Amyotrophic Lateral Sclerosis (ALS) TDP-43 Proteinopathy Research Model - B6-hTARDBP Mice

This issue introduces the TDP-43 proteinopathy research model - B6-hTARDBP mice (Product Number: C001418). In amyotrophic lateral sclerosis (ALS), pathological abnormalities of TDP-43 protein are almost universally present, regardless of whether the TARDBP gene is mutated. This indicates that TDP-43 plays a key role in the progression of ALS, serving as a widespread pathological marker and a promising therapeutic target. The B6-hTARDBP mouse model expresses humanized TARDBP gene and protein, making it an ideal platform for studying TDP-43 protein-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS), commonly known as "Lou Gehrig's disease," is a fatal neurodegenerative disorder characterized by the gradual degeneration and death of upper and lower motor neurons (MNs) within the central nervous system that control muscle movement and breathing, which leads to progressive muscle weakness and atrophy. Most ALS patients die within three to five years of the onset of symptoms due to the inability to breathe independently.^[1-2] About two-thirds of familial ALS cases and one-tenth of sporadic ALS cases are associated with genetic mutations, including genes such as SOD1, TARDBP, ALS2, C9ORF72, and FUS.^[3]

Figure 1. Classification of ALS subtypes and stages of development for FTD based on different types of functional impairments in upper and lower motor neurons (UMN & LMN) ^[3].

The Relationship Between TDP-43 Protein Aggregation and ALS

Transactive response (TAR) DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, is a nuclear protein involved in the processing, transport, and metabolism of RNA, as well as the regulation of protein entry into the nucleus, circadian rhythms, and protein stability. TDP-43 is typically localized to the nucleus and can shuttle between the nucleus and cytoplasm. In some ALS and FTD patients, mutations in the TARDBP gene cause TDP-43 protein to relocate from the nucleus to the cytoplasm and form inclusion bodies. These cytoplasmic inclusion bodies interfere with the normal functions of TDP-43, produce toxicity, and hinder cellular processes - ultimately leading to cell death.^[4-5] Importantly, pathological abnormalities of TDP-43 protein can be observed in nearly all ALS cases, regardless of the presence of TARDBP mutations. In fact, TDP-43 protein is a major component of protein aggregates in post-mortem tissues of ALS patients, indicating that TDP-43 plays a key role in various forms of ALS - serving as a widespread pathological marker and a promising therapeutic target.^[5]

Figure 2. Potential mechanisms by which TDP-43 protein dysregulation leads to motor neuron (MN) functional impairment and neuromuscular junction (NMJ) disruption.^[5]

TDP-43 Targeted Therapy

Similar to the current status of treatment for other neurodegenerative diseases, drug development for ALS faces significant challenges. Currently, one focus of research is on the TARDBP gene or TDP-43 protein, but no therapy targeting TDP-43 has yet to successfully slow or reverse the disease progression. Strategies for TDP-43 targeted therapy primarily involve two approaches: one is to inhibit the formation of TDP-43 aggregates or clear existing aggregates, and the other is to induce the degradation of pathological TDP-43. Additionally, there are related strategies such as stress granule (SG) dynamics regulation, autophagy activation, regulation of TDP-43 nucleocytoplasmic shuttling, repair of RNA metabolism, and correction of abnormal splicing events.^[6-8] Emerging research directions include using small nucleic acid drugs to correct pathogenic mutations in the TARDBP gene, delivering correct copies of the TARDBP gene via AAV vectors, and employing gene therapy techniques like CRISPR.^[9-11]

Figure 3. TDP-43 targeted ALS therapies that have entered the clinical phase.^[6]

Before clinical trials can be initiated, each therapeutic approach must undergo rigorous preclinical animal evaluations, and different treatment methods require different types of animal models. In the field of TDP-43 proteinopathy and amyotrophic lateral sclerosis (ALS) research, Cyagen offers a variety of transgenic, overexpression, or point mutation disease models to meet the research and development evaluation needs for traditional drugs such as antibodies, small molecules, and peptides. To accommodate research needs for emerging therapies such as CRISPR, ASOs, siRNA, and miRNA, Cyagen has successfully developed the B6-hTARDBP mouse model with humanized Tardbp gene (Product Number: C001418), which can be used to construct humanized models with popular pathogenic point mutations for preclinical research. Below are the detailed specifications of this model.

B6-hTARDBP Mice Successfully Express Humanized TARDBP

RT-qPCR results show that, compared to wild-type mice, B6-hTARDBP mice successfully express the humanized TARDBP gene and do not express the mouse Tardbp gene.

Figure 4. Detection of gene expression in wild-type mice (WT) and B6-hTARDBP mice (hTARDBP).

The B6-hTARDBP mice successfully express the human TDP-43 protein in their brains

Western Blot results indicate significant expression of human TDP-43 protein in the brains of B6-hTARDBP mice. The expression level is comparable to that in Tg(TARDBP*A315T) transgenic mice, which overexpress the TARDBP*A315T mutation gene in the nervous system.

Figure 5. Detection of human TDP-43 protein (hTDP-43) expression in brain tissue.

Human TDP-43 protein is widely distributed in the spinal cord of B6-hTARDBP mice

Immunohistochemistry (IHC) results show that the spinal cord of wild-type (WT) mice does not express human TDP-43 protein, while significant expression and widespread distribution of human TDP-43 protein can be detected in the spinal cord of both B6-hTARDBP mice and Tg(TARDBP*A315T) mice.

Figure 6. Detection and distribution of human TDP-43 protein in spinal cord tissue.

Conclusions

The B6-hTARDBP mouse model (Product Number: C001418) is a mouse Tardbp gene humanized model which effectively expresses the human TARDBP gene without expressing the mouse's endogenous Tardbp gene. Significant expression of human TDP-43 protein is observed in both the brain and spinal cord, with expression levels and distribution patterns similar to those in classic transgenic models. Therefore, the B6-hTARDBP mouse model can be used for studying TDP-43 protein-related neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

Furthermore, Cyagen’s proprietary TurboKnockout and BAC recombination technologies can provide murine models of disease featuring popular point mutations. We can provide customized services tailored to specific point mutations to meet the extensive preclinical research and development needs for drug screening and pharmacology experiments related to ALS, FTD, and other neurological disease studies.

Recommended Models for Neurodegenerative & Neuromuscular Disease Research:

References:

[1] Hardiman O, Al-Chalabi A, Chio A, Corr EM, Logroscino G, Robberecht W, Shaw PJ, Simmons Z, van den Berg LH. Amyotrophic lateral sclerosis. Nat Rev Dis Primers. 2017 Oct 5;3:17071.

[2] Gregory, J.M., Fagegaltier, D., Phatnani, H. et al. Genetics of Amyotrophic Lateral Sclerosis. Curr Genet Med Rep 8, 121–131 (2020).

[3] Feldman EL, Goutman SA, Petri S, Mazzini L, Savelieff MG, Shaw PJ, Sobue G. Amyotrophic lateral sclerosis. Lancet. 2022 Oct 15;400(10360):1363-1380. 

[4] Suk TR, Rousseaux MWC. The role of TDP-43 mislocalization in amyotrophic lateral sclerosis. Mol Neurodegener. 2020 Aug 15;15(1):45.

[5] Lépine S, Castellanos-Montiel MJ, Durcan TM. TDP-43 dysregulation and neuromuscular junction disruption in amyotrophic lateral sclerosis. Transl Neurodegener. 2022 Dec 27;11(1):56.

[6] Hayes LR, Kalab P. Emerging Therapies and Novel Targets for TDP-43 Proteinopathy in ALS/FTD. Neurotherapeutics. 2022 Jul;19(4):1061-1084.

[7] Francois-Moutal L, Scott DD, Khanna M. Direct targeting of TDP-43, from small molecules to biologics: the therapeutic landscape. RSC Chem Biol. 2021 Jun 21;2(4):1158-1166.

[8] Buratti E. Targeting TDP-43 proteinopathy with drugs and drug-like small molecules. Br J Pharmacol. 2021 Mar;178(6):1298-1315.

[9] ALS News Today. (n.d.). ALS Gene Therapy SOL-257 Targeting TDP-43 Shows Promise in Mouse Model. Retrieved from https://alsnewstoday.com/news/als-gene-therapy-sol-257-targeting-tdp-43-shows-promise-mouse-model/

[10]  ALS News Today. (n.d.). Vectory Raises Millions to Advance ALS Antibody Therapy TDP-43. Retrieved from https://alsnewstoday.com/news/vectory-raises-millions-advance-als-antibody-therapy-tdp-43

[11]  ALS News Today. (n.d.). Takeda Acquires License for ALS Therapy Aimed at Toxic TDP-43. Retrieved from https://alsnewstoday.com/news/takeda-acquires-license-als-therapy-aimed-toxic-tdp-43/