Iron Gone Wild in Mouse Model of ALS

The human body maintains a precisely controlled postal system to shuttle iron from gut to brain, from cell to cell, and in and out of the cellular warehouse, the iron-storage molecule ferritin. Iron is an essential cofactor in many metabolic processes, but it can also be a dangerous parcel: the ferrous form is a highly reactive oxidant. Researchers at McGill University in Montréal, Quebec, have found that the iron regulatory system loses control in a mouse model of amyotrophic lateral sclerosis (ALS), and that an iron-clearing molecule delays disease in the animals. They report their results in the January 21 issue of the Journal of Neuroscience.

This paper is the first report of iron accumulation in an ALS model. “Our work now provides a strong rationale for the inclusion of iron chelator therapy for the treatment of ALS,” wrote first author Suh Young Jeong, principal investigator Sam David, both of McGill, and their coauthors, in the paper. David is already seeking chelators that might be useful in people.

Not so fast, other experts say. “Right now, this is premature,” said Richard Bedlack of the Duke ALS Clinic in Durham, North Carolina. Chelation therapy, which is standard for heavy metal toxicity, is controversial with respect to other conditions. For example, last fall the National Institutes of Health cancelled a chelation trial for autism (see Mitka, 2008). Some fringe clinics offer chelation to people with ALS, but it is not recommended by the majority of physicians.

Iron homeostasis is altered or damaged in a number of neurodegenerative diseases (for review, see Zecca et al., 2004). Mutations in iron regulators cause diseases such as Friedreich ataxia, and iron builds up in the affected regions of the brains of people with Alzheimer and Parkinson disease. Drugs that mop up wayward metals have shown promise in clinical trials for Alzheimer’s (see ARF related news story). Imaging studies in the 1990s suggested iron accumulation in the brains of people diagnosed with ALS, although the results were not definitive (Oba et al., 1993; Imon et al., 1995). But in most ALS research, the role of iron has been overshadowed by interest in copper and zinc, cofactors for the enzyme superoxide dismutase 1 (SOD1), which is mutated in some inherited forms of the disease.

Jeong and colleagues discovered abnormally high iron levels in the spinal cord of one-year-old mice overexpressing the human SOD1G37R gene. Using histochemistry, the researchers found small, round, iron-positive inclusions in the cell body of motor neurons in the large ventral horn. They also observed diffuse iron staining in some glial cells. Similar staining was not present in younger mice, nor in wild-type control animals.

The different patterns suggested to David that the iron was building up in distinctive locations in the different cell types, perhaps via diverse mechanisms. The scientists analyzed three possible ways the iron postal system could go wrong: dysregulation of iron delivery and storage throughout the central nervous system, excess iron in mitochondria, and disabled iron transport along axons.

The scientists used quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) to examine mRNA levels for iron’s delivery vehicles (divalent metal transporter 1, transferrin receptor 1, and ferroportin), the iron storage protein ferritin, and enzymes that convert the metal to a less reactive form (ceruloplasmin and hephaestin). They found that in four-month-old animals, several of these genes were upregulated above wild-type levels in the lumbar part of the spinal cord, probably because the cells contained more iron, and thus needed more of the proteins that import, export, and store it. But by 12 months, the researchers observed the highest levels of these mRNAs in the cervical spinal cord. That tail-to-head progression mimics the course of the disease, David said, suggesting that the earliest affected neurons are first to fight for control of iron, and first to lose it.

Jeong and colleagues also saw iron dysregulation within the mitochondria. These organelles, which have been implicated in ALS (for review, see Dupuis et al., 2004), possess their own ferritin and use iron to assemble the heme complexes that are essential for respiration. The scientists found a twofold increase in mitochondrial ferritin in the spinal cord of mSOD1 animals, compared to wild-type controls. Since ferritin levels usually mirror iron levels, David said, that suggests the mitochondria are also dealing with excess iron.

Axonal transport is disrupted in ALS, so the scientists examined this process as well. They ligated only one sciatic nerve, leaving the nerve on the other side of the body untouched, in wild-type animals. On the tied-off side, large ventral horn motor neurons amassed more granular iron, compared to cells on the non-injured side. “This nicely demonstrates that trafficking impairment can lead to metal accumulation,” wrote Ashley Bush of the University of Melbourne in Victoria, Australia, who was not involved with the study, in an e-mail to ARF.

The iron delivery system was out of whack everywhere the scientists looked. “This suggests there are many different mechanisms that might be playing a role,” David said. But the clincher that iron causes problems was that clearing the metal from the nervous system slowed disease. The researchers treated 12 animals with the iron chelator SIH, twice a week starting at eight months, and found that they developed disease five weeks later than 18 untreated control animals. A dozen animals receiving the therapy once a week also survived longer. Histochemistry confirmed that the excess spinal cord iron disappeared with treatment. “It was just gone,” David said. And while untreated mice dropped in weight as they approached 50 weeks of age, the chelator-treated mice did not. That finding suggests that the five-week gain in lifespan may be a relatively healthy one.

The scientists treated the animals before symptoms developed. That kind of therapy might not be so useful for people, Bedlack noted. “We really don’t care much about therapies that delay disease onset.” By the time people with ALS are diagnosed and in need of treatment, the symptoms are already progressing.

Additionally, it is not clear if results obtained with mSOD1 mice will apply to the majority of people with ALS, 98 percent of whom do not carry SOD1 mutations. This problem is common to studies on mSOD1 mice; therapeutics that showed promise in those animals have been uniformly unsuccessful in clinical trials. Outside experts also noted the small number of animals used in the study; David responded, “The point is that we have statistical significance with the numbers we used.”

Bush called the rescue “impressive,” but noted that chelators often cross-react with more than one metal. It is not clear, then, whether iron clearing, or perhaps copper chelation, is responsible for the enhanced survival. And Maria Teresa Carrì, of the University of Rome in Italy, noted that the animals may have gained five additional weeks of life—which is a high number in mSOD1 mouse studies—simply because the G37R mice have a very slowly progressing disease, compared to other ALS models.

The iron accumulation is not a general feature of disease in mSOD1 mice; the scientists found no increased iron in mice carrying the SOD1G93A mutation. Those animals live only four months, and David suggested they did not survive long enough for iron to build up. “Iron accumulation is not the primary cause of the disease, obviously, but somewhere along the course of the disease, something is triggering this,” he said. “Any kind of dysregulation, in the sense of increased reactive iron in the cell, would then be a prime candidate for generating free radical damage.” Chelation therapy, he suggested, might ameliorate this portion of the disease.

David is currently considering how to translate this research finding into a treatment for people with ALS (see Q&A, below), but Bedlack suggested it is too soon to even think about a clinical trial. “It’s possible that there’s iron dysfunction in the animals, but certainly more studies will be needed to establish that,” he said. And chelation can have dangerous side effects, Bedlack said, including arrhythmias, kidney or respiratory failure, seizures, and even death. “We definitely do not recommend that patients with ALS seek chelation,” he said.

Clearly, many questions must be answered before chelation has a chance at becoming a mainstream ALS treatment. “More evidence of iron or copper abnormalities in sporadic ALS is needed before I would feel that such treatment is justified,” Bush wrote. “Nevertheless, I think that we need to understand the nature of the iron lesion that has been uncovered by the authors. It is potentially the source of much damage, and therefore I think this is an important discovery.”—Amber Dance

Q&A with Sam David. Questions by Amber Dance.

Q: How, hypothetically, might iron chelation work in people with ALS?
A: There is evidence in the literature that iron accumulation occurs in the central nervous system (CNS) of people with ALS. If this increase in iron in ALS has effects similar to what we have seen in the SOD1G37R transgenic mice, then the iron chelation therapy can be expected to reduce iron accumulation and clear the iron that has already accumulated in the spinal cord and brain.

Q: Has chelation been tried before in ALS patients, or in other animal models? What happened?
A: To my knowledge, iron chelation has not been tried in humans. Chelators of other metals (such as copper) have been tried with success in other SOD1 transgenic mouse strains.

Q: Are there validated uses of chelation therapy aside from heavy metal toxicity?
A: Iron chelation is used for conditions such as thalassemia, myelodysplastic syndromes, and sickle cell disease.

Q: What questions need to be answered before trying this therapy in people?
A: The chelator we used is not approved for use in humans. Therefore, any lipophilic iron chelators that are on the market would likely need to be tested in animal models to see if they can reduce iron accumulation in the CNS before trying in humans.

Q: Why did you treat the mice before they developed symptoms? That wouldn’t really be applicable in the clinic, where the majority of people’s disease is progressing by the time they are diagnosed.
A: The reason for this is that in all the ALS mouse models, the time between onset of symptoms and endstage is extremely short, but yes, it would be worth looking into that.

Q: If the therapy gave mice five additional weeks of life, do you have any sense of what the effect might be in people?
A: This is difficult to extrapolate since metabolism in rodents and humans is very different. However, our results with the iron chelator were among the best of the other single therapies tested in this transgenic mouse line.

Q: Do you have any specific candidates for the chelator in mind?
A: Not yet.

Q: Do chelators cross the blood-brain barrier?
A: The lipophilic chelators like the one we used in our animal studies appear to cross the blood-brain barrier.

Q: Is there any way to target chelators specifically to the CNS?
A: I am not sure of this yet.

Q: What might be potential side effects of chelation therapy for ALS?
A: Anemia and other side effects.

Q: When do you envision starting clinical trials for this kind of treatment?
A: We are looking into various iron chelators that are currently available to see if any of them would be suitable.

Comments

  1. Comment by Gang Liu, Xiongwei Zhu, Barney Dwyer, Rudy J. Castellani, Hyoung-gon Lee, George Perry, Mark A. Smith

    Chelation and Neurodegenerative Diseases: Nanoparticles as Proof of Concept
    The notion that iron dyshomeostasis contributes to the pathogenesis of a number of neurodegenerative disorders, now including amyotrophic lateral sclerosis (Jeong et al., 2009), is well established (Castellani et al., 1995; Smith et al., 1997; Castellani et al., 2000; Sayre et al., 2000; Petersen et al., 2005; Smith, 2006). In parallel, changes in a number of iron homeostasis proteins are also altered, including heme oxygenase-1 (Smith et al., 1994; Schipper et al., 1995), ferritin (Connor et al., 1992), and iron regulatory proteins (Connor et al., 1992; Smith et al., 1998).

    While clinical trials in neurodegenerative diseases using chelation therapy have been limited, such an approach has shown promise (McLachlan et al., 1993). Unfortunately, limited blood-brain barrier permeability and/or cytotoxicity with resultant side effects have limited the interpretation of such trials. In contrast to existing chelators, we recently developed a novel nanoparticle-chelator conjugate that shows potential for allowing free passage into and out of the brain without changing chelation properties (Liu et al., 2005; Liu et al., 2006; Liu et al., 2009). While still at a preclinical stage of development, the utility of such nanoparticle conjugates obviously merits serious consideration for future clinical trials to evaluate the potential of this strategy for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis.

    References:

    Castellani R, Smith MA, Richey PL, Kalaria R, Gambetti P, Perry G. Evidence for oxidative stress in Pick disease and corticobasal degeneration. Brain Res. 1995 Oct 23;696(1-2):268-71. PubMed.

    Castellani RJ, Siedlak SL, Perry G, Smith MA. Sequestration of iron by Lewy bodies in Parkinson's disease. Acta Neuropathol. 2000 Aug;100(2):111-4. PubMed.

    Connor JR, Menzies SL, St Martin SM, Mufson EJ. A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains. J Neurosci Res. 1992 Jan;31(1):75-83. PubMed.

    Connor JR, Snyder BS, Beard JL, Fine RE, Mufson EJ. Regional distribution of iron and iron-regulatory proteins in the brain in aging and Alzheimer's disease. J Neurosci Res. 1992 Feb;31(2):327-35. PubMed.

    Jeong SY, Rathore KI, Schulz K, Ponka P, Arosio P, David S. Dysregulation of iron homeostasis in the CNS contributes to disease progression in a mouse model of amyotrophic lateral sclerosis. J Neurosci. 2009 Jan 21;29(3):610-9. PubMed.

    Liu G, Garrett MR, Men P, Zhu X, Perry G, Smith MA. Nanoparticle and other metal chelation therapeutics in Alzheimer disease. Biochim Biophys Acta. 2005 Sep 25;1741(3):246-52. PubMed.

    Liu G, Men P, Harris PL, Rolston RK, Perry G, Smith MA. Nanoparticle iron chelators: a new therapeutic approach in Alzheimer disease and other neurologic disorders associated with trace metal imbalance. Neurosci Lett. 2006 Oct 9;406(3):189-93. PubMed.

    Liu G, Men P, Perry G, Smith MA. Metal chelators coupled with nanoparticles as potential therapeutic agents for Alzheimer's disease. J Nanoneurosci. 2009 Jun 1;1(1):42-55. PubMed.

    McLachlan DR, Smith WL, Kruck TP. Desferrioxamine and Alzheimer's disease: video home behavior assessment of clinical course and measures of brain aluminum. Ther Drug Monit. 1993 Dec;15(6):602-7. PubMed.

    Petersen RB, Siedlak SL, Lee HG, Kim YS, Nunomura A, Tagliavini F, Ghetti B, Cras P, Moreira PI, Castellani RJ, Guentchev M, Budka H, Ironside JW, Gambetti P, Smith MA, Perry G. Redox metals and oxidative abnormalities in human prion diseases. Acta Neuropathol. 2005 Sep;110(3):232-8. PubMed.

    Sayre LM, Perry G, Harris PL, Liu Y, Schubert KA, Smith MA. In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: a central role for bound transition metals. J Neurochem. 2000 Jan;74(1):270-9. PubMed.

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    Smith MA. Oxidative stress and iron imbalance in Alzheimer disease: how rust became the fuss!. J Alzheimers Dis. 2006;9(3 Suppl):305-8. PubMed.

    Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9866-8. PubMed.

    Smith MA, Kutty RK, Richey PL, Yan SD, Stern D, Chader GJ, Wiggert B, Petersen RB, Perry G. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer's disease. Am J Pathol. 1994 Jul;145(1):42-7. PubMed.

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    View all comments by Gang Liu

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References

News Citations

  1. Anti-Amyloid Drug Clears Phase 2a Hurdle

Paper Citations

  1. Mitka M. Chelation therapy trials halted. JAMA. 2008 Nov 19;300(19):2236. PubMed.
  2. Zecca L, Youdim MB, Riederer P, Connor JR, Crichton RR. Iron, brain ageing and neurodegenerative disorders. Nat Rev Neurosci. 2004 Nov;5(11):863-73. PubMed.
  3. Oba H, Araki T, Ohtomo K, Monzawa S, Uchiyama G, Koizumi K, Nogata Y, Kachi K, Shiozawa Z, Kobayashi M. Amyotrophic lateral sclerosis: T2 shortening in motor cortex at MR imaging. Radiology. 1993 Dec;189(3):843-6. PubMed.
  4. Imon Y, Yamaguchi S, Yamamura Y, Tsuji S, Kajima T, Ito K, Nakamura S. Low intensity areas observed on T2-weighted magnetic resonance imaging of the cerebral cortex in various neurological diseases. J Neurol Sci. 1995 Dec;134 Suppl:27-32. PubMed.
  5. Dupuis L, Gonzalez de Aguilar JL, Oudart H, de Tapia M, Barbeito L, Loeffler JP. Mitochondria in amyotrophic lateral sclerosis: a trigger and a target. Neurodegener Dis. 2004;1(6):245-54. PubMed.

Other Citations

  1. see Q&A, below

Further Reading

Papers

  1. Kiaei M, Bush AI, Morrison BM, Morrison JH, Cherny RA, Volitakis I, Beal MF, Gordon JW. Genetically decreased spinal cord copper concentration prolongs life in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci. 2004 Sep 8;24(36):7945-50. PubMed.
  2. Rodríguez-Rodríguez C, Sánchez de Groot N, Rimola A, Alvarez-Larena A, Lloveras V, Vidal-Gancedo J, Ventura S, Vendrell J, Sodupe M, González-Duarte P. Design, selection, and characterization of thioflavin-based intercalation compounds with metal chelating properties for application in Alzheimer's disease. J Am Chem Soc. 2009 Feb 4;131(4):1436-51. PubMed.
  3. Liang LP, Jarrett SG, Patel M. Chelation of mitochondrial iron prevents seizure-induced mitochondrial dysfunction and neuronal injury. J Neurosci. 2008 Nov 5;28(45):11550-6. PubMed.
  4. Avramovich-Tirosh Y, Amit T, Bar-Am O, Weinreb O, Youdim MB. Physiological and pathological aspects of Abeta in iron homeostasis via 5'UTR in the APP mRNA and the therapeutic use of iron-chelators. BMC Neurosci. 2008;9 Suppl 2:S2. PubMed.

Primary Papers

  1. Jeong SY, Rathore KI, Schulz K, Ponka P, Arosio P, David S. Dysregulation of iron homeostasis in the CNS contributes to disease progression in a mouse model of amyotrophic lateral sclerosis. J Neurosci. 2009 Jan 21;29(3):610-9. PubMed.

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