Autopsies of nearly every patient with the lethal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), and many with frontotemporal dementia (FTD), show pathologists telltale clumps of a protein called TDP-43. Now, working with mouse and human cells, Johns Hopkins researchers report they have discovered the normal role of TDP-43 in cells and why its abnormal accumulation may cause disease.
In an article published Aug. 7 in Science, the researchers say TDP-43 is normally responsible for keeping unwanted stretches of the genetic material RNA, called cryptic exons, from being used by nerve cells to make proteins. When TDP-43 bunches up inside those cells, it malfunctions, lifting the brakes on cryptic exons and causing a cascade of events that kills brain or spinal cord cells. “TDP-43’s role in both healthy and diseased cells has long been a mystery, and we hope that solving it will open new pathways toward preventing and treating ALS and FTD,” says Philip Wong, Ph.D., a professor of pathology at the Johns Hopkins University School of Medicine and senior author of the study.
Almost a decade ago, Wong says, scientists first described the clumps of TDP-43 that commonly appear in the degenerated brain or nerve cells of those with FTD or ALS. But whether the clumps were a cause or an effect of the diseases, and exactly what they did, was unknown.
“Some people thought that the aggregates themselves were toxic,” says Jonathan Ling, a graduate student in Wong’s lab and first author of the new paper. “Another theory is that the aggregates were just preventing TDP-43 from doing what it should be doing, and that was the problem.”
To figure out which theory might be right, Ling deleted the gene for TDP-43 from both lab-grown mouse and human cells and detected abnormal processing of strands of RNA, genetic material responsible for coding and decoding DNA blueprint instructions for making proteins. Specifically, they found that cryptic exons — segments of RNA usually blocked by cells from becoming part of the final RNA used to make a protein — were in fact working as blueprints. With the cryptic exons included rather than blocked, proteins involved in key processes in the studied cells were abnormal.
When the researchers studied brain autopsies from patients with ALS and FTD, they confirmed that not only were there buildups of TDP-43, but also cryptic exons in the degenerated brain cells.
In the brains of healthy people, however, they saw no cryptic exons. This finding, the investigators say, suggests that when TDP-43 is clumped together, it no longer works, causing cells to function abnormally as though there’s no TDP-43 at all.
TDP-43 only recognizes one particular class of cryptic exon, but other proteins can block many types of exons, so Ling and Wong next tested what would happen when they added one of these blocking proteins to directly target cryptic exons in cells missing TDP-43. Indeed, adding this protein allowed cells to block cryptic exons and remain disease-free.
“What’s thought provoking is that we may soon be able to fix this in patients who have lots of accumulated TDP-43,” says Ling.
Ling cautions that questions remain about the role of TDP-43 in ALS and FTD. “We’ve explained what happens after TDP-43 is lost, but we still don’t know why it aggregates in the first place,” he says. Wong’s group is planning studies that may answer these questions, as well as additional tests on how to treat TDP-43 pathology in humans.
The ALS Association estimates that as many as 30,000 Americans are living with ALS at any given time, and more than 5,000 people are newly diagnosed each year. There is no cure for the disease, and most people live two to five years after diagnosis. Similarly, FTD — thought to affect around 50,000 people in the U.S. — has no treatment and shortens life span.
Other authors on this study are Juan C. Troncoso and Olga Pletnikova from the Johns Hopkins University School of Medicine.
This work was supported in part by the Robert Packard Center for ALS Research, the Muscular Dystrophy Association, the Amyotrophic Lateral Sclerosis Association, Target ALS, the Johns Hopkins University Neuropathology Pelda Fund, the Johns Hopkins Alzheimer’s Disease Research Center (NIH grant number P50AG05146) and the Samuel I. Newhouse Foundation.
Packard scientist Phil Wong and colleagues have published a new study in Science that outlines the normal function of the ALS-linked protein TDP-43 and spells out more clearly how loss of this function contributes to disease. Unlike other RNA-binding proteins, TDP-43 does not mainly control splicing of normal exons, but rather keeps potentially damaging ‘hidden’ exons from being expressed. Returning normal TDP-43 functioning to cells may be a good drug target for ALS, Wong says.
Although the links between TDP-43 and ALS were discovered almost ten years ago, researchers still had little idea about precisely what the protein normally did in the cell. Understanding this role is key, since nearly all ALS patients and up to half of those with frontotemporal dementia (FTD) have aggregations of TDP-43 in the cytoplasm. This means that TDP-43 isn’t in the nucleus to do its normal job. The problem was that no one could be sure of what this normal job actually was, or how the loss of this function contributed to ALS and FTD.
Previous work showed that TDP-43 bound to RNA and helped to regulate alternative splicing, a process that allows a single gene to code for multiple proteins. But most TDP-43 bound to intronic regions far away from where they would be needed to regulate alternative splicing. To figure out what TDP-43 did, Jonathan Ling, a graduate student of Wong’s, removed all TDP-43 from mouse or human cells and measured how gene expression changed.
The researchers discovered that when mouse cells lacked TDP-43, there was the appearance of cryptic exons. These exons are normally buried within introns and are typically spliced out during normal mRNA processing. Further experiments revealed that TDP-43 binds directly to these cryptic exons, which enables them to be spliced out. When this didn’t happen, the mRNAs oftentimes were degraded, which left the cells without important proteins and contributed to premature cell death. Replacing this function of TDP-43 with a different repressor that spliced out cryptic exons restored cell survival.
Using cultured human cells, Ling identified another set of cryptic exons regulated by TDP-43, showing that this function wasn’t limited to mice. Importantly, Wong and colleagues found cryptic exons in post-mortem brains of ALS and FTD patients but not in controls, showing that loss of this normal TDP-43 function could contribute to disease in ALS.
These results provide not just a greater knowledge of what TDP-43 normally does, but also yield new biomarkers of disease and potential drug targets.
Money raised from the Ice Bucket Challenge that went viral last year has significantly boosted research into ALS, scientists at Johns Hopkins say.
They say it has helped them to understand more about a dysfunctional protein - TDP-43 - a mystery scientists have been studying for decades.
ALS is a rare condition affecting the nervous system.
Social media was awash with videos of people pouring cold water over their heads to raise money for ALS last year.
More than 17 million people uploaded videos to Facebook, including many celebrities who rose to the challenge, which were then watched by 440 million people worldwide.
A new study published by Johns Hopkins researchers in Science journal last week credits the Ice Bucket Challenge with helping them to unravel the mystery behind a protein called TDP-43, which in more than 90% of ALS cases is dysfunctional.
"For the past decade we've been trying to figure out exactly what it is doing, and now I think we have finally figured it out," Jonathan Ling, of Johns Hopkins Medicine, said in a YouTube video explaining the university's latest breakthrough.
Ice bucket challenge: What's happened since?
"The best part is it can be fixed, so with any luck this could lead to the possibility of a cure or at least a slowing down of this terrible disease," he continues.
"The money came at a critical time when we needed it," professor Philip Wong added.
However, they warned that the work was ongoing and many current ALS sufferers would not necessarily see the benefits of the research.
In the US, the ALS Association - which represents people with amyotrophic lateral sclerosis (also known as Lou Gehrig's disease) and all motor neurone disorders - received $115m in donations during the months of August and September, when the challenge was at its peak.
The American charity says the money helped triple the amount it spends on research every year.
Up to 15,000 people in the US are reported to be suffering from ALS.
What is motor neurone disease (MND)?
- fatal, rapidly progressive disease that affects the brain and spinal cord
- attacks nerves that control movement so muscles refuse to work (sensory nerves are not usually affected)
- can leave people locked in a failing body, unable to move, talk and eventually, breathe
- affects people from all communities
- kills around a third of people within a year of diagnosis and more than half within two years
- there is no cure
Running for Jim has received 4 award nominations at St. Tropez Film Festival to take place May 13-18, 2014.
Best Documentary Producing
Best Documentary Editing
Best Documentary Director
Best Feature Documentary