Astrocytes’ Bad Role Is Surer
Admirable finger-pointing from a new stem-cell-related study
photo credit: Edward Nyatia and Dirk Lang
It’s the second in an unholy trinity of unanswered questions in ALS: What causes the disease? Where does it start? What keeps it going? Answering any of them would move us significantly farther down the therapeutic path.
Yet while very recent investigations have especially advanced the search, the questions still stand out as some of medical science’s major frustrations.
Now new research by Sophia Papadeas, her mentor, Packard scientist Nicholas Maragakis, and their Johns Hopkins colleagues tackles the where question, at least, with a new precision. Their work uses stem cell-derived cells to study ALS, a tactic that confers sharper focus and more control than earlier studies.
The results? New findings about what cells are important in the disease and some confirmations of the old, but with extra clout.
The basic approach — choose a central nervous system cell type, give it familial ALS genes and see how it behaves in a “neighborhood” of normal cells — has been around more than a decade. Back in 2008, Maragakis sat in Packard symposia, watching then-fellow grantee Don Cleveland and colleagues as they ticked off studies on the list of cells most suspected as the “home” of ALS: the motor neurons, surrounding glial cells and the microglia that are the immune system’s outpost in brain and spinal cord.
Cleveland worked with chimeric mice — those tailored to juxtapose normal, healthy cells with suspect cells carrying the mutant SOD1 gene for familial ALS. He found, for example, that mutant SOD1 motor neurons surrounded by healthy non-neuron cells survive a long time. But healthy motor neurons flanked by cells carrying the SOD1 mutant gene — the molecular equivalent of living on Skid Row — didn’t fare as well. The animals died early with all the signs of ALS.
The startling suggestion then was that ALS isn’t solely a disease of motor neurons. Other cells are necessary.
The Light Switch and Beyond
Cleveland’s team refined that result with an ingenious inducible system that allowed the mutant SOD1 gene to be turned on or off. One insight from this light switch approach was that along with motor neurons, astrocytes — common non-neuronal cells — are now considered major players in ALS.*
Cell studies by Packard scientists and others back that up. They show that astrocytes change early on in the disease. Animal models develop abnormal buckshot-like inclusions; they lose the ability to clear away toxic amounts of the neurotransmitter glutamate; their membranes change permeability. There’s also evidence that mutant SOD1 astrocytes release an agent that harms neurons.
That’s why, in part, Papadeas and Maragakis focused their work on the cells. “We wanted to expand on and refine the earlier studies,” says Papadeaus, “but only after we first set up a live-animal system to show how mutant SOD1-carrying astrocytes interact with neighboring wild type (normal/healthy) cells.”
“We also wanted to set up a stable platform, she adds, to study astrocyte biology over a long period of time rather than try to dissect out what’s important in a model that’s progressively declining.”
What They Did
Maragakis has spent a decade exploring how fully-mature astrocytes appear from more primitive stem cells. In glial restricted precursors (GRPs), he and co-researchers found cells that lie part-way on that path. GRPs are immature enough to keep dividing, but also have the bona fide potential to become astrocytes, given the right handling and environment. With considerable time, Maragakis’ lab team honed techniques, first, to create the GRPs and then to make certain that the astrocytes that can result from them are genuine.
In this latest study, Papadeas led the group to generate GRPs carrying the mutant SOD1 gene, and then inject them into the spinal cords of, healthy (wild type) rats. GRPs typically survive being eased into adult nervous systems. And since the researchers know exactly where the “bad” GPRs are — and can precisely track the astrocytes that result from them — that gives an advantage over earlier studies. “We can follow the course of disease spread from the site of introduction outward,” Papadeas explains.
So the scientists introduced mutant GRPs — 150,000 of them —at each of three sites in the cervical spinal cord, targeting the phrenic motor neurons that affect the diaphragm and those that innervate forelimbs. Control animals were injected with GRPs having unmutated SOD1 genes.
By three months, the GRPs formed mature astrocytes that hadn’t strayed far from the sites of the injections. As is typical, those cells interacted with the rats’ existing motor neurons.
“Several things were notable, though,” says Papadeas. Early on, the motor neurons surrounded by the mutant SOD1 astrocytes accumulated abnormal clumps of protein — inclusions — typical of ALS. And at three months, many motor neurons around the injections had died — both those next to the mutant astrocytes and those slightly farther away. “The loss was significant,” she adds; “about 50 percent at three months.” The researchers also noted astrocytosis, an abnormal outpouring of astrocytes that’s typical of progressing ALS.
And at one month, rats with the mutant astrocytes experienced a decline in the front leg/paw gripping abilities. Biochemical changes were also noted.
The short of it is that healthy motor neurons died in a specific area of spinal cord injected with mutant-containing GRPs and exposed to the astrocytes that followed. “Though the rats didn’t go on to experience full ALS and though they weren’t completely paralyzed,” Papadeas says, “we saw changes characteristic of the disease in the targeted area.”
With that work done, she and colleagues now turn to explaining how the mutant astrocytes spark the downhill changes.
– Marjorie Centofanti
*microglia are also singled out with an ALS role as are muscle cells, perhaps, and most recently, oligodendrocytes.