Ice Bucket Donations at Work: New TDP-43 Mouse Models Shed Light on ALS Disease Pathways

Researchers funded by The ALS Association, through donations from the ALS Ice Bucket Challenge, discovered new evidence on the role that mutant TDP-43 plays in development and progression of the disease. This important work sheds light on novel aspects of TDP-43 biology and provides valuable tools to gain insight into early stages of ALS disease progression and could lead to the development of new therapies.

Mutations in TDP-43 protein account for approximately five percent of inherited ALS cases. The mis-regulation of the mutated protein is central to disease pathways of ALS, but how these mutations cause ALS is largely unknown: both the loss of TDP-43’s function and the gain of its function has been connected to disease development and progression.

Dr. Elizabeth Fisher from the MRC Centre for Neuromuscular Disease in London and colleagues set out to better understand how TDP-43 causes ALS. They describe new TDP-43 mouse models that demonstrate significant and opposite effects on splicing events, which is validated in cells derived from people with ALS.

“This exciting new mouse model expressing physiologically relevant levels of the mutant protein with an ALS-like disease phenotype is an invaluable tool for the research community to better understand disease mechanism and explore novel therapeutic approaches,” stated Dr. Lucie Bruijn, chief scientist of The ALS Association.

What is TDP-43?

TDP-43 is a protein present in protein aggregates (clumps of protein) found in the brain and spinal cord of almost all people with ALS and other neurodegenerative diseases, such as frontotemporal dementia (FTD) and Alzheimer’s disease (AD), among others. This accumulation of TDP-43 occurs in the cytoplasm of cells, thereby depleting TDP-43 from the nucleus where it normally resides.

Gain of Function vs. Loss of Function

How TDP-43 mutations cause ALS disease is largely unknown. Both the loss of its nuclear function, called loss of function, and its gain of function while residing in the cytoplasm in aggregates, called gain of function, have been identified as potential disease pathways.

What is splicing?

Simply stated, splicing is the editing of messenger RNA (mRNA), the instruction book to make protein. Before splicing, mRNA is made up of exons (the part of mRNA that is made into protein; also known as the coding region) and introns (the non-coding region). During splicing, the introns are removed or spliced out and the exons are joined together, readying the mRNA to be made into protein.

The study

Here, Dr. Fisher and colleagues developed new ALS mouse models that express endogenous TDP-43 mutations, meaning the mutations are in the existing TDP-43 mouse genome. This method allows analysis of observable characteristics (phenotypes) in a more physiological setting. This contrasts with overexpressing the large amounts of TDP-43 gene copies into mice, called TDP-43 transgenic mice. TDP-43 overexpression can produce confounding effects, which hamper mice phenotype analysis.

The mutations they studied were the following:

• RRM2 mut F210I located in the RNA recognition motif: lived to embryonic day 18.5
• LCD mut M323K mutation located in the C-terminal low complexity glycine-rich domain (where many ALS disease causing mutations reside within TDP-43): lived over two years

Note that when a researcher deletes out the full TDP-43 gene, called TDP-43 null, the mice do not live beyond embryonic day six.

The Results

• Identified a gain of function effect in a novel mouse model with the TDP-43 LCD mutation
  • Uncovered for the first time, a new splicing event, called “skiptic exons,” meaning a set of consecutive exons are skipped during splicing, which impacts the final protein product
  • Found this gain of function mutation in mice causes adult-onset neuromuscular characteristics, along with motor neuron loss and neurodegenerative changes
  • Identified splicing gain of function and skiptic exons in ALS patient-derived cells that carry TDP-43 ALS causing mutations

• Identified a loss of function effect in novel mouse model with the TDP-43 RRM2 mutation
  • Resulted in inclusion of normally excluded exons during splicing, called cryptic exons
  • Provides a novel tool to study chronic loss of function in mice that was not possible before

Why This is Important

This study identified a new disease pathway that highlights how TDP-43 gain and loss of function create their own splicing signature in an opposite manner. Importantly, this was validated in both a novel TDP-43 mouse model and in ALS patient-derived cells. This opens the possibility that the opposing gain and loss of function characteristics identified in the mice represent different stages of disease early and late stages, respectively. Together, these results represent potential therapeutic targets and furthers our understanding of ALS disease processes.

Paper citation
Pietro Fratta, et al. Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis. The EMBO Journal. Published online May 15, 2018.
DOI: 10.15252/embj.201798684
This paper is open- access.