Research ranging in scale from cells to populations is rapidly closing in on what goes awry in the body in 'non-familial' ALS, and what environmental factors might contribute.
The first thing Ed Tessaro noticed was a weakness in his left ankle when he was running one day. It soon spread to his lower right leg. After a misdiagnosis of spinal stenosis, he was finally referred to a neurologist who diagnosed amyotrophic lateral sclerosis (ALS) in 2009, when Tessaro was 62. Retired from a US retail company, Tessaro lives in Atlanta, Georgia, with his wife. He must use a wheelchair and other mobility aids, but that hasn't prevented him from travelling on seven continents. “There's always a way to do those things that I love to do,” Tessaro says.
ALS causes progressive degeneration of motor neurons in the brain and spinal cord, but beyond that common element, patients' experience of the disease varies widely. Whereas most people struck by ALS are around Tessaro's age (incidence rises with age and peaks in the 60 to 69 age group), there are some cases in older age groups and in younger people (British physicist Stephen Hawking was diagnosed in his 20s). The disease usually starts in the arms or legs, as in Tessaro's case, but in one-third of ALS cases, the first signs are problems in chewing, speaking or swallowing. In some, the disease may lead to a type of neural degeneration called frontotemporal dementia (FTD).
People usually die within three to five years of disease onset because the muscles controlling their breathing fail. Remarkably, Tessaro has lived with the disease for nine years. “I'm a bit of a question mark, an oddball,” he says. In the early-onset type of ALS affecting Hawking, however, survival is sometimes much longer.
As in most cases of ALS, the disease did not seem to run in Tessaro's family. None of his relatives had ALS or FTD. In fact, only about 10% of ALS sufferers are estimated to have a family history of the disease1. That figure is probably an underestimate, according to Ammar Al-Chalabi, director of the King's MND Care and Research Centre at King's College London. He points out that, in previous generations, ALS was often misdiagnosed or unrecorded, and FTD was not recognized as related to ALS. Even so, the vast majority of ALS cases have no known cause.
ALS is hard to put your finger on. There are more unknowns than knowns.
ALS therefore remains not only one of the most deadly diseases, but also one of the most mysterious. “ALS is hard to put your finger on. There are more unknowns than knowns,” says Paul Mehta, principal investigator at the National ALS Registry in Atlanta, Georgia, a database of US cases, which also includes blood and tissue samples.
That state of ignorance is about to change. “Our understanding of ALS is rocketing. Everything we used to believe about ALS we don't believe any more, or it has radically changed,” says Al-Chalabi. This change is shining a bright light on the disease's causes and effects, he says.
Researchers are learning more about what happens within the motor neurons affected by ALS, including the presence of misfolded proteins similar to those seen in diseases such as Creutzfeldt–Jakob disease and bovine spongiform encephalopathy. In 1982, Stanley Prusiner confirmed that such misfolded proteins could act as infectious agents called prions that spread disease within the body by leading other proteins to misfold in a chain reaction.
While some researchers are learning more about the cell biology of ALS, others are identifying an increasing number of genes associated with the disease, in non-familial as well as familial ALS. There are also exciting new ways to study disease mechanisms in vitro using induced pluripotent stem cells (iPSCs) and induced neuronal (iN) cells. And techniques for analysing 'big data' are being applied to large national and international registries of people with ALS, which bring together cases of this rare disease to create a critical mass for epidemiological research into possible environmental triggers. “It's only in the last year or two that we've started to get a handle on the environmental risk factors, because we're only now getting the environmental data that makes sense and helps us to understand these things,” says Al-Chalabi. When insights from genetics, cell biology and epidemiology can be brought together, this disease will give up its secrets, researchers say.
Many causes, many effects
ALS is turning out to be much more complex than previously thought. Not only does it have a wide range of effects on the body — from limb problems to difficulty in speaking and swallowing to FTD — but it seems to have many causes. Researchers are finding a growing number of genetic mutations and environmental factors that have some role in its onset. Even findings in neurons are not consistent, although clumped proteins in motor neuron cells are present in almost all cases. “You've got a kind of web, in one direction of multiple causes and in the other of multiple clinical features,” says Al-Chalabi. Finding a single strand that will knit this web together — possibly the abnormal proteins — is the ultimate goal of ALS research, because it will open avenues to preventing and treating the disease.
Since 1990, researchers have linked ALS to 120 mutations in 25 genes2. “There's a gene discovery every four years,” says Al-Chalabi. Many of the genetic mutations have been found in both familial and non-familial ALS, raising the possibility that the damaged DNA may in some cases be the result of ageing or exposure to toxins, rather than being inherited. Some genetic mutations are clearly associated with how old the patient is at disease onset, which nerves or muscles the disease first affects, or how long it takes the disease to progress2. But specific genes do not always map directly to particular clinical outcomes. Even people in the same family with ALS can have different natural histories of disease, says Al-Chalabi.
In cellular studies, researchers are puzzling over the many and complex pathways affected within ALS motor neurons1,2. One of the chief discoveries in the past 15 years is abnormal clumps of protein in the motor neurons of patients. Two of the proteins involved — TDP-43 and FUS — are normally involved in many functions within the cell. But the clumping found in ALS causes problems. The proteins are now located in the wrong place in the cell: in the cytoplasm rather than in the nucleus, where they normally function. They are also often attached to a small protein called ubiquitin, which typically marks proteins for degradation. And the clumped proteins are misfolded, like prions. The misfolded proteins also propagate themselves and spread from cell to cell like prions, suggesting one way in which the disease could slowly engulf motor neurons. The abnormal protein clumping is toxic to the neurons, resulting in inflammation and leading them to cease functioning and to degenerate.
Other causes of motor neuron degeneration in ALS have been found. One involves mutations in the C9ORF72 gene (see page S106) that, among other things, can result in the production of abnormal C9ORF72 proteins that are toxic to neurons, leading to neuronal self-destruction (autophagy). Another is an abnormality in the axons of motor neurons — the cellular extensions through which nerve signals are transmitted to muscles. Axonal structural integrity is maintained by an internal protein cytoskeleton, made up mainly of microtubules. In some types of ALS, the microtubules are impaired or behave abnormally — apparently as a result of mutations in genes that code for cytoskeletal proteins.
To prove a link between a genetic mutation and motor neuron defects, researchers can sometimes replicate the mutation in animal models. But in many cases of non-familial ALS, no mutations are known. Models that use iPSCs derived from the patient's cells to reproduce the malfunctions might get over that hurdle, says neuroscientist Ludo Van Den Bosch at the VIB-KU Leuven Center for Brain and Disease Research in Belgium. iPSCs are created by converting adult cells into stem cells in culture, from which new neurons can be derived. iPSCs are already used to study neuronal defects in people with identified mutations, and the next step would be to use them to investigate patients with no known mutations. This is currently the only way to create a model for ALS not associated with a genetic mutation, says Van Den Bosch. “How do you model a disease that there is no known cause for?”
Also on the horizon are cultured cells called iN cells, neuronal cells that are generated directly from patients' cells without going through the stem-cell stage. Van Den Bosch hopes that research based on iPSCs and iN cells will answer outstanding questions, including whether the many biological pathways identified “are cause or consequence of the disease”.
Strength in numbers
Answers may also come from finding commonalities among patients. Clinicians have long noted similarities between people with ALS, but epidemiological research to tease out true linkages has been bedevilled by the small number of cases. If there are too few patients, statistical analyses cannot determine whether associations with genetic or environmental factors are due to chance alone. Studies with small numbers of patients typically compare each ALS case with a healthy control. But there are pitfalls with this design, including patients' poor or biased memories of potential causative factors. “For that reason, whenever possible we prefer to have prospective studies — large cohorts that are followed over time,” says Marc Weisskopf, who studies environmental and occupational epidemiology at the Harvard School of Public Health in Boston, Massachusetts.
The Scandinavian countries, for example, have registries containing full health data for all their citizens. “This is the only part of the world where we are able to do this type of study” looking at an entire population, says Johnni Hansen at the Danish Cancer Society Research Center in Copenhagen, who works on ALS as well as cancer. Although Denmark has only around 6 million people, the country has collected data on its entire population since 1977. “Because we can collect all cases in the country over many years, we can have a relatively large study,” says Hansen. Denmark also has a database of all citizens' jobs since 1964, enabling correlations between health and occupational data.
Studies based on the Danish data have associated a higher risk of ALS with a variety of factors, including extremely-low-frequency electromagnetic fields, cardiovascular disease, physical trauma, exposure to formaldehyde and military service. The link to formaldehyde has been confirmed in several studies, after it was first uncovered in a US study intended to look at a possible link between pesticide and herbicide exposure and ALS.
Other countries have set up registries specifically for people with ALS. European countries have pooled their ALS data as part of the EURALS consortium, launched in 2004, which includes population-based ALS registries in Italy, Scotland, Ireland, Serbia and some areas of England and France; the Netherlands is planning to join3.
In the United States, the National ALS Registry, set up in 2010, “does more than count cases”, says Mehta. As part of the Agency for Toxic Substances and Disease Registry, it funds research and helps researchers recruit patients for association studies and clinical trials. For example, the registry is funding research into harmful 'algal blooms' caused by cyanobacteria, after a study had shown that exposure to cyanobacteria and their neurotoxins increased the risk of ALS. Since 2016, the registry has also collected blood and tissue samples from people with ALS, both living and deceased.
One of the most intriguing risk factors to emerge from the population-based registries is military service. The higher incidence of ALS among service personnel has been shown in study after study, and the incidence is higher even among those who have not been in combat zones. Tessaro served in the US Army's air defence during the Vietnam War, but was not in combat. Studies of US veterans from the 1991 Gulf War, for example, have shown rates of ALS of up to double that in the general population. What is it about military life that underlies the association? “The bottom line is that we don't know,” says Weisskopf. “We need much more data about what people are doing in the military.” That research needs to look at risk factors identified in other studies, such as extreme physical exertion, physical trauma and lead exposure, he says.
Physical exertion and trauma might also be implicated in the high rate of ALS among professional American-football players — four times as high as in the general population. But this association may be illusory. Some researchers think that a recently recognized condition caused by repeated head trauma — chronic traumatic encephalopathy (CTE) — has in the past been mistaken for ALS. Weisskopf, who is also an adviser to a study on football players, says that the relationship is not clear: CTE may have been misdiagnosed as ALS; CTE may lead to ALS; or trauma could cause both conditions independently. Al-Chalabi thinks that misfolding proteins could be the link, because traumatic brain injury of the kind seen in American footballers leads to neurological changes that include misfolded proteins.
Step by step
The next frontier in studying the causes of ALS will be research into how the factors revealed by epidemiology, genetic mutations and cell-biology pathways might work together.
“We need to take some of these things that we're finding in the epidemiology back to the lab and look at them in animal models,” says Weisskopf. “Are they doing things to the cells? Do they match up with some of these things the basic biology is finding?” Working in the other direction, from patients' tissues, iPSCs and iNs should be able to recreate the disease processes in cells.
Protein misfolding may be the thread that pulls together the web of causes and effects in non-familial ALS. It could result not only from genetic mutations, but also from environmental factors that affect cellular processes that maintain protein integrity. “If any of those processes are not working, they might result in protein misfolding,” says Al-Chalabi. Referring to one environmental contaminant implicated in ALS, Weisskopf says, “formaldehyde has the potential to do strange things to proteins”.
Key to this research are today's patients with ALS, whose data, and increasingly their blood and tissue samples, are held in the registries. Although the research will come too late to help them, they are making these discoveries possible and should not be forgotten, emphasizes Mehta. Tessaro took part in a research study of neural stem-cell transplantation and has enrolled in the US registry. He and his wife of 48 years, Judy, have helped the Muscular Dystrophy Association to raise millions of dollars for ALS research. “Just being part of something that's bigger than yourself — that's what it's all about,” he says.
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Brown, C. Non-Familial ALS: A tangled web. Nature 550, S109–S111 (2017). https://doi.org/10.1038/550S109a
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