TARDBP (1-414) Human

What is TARDBP and what protein does it encode?

TARDBP is the gene located on chromosome 1 that encodes the TAR DNA-binding protein 43 (TDP-43), an RNA-binding and DNA-binding protein with multiple cellular functions. TDP-43 is a major component of ubiquitinated inclusions that characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) . The full-length human TDP-43 protein consists of 414 amino acids and contains several functional domains that mediate its various roles in RNA processing, gene expression regulation, and protein-protein interactions .

What are the functional domains of TDP-43 protein?

The TDP-43 protein contains several distinct functional domains, most notably the highly conserved glycine-rich C-terminal domain. This C-terminal region is involved in protein-protein interactions, particularly with heterogeneous ribonucleoproteins (hnRNPs), and is necessary for the splicing inhibitory activity of TDP-43 for certain RNA transcripts . This domain also influences the solubility and cellular localization of TDP-43 . Additionally, TDP-43 contains RNA recognition motifs (RRMs) that are critical for its RNA binding functions, with evidence suggesting that only TDP-43 products containing an intact RRM can cause toxicity in certain model systems .

What neurodegenerative conditions are associated with TDP-43 pathology?

TDP-43 pathology defines a growing class of neurological diseases collectively referred to as TDP-43 proteinopathies. While most prominently associated with ALS and FTLD-U, TDP-43 pathology has been observed to varying degrees in multiple conditions including Lewy body disease, parkinsonism-dementia complex of Guam, corticobasal degeneration, Alzheimer's disease, and hippocampal sclerosis . This widespread involvement across different neurodegenerative conditions underscores TDP-43's importance as a central player in neurodegeneration mechanisms .

What types of TARDBP mutations have been identified in ALS patients?

Over 40 TARDBP mutations have been discovered in patients with ALS, with the majority located in exon 6, which encodes the highly conserved, glycine-rich, C-terminus of TDP-43 . Specific mutations identified include p.Gly290Ala, p.Gly298Ser, p.M337V, p.N345K, p.I383V, p.G348C, p.R361S, p.N390D, and p.N390S among others . These mutations provide conclusive evidence that TDP-43 plays a direct role in neurodegeneration. Notably, three mutations discovered by researchers (p.M337V, p.N345K, and p.I383V) were found exclusively in familial ALS patients at a frequency of approximately 3.3% in this subpopulation .

How do researchers screen for TARDBP mutations in patient populations?

Researchers employ DNA sequencing to screen for TARDBP mutations. In comprehensive screening approaches, all coding exons (typically 5 coding and 2 non-coding exons) of the TARDBP gene are sequenced in patient cohorts . This process involves:

• Collection of DNA samples from patients with clinical diagnoses of conditions like ALS, FTLD, or FTLD-ALS, as well as from control individuals

• PCR amplification of TARDBP exons

• Direct sequencing of PCR products

• Validation of identified mutations using additional control populations to ensure they are not common polymorphisms

In one study, sequencing analyses of TARDBP in 296 patients revealed 3 heterozygous missense mutations (c.1009 A>G, c.1035 C>A, and c.1147 A>G) in 3 of 116 ALS patients (2.6%) . The researchers confirmed the pathogenic nature of these mutations by demonstrating their absence in hundreds of control individuals .

Why are TARDBP mutations mostly concentrated in the C-terminal domain?

The concentration of TARDBP mutations in the C-terminal glycine-rich domain reflects this region's critical functional importance. This domain regulates gene expression and mediates protein-protein interactions, particularly with heterogeneous ribonucleoproteins (hnRNPs) . The C-terminal domain is necessary for TDP-43's splicing inhibitory activity for certain RNA transcripts and influences the protein's solubility and cellular localization . The clustering of disease-causing mutations in this specific region strongly suggests that disruption of these functions is central to pathogenesis. Researchers postulate that mutations in this domain may cause neurodegeneration through both gains and losses of function, altering TDP-43's normal role in regulating gene expression .

What cellular models are used to study TARDBP mutations?

Researchers employ several cellular models to study TARDBP mutations, with lymphoblastoid cell lines derived from mutation carriers being particularly valuable. These patient-derived lymphoblastoid cells allow for the biochemical analysis of mutant TDP-43 under various conditions . Additionally, induced pluripotent stem cells (iPSCs) derived from patient lymphoblasts can be differentiated into neurons, creating disease-relevant cell types for studying mutation effects . Other cellular models include yeast systems, which have established connections between TDP-43 aggregation and toxicity, showing that only TDP-43 products that form aggregates and contain an intact RNA recognition motif (RRM) are toxic .

How do researchers analyze TDP-43 protein biochemistry in experimental models?

Biochemical analysis of TDP-43 typically involves:

• Treatment of cells with proteasome inhibitors (e.g., MG-132 or PSI) to induce TDP-43 accumulation

• Cellular fractionation to separate detergent-soluble and detergent-insoluble protein fractions

• Western blot analysis to detect full-length TDP-43 and truncation products

• Immunocytochemistry to visualize TDP-43 localization and aggregation

• Splicing assays (e.g., CFTR exon 9 skipping) to assess functional effects on TDP-43's RNA processing role

Using these approaches, researchers have observed that proteasome inhibition leads to increased accumulation of detergent-insoluble TDP-43 fragments of approximately 25 and 35 kDa in lymphoblastoid cell lines derived from patients with TARDBP mutations . These methods also revealed that TDP-43 is a caspase substrate, and that proteolytic cleavage of TDP-43 leads to its redistribution from the nucleus to the cytoplasm and generates insoluble C-terminal fragments similar to those found in diseased brains .

What mechanisms connect TARDBP mutations to neurodegeneration?

The pathogenic mechanisms linking TARDBP mutations to neurodegeneration involve both gain- and loss-of-function pathways:

Loss-of-function mechanisms:

• Proteolytic cleavage of TDP-43 generates fragments that are more aggregation-prone

• These fragments form inclusions more readily than full-length TDP-43

• Cleavage and subsequent aggregation deplete functional TDP-43 and may sequester remaining full-length protein

• TDP-43 depletion has severe consequences, as demonstrated in Drosophila models where deletion of the TDP-43 homolog results in neuromuscular junction defects, paralysis, and reduced lifespan

• In mice, TDP-43 depletion affects numerous RNA transcripts encoding neurodegeneration-related proteins

Gain-of-function mechanisms:

• TDP-43 fragments and their inclusions may themselves be toxic

• Yeast models demonstrate that TDP-43 aggregation correlates with toxicity

• C-terminal fragments lacking the N-terminal region cannot properly function in RNA splicing

• Treatment with PSI (proteasome inhibitor) leads to increased active caspase-3, promoting apoptotic cell death

These mechanisms suggest that TARDBP mutations contribute to disease through multiple pathways, affecting TDP-43's normal cellular functions while also creating toxic species.

How do researchers differentiate pathological TDP-43 from normal TDP-43?

Researchers differentiate pathological TDP-43 from normal TDP-43 through several biochemical and histological characteristics:

• Subcellular localization: Normal TDP-43 is predominantly nuclear, while pathological TDP-43 shows cytoplasmic mislocalization and nuclear clearing

• Post-translational modifications: Pathological TDP-43 is hyperphosphorylated, ubiquitinated, and abnormally cleaved

• Solubility: Pathological TDP-43 shows increased detergent insolubility compared to normal TDP-43

• Fragmentation patterns: Specific C-terminal fragments (25-35 kDa) are characteristic of pathological conditions

• Immunohistochemical properties: Specialized antibodies can distinguish pathological forms from normal TDP-43

These characteristics have been observed in both patient tissues and experimental models. For instance, lymphoblastoid cell lines from TARDBP mutation carriers (M337V, N345K, and I383V) show increased accumulation of detergent-insoluble TDP-43 fragments of approximately 25 and 35 kDa when treated with proteasome inhibitors .

What is the significance of TDP-43 fragmentation in disease pathogenesis?

TDP-43 fragmentation plays a critical role in disease pathogenesis through several mechanisms:

• Enhanced aggregation: TDP-43 fragments, particularly C-terminal fragments, are more aggregation-prone and form inclusions more readily than full-length TDP-43

• Loss of function: C-terminal fragments lacking the N-terminal region (e.g., fragments 208-414, 218-414, and 220-414) cannot properly function in RNA splicing, as demonstrated in CFTR exon 9 skipping assays

• Toxic gain of function: The fragments themselves may be toxic species

• Sequestration: Aggregating fragments may sequester remaining full-length TDP-43, further depleting the functional pool

• Caspase activation: The generation of TDP-43 fragments is linked to caspase-3 activation, promoting apoptotic cell death

Studies have shown that proteasome inhibition in lymphoblastoid cells from patients with TARDBP mutations leads to increased production of these pathological fragments . Additionally, caspase inhibitors can attenuate both caspase-3 activation and TDP-43 fragmentation, suggesting a mechanistic link between these processes .

What neuropathological techniques are used to identify TDP-43 pathology in tissue samples?

Researchers employ several neuropathological techniques to identify and characterize TDP-43 pathology in tissue samples:

• Immunohistochemistry (IHC): Using antibodies specific to TDP-43 to visualize its distribution and aggregation in tissue sections

• Double immunofluorescence: Combining TDP-43 antibodies with markers for ubiquitin or other proteins to assess co-localization

• Biochemical fractionation: Separating proteins based on solubility in detergents to isolate aggregated TDP-43

• Western blotting: Analyzing protein expression patterns, including full-length TDP-43 and disease-specific fragments

• Mass spectrometry: Identifying post-translational modifications and protein interactions

These techniques have confirmed TDP-43 neuropathology in CNS tissue from patients with TARDBP mutations. For example, in one family carrying the p.Gly298Ser mutation, TDP-43-positive inclusions were observed in many areas of the CNS, including remaining anterior horn cells in diseased spinal cord .

How do familial and sporadic ALS cases compare in terms of TDP-43 pathology?

TDP-43 pathology is present in both familial and sporadic ALS cases, with certain similarities and differences:

Similarities:

• Both show TDP-43-positive inclusions in affected CNS regions

• Both demonstrate cytoplasmic mislocalization of TDP-43 with nuclear clearing

• Both exhibit hyperphosphorylated and ubiquitinated TDP-43 species

Differences:

These observations suggest that while the initial triggers may differ between familial (mutation-driven) and sporadic ALS, they may converge on common pathological mechanisms involving TDP-43 misprocessing and aggregation.

What are the current challenges in modeling TDP-43 proteinopathies?

Researchers face several challenges when modeling TDP-43 proteinopathies:

• Species differences: TDP-43 is highly conserved but differences between species may affect disease modeling

• Developmental effects: Complete loss of TDP-43 results in embryonic lethality in mice, complicating knockout approaches

• Cell type specificity: Differential vulnerability of neuronal populations to TDP-43 dysfunction is difficult to model

• Model selection: Different model systems (cells, flies, mice, etc.) show variable aspects of TDP-43 pathology

• Temporal dynamics: Acute vs. chronic effects of TDP-43 dysfunction may yield different outcomes

• Distinguishing primary from secondary effects: Separating direct consequences of TDP-43 mutation from downstream pathological cascades

Despite these challenges, multiple complementary approaches have provided valuable insights. For instance, the combination of patient-derived lymphoblastoid cells, yeast models, and Drosophila systems has helped establish both gain- and loss-of-function mechanisms in TDP-43 proteinopathies .

What therapeutic strategies are being investigated for TDP-43 proteinopathies?

Based on current understanding of TDP-43 pathophysiology, several therapeutic approaches are being investigated:

• Preventing TDP-43 aggregation: Compounds that stabilize native TDP-43 conformation or inhibit aggregation

• Inhibiting pathological TDP-43 cleavage: Caspase inhibitors that prevent the generation of toxic TDP-43 fragments

• Enhancing clearance of TDP-43 aggregates: Boosting autophagy or the ubiquitin-proteasome system

• Restoring nuclear TDP-43 function: Approaches to maintain proper nuclear localization and function

• RNA-targeted strategies: Addressing downstream RNA processing defects caused by TDP-43 dysfunction

Research has shown that caspase inhibition can attenuate both TDP-43 fragmentation and caspase-3 activation in cellular models . This suggests that targeting the proteolytic cleavage of TDP-43 might be a viable therapeutic strategy for TDP-43 proteinopathies.