“It never made sense to me that ALS spreads,” says Marc Diamond, an associate professor of neurology at Washington University in St. Louis. “Why don’t just a few cells die, and that’s it?” That’s a question that has bothered Diamond, who now has an MDA grant to study the subject.
|Marc Diamond, associate professor of neurology, Washington University, St. Louis
There are many different hypotheses as to why ALS spreads, such as inflammation and loss of trophic support (the help cells get from other cells, which often secrete beneficial proteins that their neighbors use). But those hypotheses didn’t sit well with Diamond, partly because he knew that in other diseases they didn’t hold up.
“For example, if you have a stroke, you have a massive loss of cells with all the trophic support they have to offer being gone, but you don’t kick off a progressive problem.”
In ALS, by contrast, there’s a steady, orderly progression of weakness — and often other symptoms such as spasticity — from one region of the body to another, he notes.
Mapping progression in the nervous system
John Ravits, a research associate scientist at the Benaroya Research Institute in Seattle, also has had a longtime interest in how ALS progresses.
Ravits, a neurologist and neurophysiologist who serves on MDA’s Scientific Advisory Committee, says he hesitates to use the word “spread” when talking about ALS, because spreading implies something that moves from one location to a location next to it, and he isn’t yet certain that’s the case in ALS. His research so far, however, suggests that it is.
Unlike most clinicians, Ravits has examined the nervous systems of many of his ALS patients after death and carefully compared their symptoms in life to the appearance of their spinal cords at autopsy. He says this has been difficult to do but that it’s crucial to understanding and ultimately treating ALS.
|John Ravits, research associate scientist, Benaroya Research Institute, Seattle
He’s found that ALS does appear to spread in an orderly fashion, but that the spread is obscured by the anatomy and functions of the nervous system.
“It’s an area I’ve been chipping away at for about six years,” Ravits says. “The basic observation that I was making is that, when I first see patients at the beginning of the disease, their neurological deficit is very discrete — hand atrophy [shrinkage of muscles] or shoulder weakness or something like that. Then, as you follow the patient over time, it seems to spread, from the hand up the arm and then over to the other arm. You can use the clinical exam to analyze the anatomy of the pathology.”
A few years ago, Ravits began publishing papers describing his analysis of the number of remaining motor neurons in the spinal cord and comparing those numbers to where the disease had begun in the patient. (Motor neurons are nerve cells that control muscle movement and are destroyed in ALS.)
“I was able to show statistically that there’s this sort of gradient of cell loss as a function of distance from the point of onset,” he says. In other words, the closer he got to the motor area that once sent signals to the region of the body where the disease symptoms started, the fewer neurons there were remaining in the autopsy samples, as if the disease had fanned out from a source.
But correlating clinical manifestations of the disease with underlying motor neuron loss is more complex than it might seem at first glance, Ravits explains.
This is because ALS kills both lower motor neurons in the spinal cord and upper motor neurons in the brain. In the spinal cord, the motor neurons that are next to each other generally control body regions that are next to each other. But in the brain, neurons that are next to each other aren’t necessarily controlling regions of the body that are next to each other. For instance, a diagram of the brain’s motor cortex reveals that the upper motor neurons controlling the face are next to those controlling the hands.
“It gets confusing because you’re talking about two kinds of anatomy. One is your body anatomy, and the other is the motor control anatomy in the nervous system,” Ravits says.
To make matters even more complicated, the clinical signs caused by loss of upper motor neurons in the brain and lower motor neurons in the spinal cord are completely different, but they occur simultaneously in ALS and can partially obscure each other.
Loss of lower motor neurons leads to muscle weakness and atrophy, while loss of upper motor neurons generally leads to spasticity and brisk reflexes.
“When we examine a patient, we see this coexistence of upper and lower motor neuron deficits. That makes it complex, unless you separate them out. Then you see that there’s a kind of orderly, rational anatomical process to it,” says Ravits.
“It’s my opinion that the progression of ALS is predictable by the neuronal anatomy.”
Are misfolded proteins to blame?
Observing an orderly pattern of disease manifestation is one thing, but understanding why it occurs is another. Diamond is among those who believe a process known as “protein misfolding” may be at least part of the explanation.
Many neurodegenerative diseases, including Huntington’s, Alzheimer’s and Parkinson’s as well as ALS, involve protein misfolding, says Diamond. He believes that once the misfolding process gets started, it’s possible that it sets off a chain reaction that moves from cell to cell — a pattern that would fit with the spread of a disease like ALS.
Proteins have specific, often very complex, shapes into which cells coax them to fold. Other proteins, known as “chaperones,” do the coaxing; if they fail in making a protein fold into the right shape, the misshapen protein is often destroyed by the cell. If it isn’t destroyed, it often forms aggregates (clumps), and it can take on toxic properties.
In ALS, it’s now known that at least three proteins — SOD1, TDP43 and FUS — can misfold and form aggregates in motor neurons. Mutations in the genes for the SOD1, TDP43 and FUS proteins are among the causes of inherited (familial) ALS, a form of the disease that affects some 5 to 10 percent of ALS patients.
In those cases, protein misfolding and aggregation would be expected to occur, and it does. What’s somewhat harder to understand is that these proteins can misfold and aggregate in ALS-affected motor neurons even when their genes are perfectly normal.
“In some diseases, you really do need a genetic mutation to see a protein misfold and aggregate,” Diamond says, noting that Huntington’s disease and spinal-bulbar muscular atrophy (SBMA) appear to be in this category.
But a mutation doesn’t seem to be necessary in other cases, such as frontotemporal dementia and Alzheimer’s disease, in which the tau protein misfolds, and Parkinson’s disease, in which the alpha-synuclein protein misfolds. That now seems to be the case in ALS as well, although mutations may make it more certain that misfolding will occur.
“Every protein has the potential to misfold,” Diamond says. “And some are going to be more likely to do so than others. The mutations are probably just augmenting what is already an underlying propensity to misfold.”
Prions — the hardiest (and scariest) misfolders
The most dramatic cases of protein misfolding diseases are the so-called prion diseases. The word “prion,” coined in the 1980s, means “proteinaceous infectious particle,” and refers to a protein that can replicate itself like a virus.
So far, there’s been only one prion protein identified that causes human disease. It’s known as prion protein, or PrP. When it misfolds, it can cause other PrP molecules to misfold and can propagate this misfolding from cell to cell, causing bovine spongiform encephalopathy, or “mad cow disease,” in cattle, and the neurodegenerative condition Creutzfeldt-Jakob disease (CJD), as well as a few other diseases, in humans.
What has amazed scientists about prion diseases is that they’re the only known instance of transmission of infection without the use of DNA or RNA, the usual genetic materials that allow infectious agents (such as bacteria and viruses) to replicate.
“We think of prion diseases as special because they’re infectious,” Diamond says, hastening to note that no one is suggesting that ALS, or Alzheimer’s or Huntington’s disease can be transmitted from one person to another.
Prion diseases can be infectious, but they also can arise spontaneously in a person or animal, and they can be inherited, through a mutation in the PrP protein.
“You can eat a prion protein or have it implanted through a contaminated surgical instrument, and you’ll get a prion disease. That’s a very scary aspect of these diseases. But if you forget about that for a minute and just think about prion diseases as sporadic or inherited, which the vast majority of the time they are, they’re just like all the other neurodegenerative diseases.”
Diamond began to think about the similarities and differences between prion diseases like CJD and other diseases involving protein misfolding, like Alzheimer’s and ALS.
“In prion disease, you have one cell where this misfolding problem starts and then is spread to other cells that are either next door or are connected by synapses [the junctions between nerve cells],” Diamond says. “We did some experiments to test whether this could be true for misfolded tau protein. What we saw is that misfolded tau protein that’s on the outside of a cell can be gobbled up by the cell, and that when it gets inside, it interacts with the normal tau protein that’s on the inside and converts it to the misfolded form.”
Tau, it seems, is behaving a lot like a prion protein, at least in these experiments, but the difference between tau and a prion protein may be how efficiently it can carry out its misfolding program.
“I think the reason there’s only one true ‘prion protein,’ Diamond says, “is that most proteins don’t do this very efficiently. They’re easily degraded, while prions, by some quirk of fate, are extremely resistant to this type of destruction. That makes them infectious — able to move between organisms — and it probably makes them more efficient at propagating within an organism.”
If prion-like misfolding of proteins like SOD1 or TDP43 explains propagation in ALS, the proteins probably are behaving more like tau than like PrP.
ALS propagation by misfolding — plausible, but not proven
|Robert Baloh, assistant professor of neurology, Washington University, St. Louis
Robert Baloh, an assistant professor of neurology at Washington University School of Medicine in St. Louis, has been studying protein misfolding in ALS, particularly in relation to TDP43.
“It appears that the TDP43 protein contains a region that has a similar structure to one found in the prion protein,” Baloh says, “which may explain its tendency to misfold and form protein aggregates. However, we have not shown that TDP43 can act as a prion. We’re investigating whether it can propagate the misfolded form from cell to cell, but what we’ve shown thus far does not prove that.”
Baloh, who serves on MDA’s Medical Advisory Committee and has an MDA research grant to study TDP43, says the evidence that misfolded SOD1 can spread its misfolded form from cell to cell is slightly stronger than it is for other ALS proteins, but it’s still not conclusive.
Experiments to see whether a misfolded TDP43 molecule can cause normal TDP43 to misfold within one cell and then see whether it can cause such misfolding to spread to adjacent cells have yet to be done but must be done, Baloh says.
“I think the tau literature is probably a little further along in demonstrating cell-to-cell transfer of misfolded protein,” he says. “We’re just not there yet. We’re trying, but I haven’t seen it demonstrated clearly.”
On the other hand, he says, a prion-like propagation is his best guess for understanding how ALS moves from one region of the body to another.
“I think [the hypothesis] is attractive because it could explain a lot. But we have to be careful. Just because it’s a great hypothesis doesn’t mean it’s right. I think we’re all going to have to get down to the work of figuring out whether it’s true or not and then seeing if we can manipulate it for the benefit of our patients.”
Stopping misfolded proteins could be a way to stop ALS
Stopping the spread of ALS might not be as good as preventing the disease entirely, Baloh notes, but it certainly would be beneficial to patients.
“There’s no doubt it would be great if we could stop that progression — if after we see an initial symptom in one arm or one leg, we could start a treatment that would stop it or slow it down,” he says.
Strategies such as antisense (which stops protein synthesis at the genetic level) or antibodies (which can be made to stick to proteins like magnets) might stop ALS progression if prion-like propagation is its driving force. Antisense against SOD1 for people with SOD1 mutations is already being tested. (See Trial of SOD1 blocker now open, MDA/ALS Newsmagazine, March-April 2010.)
Other possibilities for stopping misfolded proteins from spreading their damaging effects could be increasing the efficiency of a “garbage disposal” method that cells use to rid themselves of misfolded proteins; or increasing the ability of chaperone proteins to either refold misfolded proteins or designate them for disposal.
“I think it’s a very reasonable hypothesis,” Baloh says of the proposal that protein misfolding underlies disease progression in ALS and that stopping misfolded proteins might stop progression. He notes that many biotech companies are working on strategies such as enhancing chaperone activity with small molecules.
“People are trying,” he says.