ALS Alert Newsletter | December

ALS CLASSROOM: Axonopathy

Why Axons Go Downhill

New work highlights axon sickness while it suggests what's behind it – even, maybe, in ALS.

Healthy axons (green) reach out, then branch (red-green) to close in on their  muscle targets.

It’s like discovering your right arm is slightly foreign, that it isn’t made of quite the same stuff as the rest of your body and, worse, that it’s more prone to injury — this concept of axonopathy that Packard investigator Jonathan Glass champions.

For the better part of a decade, Glass and his colleague Lindsey Fischer have explored the idea that ALS is a disease whose major insult is directed at the motor nerve cell’s axon processes that extend out to muscle. The thought is that the downhill path begins in axons, which are, for some reason, biologically more vulnerable than the rest of the motor neuron.

The suggestion that neurodegenerative diseases, in general, target axons has been around a while. Glass and Fischer, however, were quick to apply that idea to ALS. In 2004, their analysis of ALS model mice at various stages of the disease fairly shouted axonopathy. Since then, that work has drawn others into the field; today there’s a new push to understand what goes on in far reaches of the motor neuron.

Now a new study from Glass’s lab — it came out last month — appears to strengthen the case for ALS being an axon disease while it offers a reason why. Interestingly, this new work barely mentions ALS, though that likely hovers in researchers’ minds. At this point, Glass says, the ALS connection requires a small-but-rational leap of faith.

Learn more about the study:

Gathering Evidence
What's at Work?
Lead-up to the Study
The New Study – At Last
What About ALS?
More Than Blueberries

At four months of age, axons in mice without the SOD1 antioxidant pull back from muscles and fade away.

Gathering Evidence

The idea that motor axons degenerate in ALS isn’t in question. That is well-documented. So is the fact that axons pull away from their target muscles early on in ALS — an act that makes muscles useless. The Great Unknown, however, is the source of those unfortunate effects and how they occur. Until recently, everyone assumed that dying axons result from a dying cell body. But Packard investigator Ron Oppenheim’s elegant work with ALS animal models a few years back showed that neuron cell bodies can be completely protected from death pathways and still, animals and their axons decline. Others’ studies seconded that finding. Even stem cell injections into the spinal cord that can protect motor nerve cell bodies — Clive Svendsen’s Packard-supported work — fail to save axons in ALS model mice or the mice themselves.

More support for axonopathy: Axons degenerate early in ALS, before cell bodies do — a phenomenon seen not only in ALS but in a variety of motor neuron diseases. “Even at animals’ end stages, when paralysis is severe,” Glass says, “actual motor neuron loss can be surprisingly mild. This suggests to us that what happens is more directly related to deteriorating motor axons.”

As for human evidence that ALS is an axonopathy, that’s obviously hard to come by, though autopsies of a few patients who’ve died from non-ALS reasons support the idea.

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What's at Work?

As Glass and Fischer shifted their focus to the why of axonal degeneration, a search of the scientific literature pointed to one of the earliest suspected causes of ALS: oxidative stress. “The more I look into this,” says Glass, “the more I believe in its importance.”

Oxidative stress results from the release of toxic, reactive free radicals. The molecular fragments come as a normal byproduct of metabolism in the body. Mitochondria, for example, generate streams of free radicals as a consequence of making a cell’s energy. Fortunately, before they’re released into tissue like so many shards of glass, most free radicals are made harmless by antioxidant enzymes.

One of the most potent of these is superoxide dismutase 1 (SOD1), an enzyme common in cell cytoplasm. But SOD1 also exists within mitochondria, as Packard investigator Val Culotta, and others have argued. Culotta showed how SOD1 can slip into the small organelles from cytoplasmic supplies. Why is it there? Intuitively, it makes sense that an area where the body releases most of its free radicals would also contain ways to limit their harm. Showing that has been difficult. But most scientists now believe damage control is a main reason that mitochondria import SOD1.

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Lead-up to the Study

One thing Glass and Fischer’s newest study does is build the case that oxidative stress is a real player in axon degeneration. In doing so, their research weaves together more than a decade of Packard and others’ discoveries involving mitochondria, SOD1 and the fine points of neurodegenerative disease.

For example, Giovanni Manfredi—another of the study’s authors—showed earlier that mitochondria are in shorter supply in animal models of ALS, where their axons interact with muscles. Zuoshang Xu also documented the dramatic early destruction of mitochondria in ALS. More recently, several Packard scientists have begun mapping the cascade of reactions whereby healthy SOD1 gets into mitochondria — all potential sites where disease could trip up the process and keep the antioxidant out.

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The New Study – At Last

So here’s what Glass’s team did: First, they investigated an existing mouse model of oxidative stress to learn more fully what was happening. The models stand out for their lack of SOD1 anywhere, either in motor cell cytoplasm or in mitochondria. Mice without cytoplasmic SOD1 don’t do well, it’s known, so it wasn’t such a surprise to find a progressive degeneration of motor neurons in the mice lacking SOD1 in either place.

The animals’ symptoms began with weakened hind limbs. At four months, the tibialis leg muscle had lost a third of its connections with axons. By 18 months, that climbed to two thirds. Some 40 percent of mitochondria had gone. As well, separate cultures of mouse axons confirmed readings of high oxidative stress.

“This tells us that motor axons and their mitochondria need SOD1 to survive, that they’re sensitive to oxidative stress injury,” Glass explains.

The second part of the research is more intriguing. It came about from Glass’s team asking: What if we just fix the oxidative stress in the mitochondria? Would that be useful? “Lo and behold,” he says, “it was.”

Through unusually sophisticated transgenic techniques, Manfredi introduced human SOD1 into the mitochondria of the enzyme-bereft mice — and only into mitochondria — where it stayed anchored. The tactic apparently rescued both mitochondria and motor axons. The result, Glass says, “felt just short of unbelievable.”

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What About ALS?

If replacing a missing antioxidant (SOD1) in mitochondria can rescue motor neurons from axon death, it’s hard not to conclude that mitochondria and oxidative stress are very much involved in that downhill process. That idea, the authors say, could apply to any disease where axons degenerate, whether it’s the peripheral neuropathy that diabetics get or Alzheimer’s, in which brain axons fade away.

But what about ALS? “We didn’t write an ALS paper,” says Glass who’s been with Packard from the beginning, who heads Emory University’s ALS center and who sees ALS patients daily, “but one that only speaks to the underlying reason why axons die.”

And there’s good reason for that. The present study investigates a lack of SOD1. ALS, however — at least the main familial sort studied more than a decade — is a disease of mutant SOD1. Some 100 mutations are known to exist in the human SOD1 gene, many creating a warped protein that somehow trips the disease. “Some people, then, would argue that for that reason, our study is irrelevant to ALS.”

But Glass holds it probably isn’t. “We don’t know exactly how mutant SOD1 behaves in ALS, but is it a coincidence that both a loss of SOD1 and an abundance of mutant SOD1 — we’re talking about the same molecule here — produce a similar deterioration of axons and disease appearance in animals? I suspect not. For there to be a completely different mechanism would be surprising.”

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More Than Blueberries

Therapy is never far from mind. Could you design an antioxidant drug that aims specifically at mitochondria? “Actually, there already are antioxidants out there that may already be able to do that,” Glass says. “So if we could spend more time pushing this forward, yes, it’s reasonable to think we might design something useful.” Some clinicians have already tried oral, systemic antioxidants to target oxidative stress in ALS. “But they’re likely not focused or potent enough to get to the heart of the problem,” Glass adds. “It’ll take more than eating blueberries.

“My mantra, though, is that until we understand a disease’s biology, we’ll never design a good therapy. Otherwise, you’re shooting in the dark. What we really need to know isn’t so much what makes axons die as it is what keeps them going? In the axon, you have this little piece of protoplasm way away from the cell body. It performs the complex function of carrying a signal while staying ‘attached’ to a muscle. What keeps it there? What’s necessary to keep it from falling apart? That,” Glass says, “is what we need to know.”

– Marjorie Centofanti