Products for Neurodegneration Research: TDP-43

 TDP-43 Antibodies and Kits (click catalog number for product and ordering information)

Catalog Number	Product Name	Specificity	Size
CAC-TIP-PTD-P03	Anti TAR DNA-Binding Protein 43 (TDP-43), phospho Ser409 pAb (Rabbit, Antiserum)	Human	100UL
CAC-TIP-PTD-P04	Anti TAR DNA-Binding Protein 43 (TDP-43), phospho Ser410 pAb (Rabbit, Antiserum)	Human	100UL
CAC-TIP-PTD-M01A NEW FORMAT	Anti TAR DNA-Binding Protein 43 (TDP-43), phospho Ser409/410 mAb (Clone 11-9)	Human	100UL
CAC-TIP-PTD-M01	Anti TAR DNA-Binding Protein 43 (TDP-43), phospho Ser409/410 mAb (Clone 11-9)	Human	100UL
CAC-TIP-TD-P07	Anti TAR DNA-binding protein 43 (TDP-43) Amino Acids 3-12 pAb (Rabbit, Antiserum)	Human, Rat	100UL
CAC-TIP-TD-P09	Anti TAR DNA-binding protein 43 (TDP-43) Amino Acids 405-414 pAb (Rabbit, Antiserum)	Human, Rat	100UL
CSB-E17007h	Human TAR DNA-binding protein 43 (TARDBP/TDP43) ELISA Kit	Human	1x96 rxns
PRX-MKB3512	Anti TARDBP pAb (Rabbit, Antiserum)	Mouse	100UL
BAM-70-325-EX	Anti Importin Subunit Alpha-3 (KPNA4/Qip1) mAb (Clone 3D10)	Human, Monkey, Mouse, Rat, Hamster, Dog, Cow	200UG
PRX-MKA0724	Anti Importin 13 (IPO13) pAb (Rabbit, Antiserum)	Human, Mouse	100UL
PRX-MKA0724	Anti Importin 13 (IPO13) pAb (Rabbit, Antiserum)	Human, Mouse	100UL
CAC-CE-005A	Anti Importin-4 (IPO4) mAb (Clone 3C2)	Human, Monkey, Mouse, Rat, Hamster	100UL

 About TDP-43 Biology and Molecular Biology 

TDP43 was identified in 2006 as a key component of insoluble aggregated inclusions in neuronal cells of patients suffering from ALS and FTD. ALS is a fatal late-onset neurodegenerative disorder involving progressive motor neuron dysfunction and muscular atrophy. FTD is heterogeneous clinical syndrome characterized by progressive changes in behavior, personality, and/or language. Ninety seven percent of ALS cases and ∼45% of FTD cases involve TDP43 aggregation (FTD-TDP). Most ALS cases are sporadic (sALS, 90–95%) and associated with elevated cytoplasmic TDP43. Rare familial ALS cases (fALS, 5-10%) involve inheritance of mutations in TARDBP (encoding TDP43), SOD1 (superoxide dismutase 1), FUS (fused-in-sarcoma), C9ORF72 repeat expansion, and NEK1 (NIMA-like kinase 1). Two other diseases with TDP43 pathology are primary lateral sclerosis and progressive muscular atrophy; together with ALS and FTD-TDP, these four diseases are the principal TDP43-proteinopathies. 

 From: Prasad, A., Bharathi, V., Sivalingam, V., Girdhar, A., Patel, B. (2019). Molecular Mechanisms of TDP-43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis Frontiers in Molecular Neuroscience 12(), 25.

TDP43 was first identified in 1995 as a 414 amino acid repressor regulating HIV-1 gene expression. It is a highly conserved and ubiquitously expressed RNA/DNA-binding protein belonging to the large heterogeneous nuclear ribonucleoprotein (hnRNP) family. Its expression level is tightly autoregulated as it shuttles continuously between the nucleus and cytoplasm. Characteristically, its distribution shifts from predominantly nuclear in normal cells to cytoplasmic in diseased cells. It appears to be involved in multiple aspects of RNA metabolism, including transcription, splicing, transport and stabilization. The pathological hallmarks of TDP43 proteinopathies include defects in RNA metabolism and nucleo-cytoplasmic transport, C-terminal fragmentation, aggregation and deposition into cytoplasmic inclusion bodies and perturbed mitochondrial structure and function. Mutation, post-translational modification (PTM) and phase transition at liquid-liquid and liquid-solid interfaces play complex roles in disease course initiation and evolution.

TDP43 comprises an N-terminal region (aa1-102) with a nuclear localization signal (NLS, aa82–98), two RNA recognition motifs RRM1 (aa104-176) and RRM2 (aa192-262), a nuclear export signal (NES, aa239-250), a C-terminal region (aa274-414) which encompasses a prion-like glutamine/asparagine-rich (Q/N) domain (aa345-366) and a glycine-rich region (aa366-414). Mitochondrial TDP43 localization depends on internal motifs M1 (aa35-41), M3 (aa146-150) and M5 (aa294-300). Some TDP43 mutations (G294V, A328T, G348C and S393L) occur in both sporadic and familial ALS. One mutant (G295S) occurs in sALS, fALS and FTD. Of interest, a fALS-associated phosphorylation-prone TDP43 mutation (G298S) in the mitochondrial localizing internal motif M5 exhibited elevated mitochondrial import.

TDP43 interacts with nuclear and cytoplasmic proteins involved in RNA metabolism and localizes to sites of transcription and splicing. Genome-wide RNA immunoprecipitation approaches such as CLIP-seq identified more than 6000 TDP43-associated mRNA targets — nearly 30% of the entire transcriptome. Functional clustering suggested key roles in neuronal development, axon guidance and synapse structure/function. In the cytoplasm TDP43 bound with high specificity to UG-rich RNA sequences and associated with the 3′ UTRs of mRNAs and pre-mRNAs. TDP43 nuclear depletion resulted in mRNA splicing aberrations, while TDP43 overexpression titrated limiting binding partner proteins and lead to formation of dysfunctional protein complexes and neuronal cell damage. TDP43 regulated the mRNA splicing patterns of several important genes, including TARDBP, APP, SNCA (encoding α-synuclein), HTT (encoding Huntingtin), FUS and CFTR (encoding cystic fibrosis transmembrane conductance regulator). TDP43 also regulated the half-life of several mRNAs (including its own) by binding regulatory 3′ UTR sequences. This effect could be either positive (as observed for human low molecular weight neurofilament mRNA) or negative (as documented for VEGF and progranulin mRNA transcripts). TDP43 assisted in assembling bound RNA molecules into ribonucleoprotein (RNP) granules for transport on microtubules to distant axonal locations and ALS-associated TDP43 mutations impaired RNP granule transport.

TDP43 also promoted biogenesis and processing of noncoding RNAs, such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). In HEK293 cells cytoplasmic TDP43 interacted with the Dicer complex and promoted precursor miRNA (pre-miRNA) processing. And in cultured HeLa cells, rodent neurons and induced pluripotent stem cell-derived human neurons, TDP43 downregulation lead to altered miRNA expression. In genome-wide studies TDP43 bound several lncRNAs, including NEAT1 (Nuclear enriched abundant transcript 1) and MALAT1 (Metastasis associated in lung adenocarcinoma transcript 1). Interestingly, NEAT1 and MALAT1 levels were elevated in FTD-TDP. Together, these findings suggest a broad role for TDP43 in maintaining mRNA stability, maturation and transport.

Most ALS-associated TARDBP mutations occur in exon 6, encoding the glycine-rich C-terminus where they increased the propensity of short α-helices to transition toward aggregation-permissive β-sheets. Recombinant TDP43 harboring ALS-linked C terminal mutations Q331K, M337V, Q343R, N345K, R361S or N390D exhibited increased fibrillar aggregation in vitro and cytotoxicity in yeast cells. Degenerating neurons of ALS and FTLD patients contained sarkosyl-insoluble TDP43 aggregates; and similar sarkosyl-insoluble inclusions were generated in a cellular seeding reaction with in vitro-prepared fibrillar TDP43 aggregates. These aggregates were revealed in TEM to be thick bundles comprised of stacks of thin fibers. Protease treatment of full-length TDP43 aggregates followed by mass spectrometry showed their fibrillar core structure to comprise distinct C-terminal fragments.

The aberrant generation of toxic C-terminal fragments (~15-35kDa) via caspase and calpain cleavage is an important TDP43 post-translational modification (PTM) and aggregation-promoting mechanism. TDP43 mutations associated with accelerated disease progression (including A315T and M337V) exhibited increased susceptibility to protease-mediated cleavage and C-terminal fragment release. For example, in vitro, calpain-I fragmented recombinant TDP43^A315T or TDP43^M337V more rapidly than TDP43^WT. Similarly, caspase-3 more efficiently cleaved TDP43^D169G than TDP43^WT. By contrast, TDP43^A90V imparted partial resistance to caspase-3 digestion. In an isogenic cell line study, TDP43 with ALS-linked mutations G298S, Q331K, or M337V exhibited longer half-lives and greater stability than WT. Thus, elevated protein half-life may have contributed to accelerated disease onset in fALS patients with TDP43 mutations D169G, K263E, G298S, A315T, M337V, Q343R, G348C, N352S or A382T.

While TDP43 is predominantly nuclear, it continuously shuttles to and from the cytoplasm, thereby engaging diverse physiological and pathological cytoplasmic functions. A prominent feature of ALS and FTD-TDP is the loss of functional nuclear TDP43 and its increased deposition in cytoplasmic inclusion bodies of brain and spinal cord neurons. NLS deletion caused TDP43^deltaNLS accumulation in cytoplasmic aggregates and sequestered TDP43^WT, thereby further depleting the nuclear TDP43 pool. By contrast and as expected, NES deletion promoted the formation of nuclear aggregates. Only one fALS-linked mutation (A90V) has been identified in the NLS. Several C-terminal mutations (G294V, A315T, M337V, G376D and A382T) enhanced TDP43 cytoplasmic mislocalization by unknown mechanisms. Factors influencing nucleocytoplasmic transport, such as the role of nuclear importins, transport-partners and effects of mutations on TDP43 conformation during transit, require further investigation.

Phosphorylation is another PTM that plays an important role in the development of TDP proteinopathies. Phosphorylation of TDP43 appears to occur via the casein kinases CK1 and CK2 and glycogen synthase kinase (GSK3). Phosphorylation is associated with increased TDP43 cytoplasmic mislocalization and aggregation in neuronal cells. Notably, TDP43 inclusions in brain cortex versus spinal cord cells of ALS and FTD patients exhibited distinct phosphorylation patterns. While affected cortex showed accumulation of phosphorylated C-terminal fragments, spinal-cord cells showed predominant deposition of phosphorylated full-length TDP43. Owing to the availability of highly specific antibodies to TDP43 in different states of phosphorylation, phospho-TDP43 positive inclusions in brain samples have been well-characterized. TDP43 has 41 serine, 15 threonine and 8 tyrosine residues that may act as phosphorylation sites. Using 39 antisera against potential phosphorylation sites Hasegawa et. al. determined that TDP43 from FTD and ALS brain cells is phosphorylated at casein kinase phosphorylated serines 379, 403/404, 409, 410, 409/410; the latter dual modification is considered a signature of ALS pathology. Validated for WB, IHC, and ELISA, six of the most relevant of these antibodies are available through CosmoBio USA.

Aggregated TDP43 in neuronal cells can possess amyloid-like features. Both WT and ALS-associated mutant TDP43 peptides efficiently formed β-sheet-rich fibrillar aggregates that stained positively with the amyloidogenic dye Thioflavin-T (ThT). A peptide from the amyloidogenic core region (aa286-366) containing ALS-linked mutation G335D exhibited amyloid-like aggregation and cross-seeding abilities. Interestingly, the shortest TDP43 peptides shown to form amyloid-like aggregates, DLII (aa247–250) and NFGAF (aa312–316), bore resemblance to the amyloidogenic core sequence of the human islet amyloid polypeptide (IAPP).

Overall net charge regulated amyloid aggregation of Aβ42 peptide, a central player in the development of AD. Similarly, a low net charge decreased TDP43 solubility and enhanced its aggregation whereas a high net charge promoted electrostatic repulsion and impeded its aggregation. Consistent with this, Hofmeister series kosmotropic anions greatly accelerated and chaotropic anions impeded the rate of amyloid-like aggregation. High resolution structural studies of TDP43 peptides from the RRM2 domain and the low complexity domain (LCD, aa312-317) revealed a common amyloid steric zipper structure. Structural polymorphism in the backbone conformation of the steric zipper structure of an RRM2 peptide (aa247-257) defined distinct amyloid aggregate types. An LCD region peptide (aa312-317) and its ALS-linked mutants (A315T and A315E) formed kinked β sheet structures that promoted the formation of phase separated droplets and hydrogels, akin to stress granules (SGs).

One mechanism that eukaryotic cells have developed to protect against oxidative stress, heat shock, viral infection and toxic chemicals is the rapid reversible formation of cytoplasmic SGs, membraneless protein/nucleic acid-containing organelles of ≤5 µm in diameter. These compartments arise from conversion of a single-liquid-phase system into one with multiple coexisting, spatially separated liquid phases (also referred to as liquid-liquid phase separation or liquid demixing). SGs are storage and sorting stations for RNA-binding proteins, translationally stalled mRNAs and arrested pre-initiation complexes. Neuronal cells are vulnerable to stress and hence susceptible to conversion of SGs into pathological inclusion bodies, as seen in ALS- and FTD-TDP-affected brains. TDP43 is involved in both assembly and maintenance of SGs as well as the expression of key SG-nucleating proteins: G3BP (rasGAP SH3 domain binding protein 1) and TIA-1 (T cell-restricted intracellular antigen-1). Under sorbitol-induced osmotic stress, TDP43G348C appeared in progressively larger SGs. Quantification of TDP43 levels in SGs revealed ALS-linked mutants D169G and R361S accumulated in larger quantities than WT. Additionally, TDP43-containing SGs in neuronal cells expressing aggregation-prone TDP43 mutants A315T or Q343R exhibited increased average size, decreased distribution density and impaired mobility. Similarly, in neuronal cell line SHSY5Y expression of TDP43 mutants D169G, G294A, Q343R, Q331K, M337V or N390D enhanced formation of TDP43-positive inclusion bodies. Interestingly, TDP43-mediated sequestration of translation factors into SGs may modulate protein translation. For example, in neuroblastoma cells, increased cytoplasmic TDP43 globally repressed protein synthesis in a fashion that was rescued by over-expression of ribosomal protein RACK1 (Receptor of activated protein C kinase 1). Importantly, in ALS patient motor neurons TDP43 interacted with and sequestered RACK1 into inclusion bodies.

Recent crosslinking experiments revealed that in normal human brain TDP43 exists in a spectrum of oligomeric species, ranging from dimers, trimers, tetramers to higher order multimers. TDP43 oligomerization is proposed to increase its affinity and specificity for RNA targets and protein partners. Cellular overexpression of an 86 kDa dimeric form of TDP43 caused the N-terminal region (aa3-183) to seed the formation of (potentially pathological) high molecular weight aggregates. Fang et al. reported that full-length TDP43 formed spheroidal and ring-like oligomeric structures cytotoxic to neuronal cells. TEM and dynamic light scattering analyses of recombinant full-length TDP43 purified by size exclusion chromatography revealed the size distribution of oligomeric TDP43 to be from 40 to 400 nm. Consistent with this, TEM analysis of gold immunolabelled FTD-TDP brain fractions revealed TDP43 oligomers to exhibit a diameter of ∼50nm. TDP43 oligomers also manifested a propensity to cross-seed Aβ42 peptide, demonstrating structural inter-convertibility among common oligomeric amyloid structures. Finally, polyclonal antibodies raised against TDP43 oligomers detected not only oligomeric aggregates obtained in vitro, but more importantly oligomers in brain sections from a TDP43 mouse model and from FTD-TDP-affected patients.

Across various experimental models, TDP43 and its disease-associated mutants significantly enhanced mitochondrial abnormalities, reflecting well-documented mitochondrial dysfunction in ALS patients. In primary motor neurons WT and mutant TDP43 over-expression reduced mitochondrial length and movement and both defects could be reversed by co-expression of mitochondrial fusion protein Mfn2 (Mitofusin-2). Similarly, TDP43 expression caused abnormal mitochondrial transport and distribution in a mouse model; enhanced mitochondrial fission and fragmentation in a fly model; and increased oxidative stress and formation of peri-mitochondrial TDP43 aggregates in a yeast model. Mitochondria are primary sites for reactive oxygen species (ROS) production and a major target of ROS-induced damage. In cell line NSC34, mutant TDP43 overexpression induced oxidative damage and increased nuclear accumulation of antioxidant response modulator Nrf2. Interestingly, despite nuclear accumulation, total Nrf2 was markedly depleted and the Nrf2/ARE pathway was impaired, resulting in reduced neurites and increased lipid peroxidation products. In D. melanogaster, TDP43 expression increased levels of protein carbonylation and glutathione S-transferase D1. Importantly, TDP43 aggregation and oxidative stress appear to mutually reinforce each other. In primary cortical neurons, glutathione depletion with ethacrynic acid elevated TDP43 insolubility and promoted its fragmentation. In COS-7 cells TDP43 modification with 4-hydroxynonenal increased TDP43 insolubility and cytosolic localization. Exposure of TDP43-expressing cells to promoters of cysteine oxidation and disulfide bond formation led to TDP43 aggregation. For example, cysteine oxidation of the RRM1 domain enhanced TDP43 aggregation and inhibited its nucleic-acid binding. By contrast, treating TDP43 mutant-expressing cells with reduced glutathione (GSH) diminished aggregate formation, ROS generation and cell death.

How TDP43 damages mitochondria is an active area of research. Mutant TDP43 expression disrupted ER-mitochondrial connectivity by perturbing ER protein VAPB (vesicle associated membrane protein) interaction with mitochondrial protein PTPIP51 (tyrosine phosphatase interacting protein). Disrupted VAPB-PTPIP51 interaction reduced mitochondrial calcium uptake and stimulated autophagy. TDP43 overexpression promoted mitochondrial fragmentation and a concomitant increase in mitochondrial fission factors Drp1 (Dynamin related protein 1) and Fis1 (Fission 1). ALS patient-derived fibroblast cells carrying TDP43 mutations exhibited increased mitochondrial recruitment of Drp1 and enhanced mitochondrial fragmentation. And, a selective Fis1/Drp1 peptide inhibitor called P110 greatly reduced mitochondrial dysfunction, thereby directly implicating Drp1 in mitochondrial toxicity. SOD1 (superoxide dismutase 1), also implicated in ALS pathology, was transported to mitochondria via the TOM (translocase of the outer membrane) complex and when mutated accumulated in the mitochondrial matrix and intermembrane space where it elicited toxicity. Misfolded SOD1 also aggregated on the outer mitochondrial membrane and was involved in mitochondria dependent apoptosis. Exogenous mutant SOD1 aggregates elicited increased TDP43 cytoplasmic mislocalization, aggregation, C terminal fragmentation and phosphorylation. Mutations in CHCHD10 (coiled helix domain containing 10) are linked to ALS. CHCHD10 was important for mitochondrial cristae morphology and bound TDP43 in the mitochondrial intermembrane space. CHCHD10 loss-of-function mutations were associated with MICOS (mitochondrial contact site and cristae organizing system) disassembly and negatively impacted respiratory chain complex assembly. TDP43 overexpression relocated CHCHD10 from mitochondria to the nucleus and CHCHD10 loss-of-function mutations induced cytoplasmic TDP43 accumulation. As TDP43 bound and stabilized mitochondrial transcript intermediates, including those encoding components of the electron transport chain, and as a considerable amount of TDP43 was transported into the mitochondria even under normal conditions, more studies are required to unearth molecular mechanisms of TDP43 function and toxicity in relation to mitochondria.

(Adapted from: Prasad, A., Bharathi, V., Sivalingam, V., Girdhar, A., Patel, B. (2019). Molecular Mechanisms of TDP43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis Frontiers in Molecular Neuroscience 12(), 25; Hasegawa M, et. al. (2008) Phosphorylated TDP43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis Ann Neurol. Jul;64(1):60-70); Prasad, A., Sivalingam, V., Bharathi, V., Girdhar, A., Patel, B. (2018). The amyloidogenicity of a C-terminal region of TDP-43 implicated in Amyotrophic Lateral Sclerosis can be affected by anions, acetylation and homodimerization Biochimie 150(), 76-87); and Mackenzie, I., Neumann, M. (2016). Molecular neuropathology of frontotemporal dementia: insights into disease mechanisms from postmortem studies Journal of Neurochemistry 138(S1), 54-70)