It was a shot in the dark — or at best, a dimly lit room: injecting a mouse with a little bit of spinal cord fluid from someone with the most common form of amyotrophic lateral sclerosis, or ALS.
Then, within a day, research scientist Jamie Wong observed her first rodent subject was acting differently. It was weak.
Maybe the mouse is weak because Wong hit one of its nerves during the injection, researchers at the Tisch MS Research Center of New York thought. An injection with saline solution disproved that theory — that mouse was fine. So if it wasn’t a nerve injury, could it be that a mouse injected with spinal fluid develops the same kind of neurological degeneration that is a hallmark of ALS?
“We found that actually the motor neurons were dying, so very much like ALS,” said Saud Sadiq, director and chief research scientist at the Tisch MSRCNY. “And then you get excited and say, OK, now let’s really do it a number of times.”
Over the early months of the Covid pandemic, when New York City was frozen to a halt, Tisch researchers faithfully reported to their lab to see what happened when sporadic ALS, a non-genetic form of this debilitating disease, manifested in mice. Their findings, published Monday in the journal Brain Communications, contain several promising breakthroughs in a disease that has historically been difficult to study, let alone cure.
ALS is an aggressive, chronic disease that carries a three-to-five-year life survival rate after diagnosis. Some people, as Stephen Hawking did, may live longer, but many don’t. Each patient’s ALS is different, clinicians say. But a consistent part of the disease is a brain-body disconnect that arises as message-carrying motor neurons in the brain and spinal cord die. The brain can’t communicate signals down to the muscles, making it difficult to move, speak, eat, and perform other basic tasks.
In 1993, a critical paper suggested a gene called SOD1 was a cause of the disease, giving scientists an entry point for research. But certainty has been elusive since then. It is estimated only 10% of ALS cases are caused by inherited genetic mutations — what’s called familial ALS; the other 90%, those sporadic cases, have an unknown cause.
Scientists surmise it might be multiple overlapping biological factors that are set off by an environmental trigger, but they really don’t know. At least 70 trials of potential ALS drugs have failed, leaving patients with just a couple of FDA-approved treatments. The last to be approved, in 2017, was edaravone, and it is the only new drug to be approved for ALS in almost three decades.
Part of the difficulty is that, before Sadiq and his team made one, no animal model existed that translated sporadic ALS, complete with motor neuron death, into adult mice. Unlike a disease of the liver or kidney, neurologists can’t take a piece of relevant tissue from a person with ALS, so there is no way to study the illness in-depth while a patient is alive. Some previous studies had analyzed the next best thing — cerebrospinal fluid — from patients with ALS and suggested there were toxic elements causing damage to the neurons. However, nobody had rigorously compared the spinal fluid of patients with familial ALS to that of people with sporadic ALS (sALS).
Sadiq, Wong, and the other researchers used spinal fluid from 11 patients with sALS, and seven with familial ALS. They also injected control groups of mice with the cerebrospinal fluid from five patients with multiple sclerosis (who Sadiq typically treats) and four healthy volunteers to make sure the loss of motor neurons wasn’t linked with just any disease.
Once the mice were injected with a sample in their cervical spinal cords, they were held by their tails above their cages and given five tries to reach out and grab the bars. If their reach was inaccurate, their grip weak, or their forepaws were clenched, that was documented. A flaccid tail also counted against them. Researchers never knew which group of mice they were testing, to ensure there was no bias in scoring.
Once they felt sure that the sALS fluid was driving motor problems and disability in their mice, they wanted to know why.
By filtering the spinal fluid numerous times, each time stripping away an element and then studying the mice’s motor skills and strength, they could cross out names from the list of thousands of potentially poisonous proteins found in human cerebrospinal fluid. Wong eventually narrowed it down to one culprit: apolipoprotein B-100.
“It’s usually not found in healthy people, in the cerebrospinal fluid,” she said. “We found it was significantly elevated in sporadic ALS cerebrospinal fluid, and not in healthy people, and also not in the familial ALS samples that we looked at.”
ApoB-100, that neurotoxic protein, is usually a concern for other specialists — cardiologists. It is a building block of low-density lipoprotein, LDL, which transports cholesterol in the blood. By administering an ApoB-100 test, doctors can usually tell how much “bad” cholesterol is in a person’s blood, and get ahead of fatty plaques that might build up in blood vessels.
The fact that the familial ALS spinal fluid did not have ApoB-100 told the researchers two things: the genetic subtypes of ALS are truly different from the sporadic form of the disease, and ApoB was a possible target for stopping sALS.
When they sacrificed the mice to examine their brains and spinal cord cells (work that is impossible to do with live animals), Wong and her colleagues found motor neuron death was consistent even 28 days after the animals were injected with the sALS spinal fluid full of ApoB. And when, with live mice, they filtered ApoB out, the mice didn’t experience the ALS symptoms.
“There’s mounting evidence in ALS that the lipid pathways and LDL pathways are important, and this is more evidence pointing toward that,” said Jason Thonhoff, an ALS researcher in the Neuromuscular Clinic at Houston Methodist Hospital’s Stanley H. Appel Department of Neurology, who was not involved in the research.
The initial study is not definitive evidence that ApoB is the source of sporadic ALS. Nor do the findings explain how ApoB arrives in the cerebrospinal fluid to begin with, or how the protein kills motor neurons. A single dose of sALS spinal fluid in the mice also does not exactly mirror the progressive, degenerative nature of the disease in humans — that is a weakness of the model, Sadiq conceded. Future study will need to explore such questions, he said.
But the paper does provide initial proof that, if ApoB is filtered out of cerebrospinal fluid, symptoms of sporadic ALS might improve, Wong said. Exactly how that would occur, or how often patients would need to get the filtration procedure, is far from clear. Researchers might also be able to look upstream and downstream to figure out how ApoB causes neuronal death, and could then develop therapies to stop the progression of sporadic ALS altogether.
Such a breakthrough could redeem early trials of regenerative stem cell therapies for ALS, which have largely failed to revive patients’ motor function. “Part of the reason may be, well, you were in a toxic environment,” Sadiq said. “You can’t plant trees in a forest fire. So once you put out the forest fire, planting new trees may regenerate the forest.”
It’s also possible that ApoB, like the SOD1 gene discovered in 1993, is just one of many ways this very complicated disease comes about.
This article was supported by a grant from Bloomberg Philanthropies.