Templated Aggregation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Seeding with TDP-43 Peptide Fibrils

Abstract

Frontotemporal lobar degeneration (FTLD)2 is the second most common dementing disorder in patients under 65 years of age and is characterized by progressive atrophy of the frontal and temporal lobes in the brain. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration. In 2006, TAR DNA-binding protein of 43 kDa (TDP-43) was identified as the major component of tau-negative, ubiquitin-positive inclusions in these diseases (1, 2). TDP-43 is a heterogeneous nuclear ribonucleoprotein of 414 amino acids, originally identified as a transcriptional repressor that binds to the transactivation-responsive region of the HIV-1 gene (3). It is expressed ubiquitously and is localized mainly in nuclei, functioning in exon splicing, gene transcription, regulation of mRNA stability and biosynthesis, and the formation of nuclear bodies. For example, it has been reported that TDP-43 binds to the junctional region between exons 9 and 10 of the gene coding cystic fibrosis transmembrane conductance regulator and causes skipping of exon 9 (4). Structurally, TDP-43 is characterized by two RNA-recognition motifs, a C-terminal tail that contains a glycine-rich region and a glutamine/asparagine (Gln/Asn)-rich region.Discovery of missense mutations in the TARBDP gene in patients with familial and sporadic ALS and FTLD (5-12) demonstrated a direct link between genetic lesions and development of TDP-43 pathology. Importantly, most of the mutations are localized in the carboxyl-third of the molecule, which shows sequence similarity to prion proteins. Furthermore, biochemical and histological analyses demonstrated accumulation of full-length and C-terminal fragments (CTFs) in hyperphosphorylated and fibrillar forms in brain and spinal cord of patients (13). Furthermore, the CTF-banding patterns are different between the diseases and are closely related to the neuropathological phenotypes, which can be classified into at least four groups (types A-D) (13,14). The CTF-banding patterns are considered to reflect the protease resistance of the assembled protein structures (15). It was recently demonstrated that the pathological TDP-43 has prion-like activity and can convert normal TDP-43 to an abnormal form in cultured cells (16). Interestingly, the CTF-banding patterns of converted host proteins resemble those of the pathological TDP-43 used as seeds, suggesting that the conversion is template-dependent.These findings strongly suggest that aggregation of C-terminal regions of TDP-43 plays a central role in the pathogenesis and progression of ALS and FTLD with TDP-43 pathologies (FTLD-TDP). The aggregation of CTFs of TDP-43 has also been demonstrated in experimental cellular models; for example, our previous study showed that expression of CTF(162-414) of TDP-43 with a GFP tag in SH-SY5Y cells induced cytosolic mislocalization of TDP-43 in phosphorylated and ubiquitinated aggregates, recapitulating the TDP-43 pathology in brains (17).

Prion‐like propagation as a pathogenic principle in frontotemporal dementia

“…Interestingly, seeded aggregation required the interaction of the peptide seed with the identical peptide sequence of the host protein (Shimonaka et al . ). This indicates, that conformation‐specific templating of TDP‐43 can lead to differentially misfolded conformers, which furthermore can cause different patterns of pathology and disease phenotypes in a strain‐like manner.…”

“…; Shimonaka et al . ), which can cluster together in large complexes with resemblance to pathological inclusions (Fang et al . ).…”

“…Moreover, seeding aggregation with in vitro aggregated synthetic TDP‐43 peptides (Shimonaka et al . ) as well as homogenates from ALS and FTD patients (Nonaka et al . ) in cell culture models resulted in various types of pathologies showing the same distinct biochemical characteristics as observed in the original seeds.…”

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons

“…This propensity has been localised to a low-complexity domain in the C-terminal region that may play a role in RNA binding, granule formation, and/or liquid–liquid phase separation to subserve transcription dynamics and mRNA transport [6, 12, 32, 34, 55, 56]. It is hypothesised that inherited or spontaneous mutation of the encoding gene, TARDBP , or of an interacting partner, upsets the balance between aggregation and dissolution of TDP-43 [14, 71], which may be differentially regulated under changing conditions, including during the cellular stress/death response [7, 30].…”

Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

“…11 More recently, it was observed that TDP-43 fibrils formed from short C-terminal derived peptides triggered the seeddependent aggregation of wild type TDP-43 (TDP-43 WT ) or TDP-43 lacking a nuclear localization signal in SH-SY5Y cells resulting in different peptides sequences of TDP-43 producing fibrils with distinct biochemical properties reminiscent of prion strains. 12 Recently a novel method for quantifying protein inclusions was described. 13 We reasoned that this method might be used to provide a quick and robust measure of prion-like propagation.…”