Tracking Post-Polio Syndrome
 by Warren Froelich

from the Salk Institute Signals / Fall 1999

In a lab once occupied by polio vaccine pioneer Jonas Salk, researchers are now tackling some unfinished business: Post-polio syndrome.

“This syndrome’s difficult to ignore, and it shouldn’t be ignored,” said Assistant Professor Samuel L. Pfaff, a developmental neurobiologist at The Salk Institute who directs the lab located on the 5th floor of the Institute’s West Building.

“It’s going to affect such a large number of people in our population,” he added. According to the best estimates, roughly 40 percent of the 600,000 polio survivors in this country, or about a quarter million people, currently suffer with post-polio syndrome.

Generally striking decades after recovering from paralytic polio, symptoms usually begin with progressive muscle weakness, followed by debilitating fatigue, loss of function and pain, especially in the muscles and joints, swallowing becomes difficult, and breathing turns labored.

Until recently, post-polio syndrome seemed to defy explanation. But during the past half dozen years or so, scientists like Pfaff have begun to unravel some of the genetic mechanisms that may one day provide new tools to treat post-polio syndrome and other similar neurological disorders including Lou Gehrig’s disease.

Most of the new insights involve molecular detective work and the search for genes responsible for creating and directing the responsible neurons to their final destinations in the body.

“What we are really after is an understanding of genes and molecules at the most fundamental level that generate these neurons and how these neurons are wired up correctly,” said Pfaff, speaking in his office overlooking seaside bluffs and the vast Pacific beyond.

“The research during the past four or five years has become quite exciting because we’ve begun to identify many genes that seem to be critical for these particular neurons.”

The basic question

To understand what happens during post-polio syndrome, a brief explanation is needed for what happens during polio itself.

It all starts with the nerves that drive muscles, aptly termed motor neurons. Physiologically speaking, these neurons consist of a cell body located in the spinal cord, and a long tentacle, or axon, which extends to the muscles. Near the tip of the axon, sprouts of tiny branches spread out and attach to muscle cells. Where the nerve and muscle connect, across a small gap called a synapse, a chemical called acetylcholine is released. This causes the muscle fiber to contract.

The distinctive characteristic of paralytic polio is the uncanny ability of the virus to attack only motor neurons, leaving adjacent nerve cells intact.

“These motor cells are important for muscle control, including breathing, which is one of the reasons why it leads to many individuals needing iron lungs as an assist,” said Pfaff.

The poliovirus infects virtually all the motor neurons in the spinal cord; the infected cells either overcome the virus or die. Those motor neurons that survive the onslaught reconnect with muscle cells abandoned by the death of their original motor neuron. Each of the survivors accomplishes this task by growing new axonal sprouts. A single motor neuron that initially simulated 1,000 muscle cells might eventually connect or innervate 5,000 to 10,000 cells.

Although no one knows for certain, it’s now generally believed post-polio syndrome results when these overly extended motor neurons start wearing down over time, the result of metabolic exhaustion.

“This probably leads to premature aging and death among, these cells and there probably aren’t enough cells to continue to compensate,” said Pfaff, who earned his Ph.D. in molecular biology from the University of California, Berkeley and completed postdoctoral fellowships at Vanderbilt University and Columbia University.

In theory, if the mechanisms could be found that set in motion the growth and guidance system used by motor neurons, it could then be possible to correct the neurological deficit resulting from their loss.

It’s as basic question asked by developmental biologists seeking to understand how these neurons are assembled in the first place.

“The question always centers on how does a particular cell type know what it is during embryonic development, since everything starts out from a pretty common origin,” said Pfaff. “And then the other key feature of the nervous system is that it has to be wired up correctly. So neurobiologists commonly ask how does that happen.”

It’s midmorning in Pfaff’s lab. The sounds of a jazz radio station are wafting in the background, and a green fluorescent light is flickering off a dime-sized opening in a check egg resting under a microscope.

Peering into the instrument, a young researcher is carefully directing the needle-thin ending of a pipette through the opening to its target: a tiny tube of developing nerve cells. The other end of the pipette, a flexible straw, is held between the researcher’s lips.

Then, with a small puff of air from his mouth, a cocktail of DNA molecules is pushed through the needle’s tip into the neural tube. It’s hoped that when this cocktail is charged with a small jolt of electricity, the results will trigger new neurons in the growing embryo.

The relatively new technique, called in ovo-electroporation, is generating excitement among researchers interested in a comparatively easy way to inserting new genes into embryonic spinal cords.

“It’s not going to be a single simple gene that’s going to be adequate to specify motor neuron identity,” said Pfaff. “It will be some combination of genes, and we’re at the stage now where we are trying different cocktails to see if we got it or not.”

On a Quest

One of the big incentives for doing basic research comes from the discovery of the unexpected. So it’s been with the search for new genes responsible for triggering neuronal growth and then guiding exonal projections to their final targets.

Take, for example, a gene called islet 1, or Ist-1, which as its name suggests (from the islets of Langerhans in the pancreas) was first identified as a kind of master regulator or transcription factor thought to contribute to insulin gene expression.

In their work, however, Pfaff and others in his lab were surprised to find Ist-1 being expressed in motor neurons, in fact, the gene is absolutely essential to generate these neurons. Further, it now turns out that probably a half dozen or more genes active in the pancreas are also expressed in motor neurons.

“There appears to be no logical reason for that to be, because the two structures are so unrelated,” said Pfaff. “But it seems to be a general concept in biology that many genes tend to be recycled over and over again in different cells and that it’s sort of the unique combination of genes that leads to the formation of different cell types.”

Indeed, determining the role of Ist-1 in motor neuron development was far from the entire story. Late last year, for example, researchers in Pfaff’s lab described how to genes, Lhx-3 and Lhx-4, act together during a brief period of development to help direct specific axons from the neural tube to their targets in the embryo.

This summer, the group turned its attention to yet another gene called Hb9, for homeobox 9. During the past couple of years, other researchers have linked this gene to hereditary sacral agenesis, a rare birth defect that results in malformations in pelvic and sacral arenas.

“It was clear this was an important gene. You need it, and it can’t be messed with too much,” said Pfaff. “It seemed to provide evidence that Hb9 may be a master regulator.”

To determine Hb9’s role in motor neuron development, the scientists engineered “knockout” mice that lacked the gene. The effects were both surprising and profound. In fact, the mutated mice formed motor neurons; however, the animals still died at birth because they couldn’t breath properly.

Somewhat like archeologists, the scientists began to unearth clues that would help them understand the role Hb9 played in the process. Instead of being critical for initiating motor neuron growth, the scientists reasoned, Hb9 must have something to do with helping axons find their way to the right muscle targets. Without proper guidance, nerve cells couldn’t be wired to the diaphragm, making breathing impossible.

Contrary to their expectations, they discovered that the gene acts by silencing the activity of other genes that turn on in nearby nerve cells called interneurons. Without this dampening activity of Hb9, other inappropriate genes are set in motion, effectively preventing the correct wiring of nerve cells to their targets in the embryo.

”Going into this, the simple expectation was once you eliminated Hb6, then you wouldn’t get motor neurons,” said Pfaff.
“This study points out that you can’t think of everything in terms of turning things on. You also have to think in terms of turning off the incorrect things as well.”

* * *

At age 38, Pfaff is too young himself to recall the fear raised by the specter of polio earlier this century. But he has spoken to others with first-hand experience, including some of the survivors who now are facing the aftermath of their disease.

“Post-polio syndrome is found in a very high percentage of these survivors, particularly in the fourth, fifth and sixth decades of their lives.” he said. “Many are reaching that stage now.”

Though medical science can offer little to these patients for the moment. Pfaff talks enthusiastically about the possibilities for the future, particularly with new advances in gene therapy that one day may direct immature stem cells into becoming mature motor neurons.

His recent experiments are giving him and other hope that many of the genetic factors needed in the process are starting to reveal themselves. In all, perhaps 25 genes have so far been identified, their roles need to be better defined and studied.

“We’re at a pretty exciting stage, where we can begin to put some of these factors back into stem cells,” he said. “If we can now control the differentiation of stem cells, that will begin to give a sort of ready access to motor neurons whenever they are needed.

“It’s a long way from therapy, unfortunately. But these things happen in small incremental steps, and at least we’ve begun to climb a few of those steps.”

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