Animal models enable researchers to track amyotrophic lateral sclerosis (ALS) pathogenesis. Erik Storkebaum, MSc, and colleagues at the Center for Transgene Technology and Gene Therapy at Flanders Interuniversity, Leuven, Belgium, took several approaches to increase supply of the neuroprotective protein vascular endothelial growth factor (VEGF) in an animal model. "When administered to rats at 60 days, which is 1 month before symptoms, it delayed onset and prolonged survival by 22 days. When we gave VEGF at the age of disease onset, which more closely mimics the human situation, the treatment still prolonged life by an average of 10 days," Storkebaum reported at the Society for Neuroscience annual meeting in October 2004
ALS begins vaguely--with a twitch or sudden clumsiness. For Jenifer Estess, whose remarkable book Tales From the Bed chronicles her battle against ALS, the first symptoms were difficulty in doing sit-ups and in standing up, and a heavy sensation when holding a towel. Wildly fasciculating thigh muscles led her to seek medical help. For soap opera actor Michael Zaslow, ALS initially garbled his speech so much that he soon lost his job--but he resurfaced on a different daytime drama, in which the powers-that-be wrote ALS into his character, calling much-needed attention to the disease.
Estess had the limb-onset form of the disease, and Zaslow the bulbar form, which affects about 25% of patients. In both forms of ALS, upper and lower motor neurons, as well as astrocytes and microglia eventually die.
"It is a neurodegenerative disease of the motor neurons, throughout the system. People die because they lose control of the upper airways," said Nicholas Boulis, MD, a neurosurgeon at the Cleveland Clinic, who is investigating ways to treat the spinal cord directly. It is a more profound disturbance than other neurodegenerative disorders. "When you steer a car, the steering wheel directly connects to the wheels, but if there is power steering, movement is modulated by circuits that make it easier. Huntington disease and Parkinson disease are like the power-steering circuits; ALS is the direct-drive shaft. No matter what, you don't get output," explained Jeffrey Rothstein, MD, PhD, director of the Packard Center for ALS Research and professor of neurology and neuroscience at Johns Hopkins University.
Despite the fact that ALS seems to combine the worst aspects of all neurodegenerative diseases, optimism and effort in the ALS community are inspiring. "We're a small disease, but we move quickly from lab to trial," said Rothstein. Boulis is hopeful for another reason: "ALS presents a defined territory that is critical to extend life: the upper airways. In many ways ALS is more accessible than a spinal cord injury or Alzheimer disease, which is a far more diffuse disorder."
Still, ALS presents daunting hurdles. Gradual onset attenuates diagnosis, but progression comes so fast that clinical trials cannot run as long as those for other neurodegenerative disorders. The mind remains relatively intact without treatment as the ability to move ebbs. Many treatments have failed, the disease's rarity does not invite investment, and the first animal model has been, by many accounts, disappointing. Perhaps the greatest challenge stems from the many mysteries surrounding ALS.
CAUSES UNKNOWN, CANDIDATES MANY
A rare, poorly understood disorder with subtypes could be a research nightmare. "ALS is a phenotype, like stroke. How did you get there? There may be a very pure form with only motor impairment and a complicated variant with cognitive or sensory problems," said Michael Strong, MD, the Arthur Hudson chair in ALS Research at the University of Western Ontario. Mary Abood, PhD, a research scientist at California Pacific Medical Center, San Francisco, agrees: "Part of the reason it is so difficult to show efficacy in clinical trials is that some people decline more rapidly than others because of a different etiology. A drug being tested may be efficacious against one form but not another."
Identifying environmental triggers and inherited susceptibilities that may underlie ALS subtypes has been problematic. For example, 2 studies found that the incidence of ALS among veterans of the first Gulf War is significantly higher than the incidence among military personnel who were not deployed to the Gulf; however, risk factors have not been isolated.1,2 While the possible roles of chemicals from burning oil fields, vaccines, and exposure to unusual pathogens were being contemplated, another, more recent study that looked at incidence in association with military endeavors found elevated rates of occurrence among persons who served in the military at any time than among those who did not.3 The association, then, may be more with military service in general than with time or place of military activity.
A clearer environmental link comes from the native Chamorro people of Guam, where the incidence of a trio of neurodegenerative diseases--ALS, Parkinson disease dementia, and a combination of these--peaked shortly after World War II, affecting nearly 10% of the population. After the NIH ruled out a clear genetic pattern, attention turned to environmental triggers. A combination of folklore and logic led, in the 1960s, to the seeds of cycad trees. Consumption of seeds from the cycad tree increased when other foods became scarce during wartime.
In the early 1980s, researchers identified a possible neurotoxin: the amino acid beta-N-methylamino-levo-alanine (BMAA), which resembles an amino acid linked to an ALS-like illness prevalent in India and is similar to the excitatory neurotransmitter glutamate. However, it was present in cycad seeds in parts per million--not enough to affect the human CNS--but biologic detective work solved the puzzle.4
In 2003, Paul Cox of the Institute for Ethnobotany in Kauai, Hawaii, and Oliver Sacks, BM, BCh, at the Albert Einstein College of Medicine in New York, painted a plausible ecologic scenario of biomagnification: cyanobacteria produce BMAA in the soil, which cycad roots tap. Then, bats called flying foxes eat cycad seeds and concentrate the neurotoxin in their tissue, just as fish concentrate mercury. World War II brought guns to the Chamorro, which enabled them to blast dozens of the delectable bats from trees--easy targets with neurotoxin-slowed reflexes. Dining on bats could have poisoned the Chamorro people.
To follow up on an ALS-environmental link, in 2004, the Agency for Toxic Substances and Disease Registry, part of the US Department of Health and Human Services, launched an effort at hazardous waste sites in 5 states to see whether ALS incidence had increased in their vicinity. The first results, reported in May 2005 from Kelly Air Force Base in Bexar County, Texas, found no additional cases.
Evidence for an infection connection also is weak. Anecdotal reports blame Lyme disease for misdiagnosis, but the differential diagnosis of Lyme disease is easily ruled out, Rothstein said. Viruses have been implicated too, at first based on the similar effects of poliovirus, and more recently, with the detection of increased levels of reverse transcriptase, a retroviral enzyme, in ALS patients.5
If environmental data are sparse, molecular clues are perhaps too diverse. ALS is associated with at least 9 different genetic variants and 3 susceptibility genes. Shortly after the SOD1 connection,6 researchers developed a transgenic mouse bearing the human mutation.7 But the problem was not due to a deficit or excess of SOD1, because altering levels of the normal enzyme in the mouse did not halt pathogenesis if the errant human gene was present.8 This situation indicates a "toxic gain-of-function"--the variant enzyme does something different.
Localizing the aberrant enzyme explains part of what may be happening; in mice and human postmortem tissues, the enzyme accumulates at the surfaces of mitochondria and in the intermembrane spaces, which may explain the structurally abnormal mitochondria in some patients.9,10 Mitochondria are the cell's headquarters for apoptosis. Here the abnormal enzyme interacts with the protein Bcl-2, which normally blocks apoptosis. With this control lifted, cells die.
The other major puzzle piece to ALS etiology is excitotoxic injury, which arises from impaired handling of glutamate in the spinal cord. Transporter proteins made in astrocytes escort glutamate across synapses, where it binds postsynaptic receptors. Calcium ions enter, triggering neuronal death. Normally, the transporters bind excess glutamate and take it into nearby astrocytes.
Evidence implicates impaired glutamate transporters and receptors in ALS. Rothstein and associates11 compared synaptosomes--vesicles that form at nerve endings when the tissue is homogenized--from spinal cord; sensory, motor, and visual cortex; striatum; and hippocampus from 13 persons with ALS, 12 with Huntington disease, 15 with Alzheimer disease, and 17 controls. The transporter effectively bound its cargo in all, but in the ALS patients, binding was slow in the spinal cord and in the motor and sensory cortexes.
A study from the University of Tokyo found that faulty RNA editing alters the glutamate recep- tor.12 Researchers compared how mRNA encodes a receptor subunit in ALS patients and in controls. In the ALS patients, a single incorrect amino acid is inserted into the protein about half the time.
The relationship between oxidative stress and excitotoxicity remains murky in regard to whether each causes a disease subtype or whether one precipitates the other. "At the present time, it is very difficult to decipher the toxic mechanism involved in ALS," said Patrick Aebischer, MD, PhD, president of the Swiss Federal Institute of Technology in Lausanne, Switzerland.
Another hypothesis regarding trauma as a risk factor for ALS resurfaced recently, when German and Italian researchers wrote about "the Italian motoneuron mystery."13 ALS was diagnosed in 33 of 24,000 top Italian soccer players between the 1960s and 1996.
ALS diagnosis is clinical; no laboratory tests are available (see box, Future ALS Diagnostics). The average neurologist will probably see only 3 or 4 ALS patients in a career; an internist may never see an ALS patient. Yet by the time of diagnosis, 50% of a patient's motor neurons may be affected.
In 1999, Aventis Pharma AG commissioned a study of the diagnostic trajectories of 201 patients in 6 countries. The average experience: a patient first consults a physician 4.6 months after symptom onset and first meets with a neurologist by 10.8 months, who diagnoses ALS within 6.9 months. That's more than 18 months from the first twitch or tumble. Nearly half of the patients were initially referred to non-neurologists, including rheumatologists, orthopedists, and chiropractors. Patients with bulbar onset and/or fasciculations tended to be referred to a neurologist faster.
Diagnosing ALS takes time. "When a patient first shows up, not even a specialist can make the diagnosis," said Rothstein, who, as head of a tertiary care center, sees more than 300 patients a year, of whom only 1 or 2 have received a misdiagnosis. "If the first symptoms are in the hands, a patient might come in thinking carpal tunnel syndrome; bulbar patients think it is a stroke," he added. After these and other possibilities have been ruled out, diagnosis requires watchful waiting. "Textbook criteria are evidence of diffuse upper and motor neuron degeneration on at least 2 levels. Signs of lower motor neuron involvement include weakness in the muscle, atrophy of the muscle, and fasciculations. Signs of upper motor neuron degeneration are brisk reflexes, hyperactive reflexes, spastic muscle tone, and some weakness," Rothstein said.
Harder to exclude is multifocal motor neuropathy, an autoimmune condition. "A patient may come in with a history of progressive, diffuse lower motor neuron signs and symptoms--weakness in one hand that has spread to the other, or footdrop--over 9 months. The patient has all the lower motor neuron signs, but no gross upper motor neuron signs," Rothstein said. He uses a blood test for anti-GM1 antibodies and an electromyogram to rule out the autoimmune condition. But continuing lack of upper motor neuron symptoms prompts some physicians to diagnose atypical ALS. In these cases, Rothstein said, upper motor neuron involvement usually is clear at autopsy.
Future ALS Diagnostics
VEGF Animal models enable researchers to track amyotrophic lateral sclerosis (ALS) pathogenesis. Erik Storkebaum, MSc, and colleagues at the Center for Transgene Technology and Gene Therapy at Flanders Interuniversity, Leuven, Belgium, took several approaches to increase supply of the neuroprotective protein vascular endothelial growth factor (VEGF) in an animal model. "When administered to rats at 60 days, which is 1 month before symptoms, it delayed onset and prolonged survival by 22 days. When we gave VEGF at the age of disease onset, which more closely mimics the human situation, the treatment still prolonged life by an average of 10 days," Storkebaum reported at the Society for Neuroscience annual meeting in October 2004.
Biomarkers Metabolon, Inc., of Durham, NC, is pursuing a route to earlier diagnosis that uses mass spectrometry to screen plasma for disease-specific, nonprotein biomarker "signatures." "In very preliminary work, we believe that we can see biomarkers that are different in sporadic versus familial ALS, and lower and upper motor neuron disease," said president and CEO John Ryals, PhD. Once signatures are confirmed, the approach will be used to track disease progression, he added.
Cognitive Signs Cognitive decline may appear early in ALS, but is not yet well enough understood to aid diagnosis. "From 30% to 70% of patients may have a behavioral syndrome of disinhibition or emotional lability and difficulty with complex sequential task performance and speech. It is fairly subtle. If you look for it, you'll find it, but it doesn't leap out at you," said Michael Strong, MD, Arthur Hudson Chair in ALS Research at the University of Western Ontario, who chaired a workshop on cognitive changes in ALS in mid-May. Florid dementia is rare, he added. The subtler impairments arise from the frontotemporal lobe, apart from the motor strip.
ALS research shifts quickly from bench to bedside (see box, Future ALS Therapeutics). Finding the glutamate link quickly led to the development of the drug riluzole (Rilutek, Aventis). It increases survival by 3 to 6 months, more in younger patients, and is more effective in bulbar-onset cases.14 But it stands alone in the ALS drug arsenal. In January 2005, Novartis stopped developing TCH346, an anti-apoptotic agent, after phase 2 clinical trials did not show change in progression or survival. Agents that have been tried and rejected include celecoxib (Celebrex, Pfizer), indinavir (Crixivan, Merck), creatine, and neurotrophic factors; still in phase 3 clinical trials is minocycline (www.clinicaltrials.gov/ ct/show/NCT00047723?order=1).
Another promising antibiotic is ceftriaxone (Rocephin, Roche), identified in a screen of 1040 drugs for hiked transcription of the gene that encodes the glutamate transporter, which normally prevents glutamate excitotoxicity.15 The screen detected 20 compounds, 15 of which are b-lactamase antibiotics--but the mechanism is not related to antibacterial activity. Because ceftriaxone increased the supply of transporters in ALS mice and delayed progression, clinical trials are ongoing For more information, you can go to: www. clinicaltrials.gov/ct/show/show/NCT00001539?order=2 or go to www.clinicaltrials.gov/ct/show/NCT00111644?order=1).
The ceftriaxone story reveals the importance of experimental design. A decade ago, trials of the drug in ALS had negative results,16 but those efforts, Rothstein said, were led by a single case report that was quickly retracted.17,18 The Italian ALS study group "conducted the trials expecting improvement, didn't use placebos in many cases, and tried the drugs for 1 to 8 weeks. In ALS, it takes 9 months to a year to see something," he added.
Another old drug that is already helping some ALS patients is the cough remedy dextromethorphan/quinidine. Although a recent New York Times article ridiculed its use as treatment for inappropriate laughing and crying,19 such emotional lability is a hallmark of ALS--and a debilitating one for patients who still work. "We've been using it off-label for years. The circuitry that controls the head-and-face musculature is tied to suppression of emotion. A patient with bulbar-onset ALS can very easily laugh and cry. The slightest hint of emotion bursts out," said Rothstein. Avanir Pharmaceuticals of San Diego is seeking FDA clearance to market the drug, which will be sold as Neurodex, for pseudobulbar affect. (See also A Cough Medicine and Neurologic Disorders, AppliedNeurology, June 2005, page 8.)
Yet another familiar compound being scrutinized for anti-ALS activity is tetrahydrocannabinol (THC). Abood and her team20 found that THC protects against oxidative stress and excitotoxicity in isolated spinal cord neurons. (See also Cannabinoids Showing Potential Value in Parkinson, Lou Gehrig Diseases, Applied Neurology, February 2005, page 38).
"Then we tried it on the SOD1 mouse, in several doses, and found that it delayed disease progression and prolonged life span by about 5%, which extrapolates to about 4Z\x extra months in humans," she said. Some physicians prescribe Marinol (Unimed) off-label for appetite stimulation in ALS patients, and Health Canada recently approved Sativex (GW Pharmaceuticals), a cannabis extract, to treat multiple sclerosis. This drug may also be useful in ALS. (See Canada Approves Cannabis, Applied Neurology, May 2005, page 13.)
Perhaps the biggest hurdle for an ALS patient is the decision to use or refuse a ventilator when breathing begins to fail. Many neurologists concur that the overwhelming majority of patients refuse this measure. Rothstein explains why: the disease doesn't stop. "You eventually lose all movements. You may get little twitches in your fingers that you can use to communicate with computers. When that goes, you can use eye movements to track infrared devices to communicate. But eventually eye movements cease, and then you are locked in. People must give up their lives to take care of you for a very unusual quality of life. As physicians, we support it if it's what a patient wants, but it is important for patients to understand what it means to be on a ventilator." This dire situation may change if clinical trials after a pilot study on a diaphragm pacing system that stimulates the phrenic nerve are positive. (See DPS and Electrostimulation Allow Patients With ALS to Breathe, Applied Neurology, June 2005, page 55.)
The rarity and lack of treatment for ALS may paint a bleak picture, but the alacrity with which basic science is transitioning to clinical trials offers hope. Don Cleveland, PhD, a professor of medicine, neuroscience, and cellular and molecular medicine at the University of California, San Diego, described the current climate as one of "stubborn optimism." "The issue isn't that we can't identify strategies that can be effective, but whether we can deliver them in an optimal way. The failures are of delivery, not the concept," he said at an ALS symposium at the Society for Neuroscience 2004 annual meeting. *
Future ALS Therapeutics
Fetal Cell Implants At Chaoyang Hospital in Beijing, Hongyun Huang, MD, has treated more than 100 patients who have amyotrophic lateral sclerosis (ALS) with injections of human fetal olfactory ensheathing glial cells into 2 sites in the subcortical white matter below the motor cortex and, in some patients, at the top of the spinal cord. The waiting list for Americans with ALS is 600 strong. "People are reporting dramatic improvement--but we've been there before, with fetal transplants and Parkinson disease (PD). The placebo effect in brain surgery is enormous," said Nicholas Boulis, MD, a neurosurgeon at the Cleveland Clinic. He was referring to the phenomenon whereby participants in clinical trials for PD treatments who undergo sham surgery sometimes report improvement. But Huang does not think this is the case for ALS. "I don't think the effect that makes the patients improve is a placebo effect. This terrible disease is quite different from PD, which is often influenced by mental state." He hopes that the treatment will patch damage and halt progression.
IGF-1 Insulinlike growth factor (IGF) (myotrophin) is in phase 3 clinical trials to slow progression of weakness in ALS. Earlier trials were negative in Europe but positive in the United States. When the sponsoring company opted out of a third try, an academic consortium took over. The clinical trial uses this recombinant protein. Preclinical models are testing gene therapies, including the piggybacking of IGF-1 to clostridial toxins to target motor neurons and the use of an adeno-associated virus as a vector that uses its retrograde axonal transport from muscle to neuron.
siRNA Patrick Aebischer, MD, PhD, president of the Swiss Federal Institute of Technology in Lausanne, Switzerland, and his team are dampening expression of the SOD1 gene while providing the normal protein. The result in mice is delayed onset of ALS and 3 additional weeks of life. The technique uses small, interfering RNAs (siRNAs). "These are small pieces of RNA that bind mRNA and are degraded by enzymes, so the protein is not produced," he said. Aebischer has videos that show ALS mice being unable to hoist themselves out of a pool before treatment and showing restoration of some swimming ability after treatment. "[ALS symptoms in] the treated mice were significantly ameliorated, their [electromyographic findings] improved, and treatment conserved many more myelinated axons," he said.
Stem Cells Ian Wilmut, PhD, a professor at the Roslin Institute in Edinburgh, and Christopher Shaw, MD, of the Institute of Psychiatry in London, received a license in February 2005 to use early human embryos to explore the etiology of various diseases. The first disease they are looking into is ALS. They will introduce the SOD1 mutation and derive embryonic stem cells, which will then be stimulated to differentiate as neural tissue.
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