.Furthermore, they speculated that the compromised ability to suppress cryptic exons observed in cases of ALS/FTD could be attributed to the  splicing dysregulation associated with TDP-43 pathologies (Fig. 4).Further, by mapping transcripts of cryptic exon containing genes, they identified that these cryptic exons occurred in non-conserved regions.Ling's team therefore demonstrated that these cryptic exon recognition sites are highly species specific.Following this discovery, impairment of TDP-43's repression of non-conserved cryptic exons impairment was also identified in post-mortem brain tissues of individuals with AD.Theses tissues displayed nuclear clearance of TDP-43 without the presence of cytoplasmic inclusions (Sun et al., 2017).
In 2019, it was discovered that the dysfunction of TDP-43 resulted in the reduction of full-length mRNA encoding stathmin-2, through the inclusion of a cryptic exon within STMN2 gene transcripts (Klim et al., 2019;Melamed et al., 2019).This was validated in neurons transdifferentiated from patient fibroblasts with TDP-43 depletion or with ALS-causing mutations in TDP-43 (Prudencio et al., 2020).One underlying mechanism involves aberrant splicing in intron 1 of STMN2 pre-mRNA, leading to premature polyadenylation and the production of a non-functional mRNA.Consistently, aberrant polyadenylation and splicing of STMN2 pre-mRNA were observed in spinal motor neurons and the motor cortex of both sporadic and C9ORF72-linked ALS patients, supporting the notion that loss of stathmin-2 function is a crucial factor contributing to motor neuron degeneration.Moreover, truncated STMN2 RNA was found to be accumulated in the frontal cortex of patients with FTLD-TDP suggesting its potential application as a candidate biomarker.
The discovery of the truncated STMN2 RNA triggered what can be considered a new cryptic era in ALS.It was further reported that TDP-43 depletion also leads to the inclusion of a cryptic exon in UNC13A mRNA, resulting in reduced UNC13A mRNA and protein levels.However, this abnormal splicing event can be partially rescued by certain hnRNPs, such as hnRNP L, which can compensate for the loss of TDP-43 and restore UNC13A mRNA levels (Brown et al., 2022;Koike et al., 2023;Ma et al., 2022).Enhancing our understanding of the co-regulators associated with TDP-43's targets will contribute to the identification of potential biomarkers and the development of targeted treatment for TDP-43 pathologies.
Prior to the discovery of the role of TDP-43 in cryptic exon splicing in neurodegeneration, risk factors for ALS/FTD had been identified in the non-coding region of UNC13A, such as rs12973192 (C>G), rs12608932 (A>C), rs56041637 (CATC(6>10)) (Ma et al., 2022).Ma and colleagues were the first to link some of the strongest of these risk factors to cryptic splicing mechanisms as they were co-localised in the same intron as the UNC13A cryptic exon.KD of TDP-43 levels shows a significant increase in UNC13A cryptic exon inclusion, indicating that despite cryptic exons being non-conserved across species, small single nucleotide polymorphisms or expansions within a species can lead to significant changes in TDP-43 binding and function.
The molecular consequences of TDP-43 dysfunction in oligodendrocytes was also defined by mRNA sequencing on isolated oligodendrocytes from TDP-43 knock-out and WT mice.Cryptic exons were found incorporated in the mRNA of the top 1% of the highest expressed genes enriched in oligodendrocytes.Many of these are involved in oligodendrocyte development and myelination, including the cryptic exon in intron 2 of ERMN transcript (Heo et al., 2022).
Additionally, TDP-43 plays a crucial role in the expression of Neurofascin (Nfasc), a glial cell adhesion molecule necessary for paranodal junction formation and maintenance, by preventing the usage of a cryptic exon during splicing (Chang et al., 2021).This study provides evidence of TDP-43's functional involvement in axon-glial interactions within the peripheral nervous system.It also demonstrates that TDP-43's dysfunction in Schwann cells leads to impaired motor behaviour and conduction velocity.Cryptic exons were also detected in C9ORF72-linked ALS/FTD patient post-mortem tissues using single cell RNA sequencing (Gittings et al., 2023).Tissue analysis confirmed nuclear depletion and cytoplasmic accumulation of TDP-43 and showed accumulation of transcripts containing cryptic exons in STMN2 and KALRN genes.
STMN2 has garnered significant attention, given its status as one of the most abundant RNA transcripts in human spinal motor neurons.Briese and colleagues demonstrated that the depletion of TDP-43 in primary motor neurons has an impact on axon growth (Briese et al., 2020).Since ALS pathology is characterised by the degeneration of both upper and lower motor neurons, leading to a substantial loss of motor function (Brown and Al-Chalabi, 2017), this axonal regression is also evident when reduced production of STMN2 protein occurs due to the TDP-43 dysfunction.In such cases, increase level of truncated STMN2 mRNA is observed (Guerra San Juan et al., 2022).These findings suggests that the proper splicing regulation of TDP-43 on STMN2 RNA makes a significant contribution to the axonal pathomechanisms in ALS.
To test for whether insufficient full-length STMN2 protein level contributes to the regression of axonal growth in vivo, Baughn et al. employed antisense oligonucleotides (ASOs) based on previously reported data to recapitulate TDP-43 binding and suppress cryptic splicing (Baughn et al., 2023).This approach successfully reduced the truncated form of STMN2 transcripts, restored axonal regeneration and stathmin-2-dependent lysosome trafficking in human motor neurons lacking TDP-43.ASO administered via injection into the cerebrospinal fluid of mice effectively corrected STMN2 pre-mRNA misprocessing and restored protein expression levels, independent of TDP-43 binding.
Recently, cryptic exons in STMN2 and UNC13A transcripts were identified in post-mortem brain tissue from AD patients.These discoveries were found to be correlated with the TDP-43 pathology burden, but not with the presence of amyloid-beta or tau deposits (Agra Almeida Quadros et al., 2024;Ayuso et al., 2023).It was observed that the processing of STMN2 pre-mRNA is more susceptible to TDP-43 dysfunction compared to UNC13A.This suggests that therapeutic interventions targeting UNC31A and especially STMN2 may have the potential to address a broader spectrum of neurodegenerative conditions associated with TDP-43 pathologies, including AD.Some researchers are now directing their attention towards the expression of cryptic-exon-encoded de novo polypeptides to see whether they can be used as biomarkers (Seddighi et al., 2024).In a study focusing on TDP-43 KD human iPSC-derived glutamatergic neurons, ribosomes were found to bind to intronic regions of mis-spliced transcripts, providing evidence that these transcripts were still being translated.65 potential trypsin-digested cryptic peptides from 12 genes were identified through this process.Among these, a 46-amino-acid cryptic peptide was discovered in HDGFL2, a protein believed to regulate chromatin and DNA repair in non-neuronal cells, potentially affecting its interacting partners and biology.Moreover, the researchers detected cryptic peptides in the CerebroSpinal Fluid (CSF) of patients with FTD-ALS spectrum disorders.They identified 18 peptides across 13 genes that were mapped to cryptic exons, indicating their potential application as biomarkers for monitoring disease progression and therapeutic responses.
Irwin and colleagues generated monoclonal antibodies (Irwin et al., 2024) to identify cryptic peptides in HDGFL2, ACTL6B, AGRN, EPB41L4A, and SLC24A3 proteins.The validity of these antibodies was confirmed using HeLa cells in which TDP-43 was suppressed using siRNA.These results consistently detected HDGFL2 cryptic peptides.Using an Enzyme-Linked ImmunoSorbent Assay (ELISA), the authors were able to explore the concentration level of those cryptic peptides in CSF collected from both pre-symptomatic and symptomatic individuals affected by familial C9ORF72-linked ALS/FTD.These findings highlight the significance of investigating cryptic peptides in understanding disease mechanisms and developing potential biomarkers for ALS/FTD spectrum disorders.

Effects of TDP-43 on circRNA
Circular RNAs (circRNA) are non-coding RNAs generated through back-splicing events during precursor mRNA processing.CircRNA was F. Cheng et al. detected in HeLa cell RNA extracts using electron microscopy in the late 1970 s (Hsu and Coca-Prados, 1979) and was referred to as a "miRNA sponge" because of its ability to reduce their mRNA silencing potential and as RBP-binding sites and scaffolds for protein complexes (Van Rossum et al., 2016).
To date, circRNAs have been reported to form through at least 3 different routes that rely heavily on canonical splicing machinery, including splice signal sites and spliceosome (Zhou et al., 2020).The first route is the intron-pairing-driven circularisation, in which the 5' splice site of intron sequences can be directly joined with its 3' splice site to form a circRNA (Nielsen et al., 2003).The second way is mediated by RBPs which can either promote and/or be involved in circRNA formation by binding specific motifs in flanking intron sequences (Conn et al., 2015).The third path is through lariat-driven circularisation in which exon skipping happens during pre-mRNA undergoing GU/AG splicing resulting in the production of lariat intermediate containing intron-exon and in turns undergoes reverse splicing to form a circRNA (Kramer et al., 2015).This category of RNA has been implicated in the pathogenesis of several diseases and shows promise as potential biomarkers (Allegra et al., 2022(Allegra et al., , 2022;;Verduci et al., 2021;Xiao et al., 2022;Yu et al., 2021).With the advancements in RNA sequencing technology, the study of circRNA has emerged as a viable candidate for serving as a biomarker for ALS.
RNA-seq analysis of both 3-and 12-month-old TDP-43 mice with forebrain-specific deletion of TARDBP gene revealed that 22 circRNAs showed significant changes in expression levels in the neocortex.Additionally, 39 circRNAs exhibited alterations specifically at the age of 3 months, while 121 circRNAs were found to be altered solely at the age of 12 months (Wu et al., 2019).Differential expression of circRNA was determined in peripheral blood mononuclear cells from patients diagnosed with sporadic ALS revealing that 274 were upregulated and 151 were down regulated between ALS patients and healthy controls.A selection from this pool, hsa_circ_0023919, hsa_circ_0063411, and hsa_-circ_0088036 were identified as potential blood-based biomarkers for ALS (Dolinar et al., 2019).hsa_circ_0060762 and its host gene CSE1L were further identified as prospective disease-causing circRNA biomarkers in peripheral blood mononuclear cells in ALS patients (Ravnik Glavač et al., 2023).The perturbance of the circular transcriptome in spinal cord tissues in ALS patients (unknown if associated with TDP-43 pathology) was described in 2022 with 92 differentially expressed circRNAs across different spinal cord tissues sectionsspinal cord cervical, spinal cord thoracic and spinal cord lumbar (Aquilina-Reid et al., 2022).
Formation of circRNA relies heavily on the splicing machinery in which TDP-43 plays an important role.Although dysregulation of circRNAs and TDP-43 splicing dysregulation have been independently associated with various neurodegenerative diseases, further in-depth understanding of their coordinated interplay will undoubtedly provide insights into new disease-specific therapeutic approach and biomarkers for TDP-43 proteinopathies.
As previously noted, circRNAs, also contribute to gene expression by partially inhibiting miRNA activity and therefore the miRNA level (Lee et al., 2019).MiRNA are small ~22-nt long non-coding RNAs that negatively regulate their target genes by binding to the 3'UTR of target messenger RNA transcripts.A large fraction of them is encoded in introns and to date, there are two established pathways for the biogenesis of miRNA, the canonical and non-canonical pathways.In the canonical pathway, TDP-43 was reported to play a role in the miRNA biogenesis in both the nucleus and cytoplasm (Di Carlo et al., 2013;Kawahara and Mieda-Sato, 2012;Zuo et al., 2021).It is interesting to note that the non-canonical pathways in the miRNA biogenesis involves the spliceosome machinery, where TDP-43 splicing regulation could play a role in the conversion of miRtrons, pri-miRNA encoded in introns coding genes, into intron lariat.The intron lariat in turns is processed by the debranching enzyme 1 (DBR1) to produce a pre-miRNA (Okamura et al., 2007;Ruby et al., 2007).

Discussion
RNA splicing is an important mechanism which assists with the regulation of gene expression and protein composition within cells.Consequently, any disruption in this process can lead to a significant reconfiguration of the transcriptome and proteome, altering the abundance and variety of gene products.In such, RNA dysregulation is reported to be a key contributor to ALS pathogenesis.
Cryptic exons, which are generated from alternative splicing, have become an important mechanism in TDP-43 associated neurodegenerative disease studies since the discovery of the inclusion of a cryptic exon within STMN2 transcripts in 2019.Advanced technology enables highthroughput screening to detect new cryptic exons, however it is still unclear how cryptic exons and peptides affect RNA structure, function, localisation and especially their binding partners in cells.
Examining post-mortem brains from individuals with FTLD-TDP revealed the accumulation of cryptic RNA in the amygdala, hippocampus and frontal cortex.Notably, these regions exhibited significant phosphorylated TDP-43 accumulation, with the highest burden observed in the amygdala and frontal cortex (Ayuso et al., 2023).Interestingly, a similar accumulation of cryptic RNA was noted in the amygdala and hippocampus of AD-TDP patients, where TDP-43 pathology is frequently observed.This implies a shared mechanism involving alterations in RNA metabolism in both conditions.A recent study highlighted that the loss of TDP-43 splicing repression is a crucial and early occurrence in TDP-43 proteinopathies (Chang et al., 2023).This phenomenon initially manifested in neurons displaying nuclear clearance of TDP-43 without the presence of neuronal cytoplasmic inclusions.Another study shows that nuclear TDP-43 pathology occurs prior to cytoplasmic aggregation and is associated with TDP-43 dysfunction measured by the presence of STMN2 cryptic RNA (Spence et al., 2023).Additionally, TDP-43 pathology was identified in spinal cord motor neurons of PSP and corticobasal degeneration (CBD) cases, suggesting potential characteristics of systemic motor neuron TDP-43 proteinopathies (Riku et al., 2022).With the increased number of neurodegenerative diseases classified as TDP-43 proteinopathies and our gradual understanding of the common mechanisms linked to TDP-43 dysfunction in RNA splicing, the knowledge of cryptic RNA associated with TDP-43 dysregulation become applicable to a wider range of neurodegenerative diseases.This includes conditions like LATE, AD-TDP, PSP and CBD.This knowledge can be an opportunity to improve early detection in TDP-43 proteinopathies and provide molecular insights into the spectrum of diseases.Beyond that, it may also hold potential for patients and clinicians to use biomarkers to categorise patients based on the presence or absence of TDP-43 pathology.Such stratification could pave the way for more personalised therapies, by specifically targeting the restoration of mis-splicing events associated with TDP-43 pathology.
In recent studies, it has been suggested that a subset of cytoplasmically retained introns found in VCP mutant cultures, where mislocalisation of TDP-43 occurs, may function as molecular sponges for miRNAs and/or potentially sequester miRNA in the cytoplasmic, impacting their nuclear function (Petrić Howe et al., 2022).This interaction results in an upregulation of the target genes governed by these miRNAs.Similarly, SFPQ-positive cytoplasmic aggregates are features of familial and sporadic ALS patients who display TDP-43 pathology and was reported that SFPQ intron 9 retention is increased in the motor cortex of ALS patients, reducing its gene expression (Hogan et al., 2021).This reduction was reported to affect the circRNAome within cells as SFPQ was proven to be a critical element in the circRNA biogenesis of the Distal Alu Long Intron (DALI) type (Stagsted et al., 2021).This intriguing discovery opens a promising avenue for future investigations to explore the connection between intron retention and circRNA activity in various disease models.
A newly highlighted aspect of TDP-43 in TDP-43 proteinopathies is its involvement in APA.Studies found that reducing TDP-43 levels leads to differential PA site usage.Neurons typically show a preference for distal PA sites, contributing to the widely recognised phenomenon where genes expressed in the central nervous system (CNS) feature transcripts with the longest 3'UTR sequence among all tissues in the body (Miura et al., 2013).As the 3'UTR contains a significant abundance of binding motifs for miRNAs and RBPs, which are crucial for RNA stability, this indicates that APA likely plays a crucial role in the post-transcriptional regulatory mechanism in neurons and other cell types within the CNS.
In addition, TDP-43 is also involved in many other pathways, including miRNA biogenesis.Although no studies demonstrated the impact of TDP-43 splicing dysregulation on miRNA processing, Agranat-Tamir and colleagues presented a compelling connection between intronic miRNA processing and splicing (Agranat-Tamir et al., 2014).Their work elucidated how alternative splicing can regulate the abundance of clustered intronic miRNAs.Through their investigation into the influence of splicing on miRNA biogenesis, they observed that alternative splicing incorporated pre-miRNA sequences into exons, thereby affecting miRNA processing and subsequent levels.MiRNAs could be excellent biomarker candidates as they can be detected in various locations, both inside the cells and in the extracellular environment, including body fluids like serum, plasma, urine, saliva, bronchoalveolar lavage fluid, amniotic fluid, and semen (Fasoulakis et al., 2020;Hanke et al., 2010;Joshi et al., 2022;Lawrie et al., 2008;H. Lee et al., 2019;Ng et al., 2009;Park et al., 2009).Specifically, in the extracellular compartment, miRNAs are present within extracellular vesicles where they remain stable.It would be interesting to investigate the potential role of TDP-43 in the miRtron synthesis as miRNA can be used as additional biomarkers for a more precise diagnostic for TDP-43 splicing dysregulation in early stage of TDP-43 proteinopathies.

Conclusion
Splicing is a critical process for various types of RNA, including mRNA, miRNA, and circRNA and TDP-43 has been shown to be important in this process.When TDP-43 WT is mislocalised or mutated, its ability to regulate splicing becomes dysfunctional, resulting in the generation of aberrant RNA.However, the precise role and molecular mechanisms of TDP-43 in the generation of these atypical RNAs remain unclear.Despite this gap in our understanding, the identification of cryptic exons and the observation of circRNA and miRNA dysregulation provide promising prospects for the development of innovative diagnostics and therapeutics for TDP-43 proteinopathies in the future.

Declaration of generative AI and AI-assisted technologies in the writing process
During the preparation of this work the authors used ChatGPT in order to improve the language and readability of content already written by the authors.After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of Competing Interest
No competing interests.

Fig. 2 .
Fig. 2. TDP-43 protein structure.Schematic representation of TDP-43 protein shows N-terminal domain, followed by two RNA recognition motifs RRM1 and RRM2 and a glycine-rich C-terminal.

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Fig. 3 .
Fig. 3. Schematic figure presenting the constitutive splicing event and seven alternative splicing events of pre-mRNA: exon skipping, alternative 5' and 3' splicing, mutually exclusive exon, intron retention, alternative polyadenylation and alternative promoter.Boxes represent exon and lines are introns.

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Fig. 4 .
Fig. 4. Schematic representing the different cryptic exon splicing in healthy and disease states.Typically, TDP-43 will bind and mask specific intronic regions of pre-mRNAs which generates in turn a mature mRNA and protein.In disease state, due to the absence of TDP-43 or loss of TDP-43 RNA binding function, the intronic region is incorporated into the mRNA, resulting in either (1) the degradation of the mRNA by nonsense mediated degradation pathwayor (2) a truncated protein due to the presence of premature stop codons or (3) the incorporation of cryptic peptide in the protein.

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Table 1
Summary of recent TDP-43 splicing dysregulation findings.