A Long Shot? Could neurodegenerative disease be caused by a cyanobacterial toxin?

Abstract

Is a cyanobacterial neurotoxin involved in amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease? If so, does that offer any hope of new treatments? And what have flying foxes and Wyoming got to do with it? Adrian Burton investigates.Jackson Hole in northwestern Wyoming, USA, might seem an unlikely place to continue a line of research that started on the tropical island of Guam. Yet, this is exactly where, nearly a decade ago, Paul Cox and colleagues set up the Institute for Ethnomedicine—“cyanobacteria central” in the search for evidence that a toxin produced by the latter organisms can trigger neurodegenerative disease in genetically vulnerable individuals.The Institute, the stated mission of which is to “search for new cures by studying patterns of wellness and disease among indigenous peoples”, boasts an on-site research staff of just four to nine scientists, but has a network of collaborators around the world. Its projects combine intensive laboratory studies with extensive fieldwork. For example, the Institute is helping to develop the anti-HIV drug prostratin, which Cox brought from Samoa in a potion made from mamala tree (Homalanthus nutans) bark given to him by two healers who used it to treat hepatitis. However, the Institute is perhaps best known for its work on cyanobacteria and their possible association with amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, a line of inquiry that started a long time ago, and a long way from Wyoming.US military medical staff stationed on Guam after World War 2 noticed that many of the local Chamorro people developed a rapidly progressive neurodegenerative disorder with symptoms akin to ALS plus parkinsonism dementia (Pd). No infectious or other cause was ever found. However, Cox and colleagues suggest that this disease, known as ALS–PdC (the C for “complex”), is triggered by the ingestion of β-methylamino-l-alanine (BMAA), a toxic, non-protein aminoacid produced by Nostoc cyanobacteria living in the specialised coralloid roots of the cycad tree Cycas micronesica. Based on years of research, their reasoning is as follows. First, Chamorro people make a flour out of the tree's seeds, the kernels and coverings of which contain BMAA; additionally, Chamorro people used to eat flying foxes (Pteropus mariannus), which eat cycad seeds, completing a biomagnification pathway for the toxin (since the Chamorros dropped bats from their diet, ALS-PdC has diminished). Second, in vitro, BMAA selectively kills subpopulations of NADPH-diaphorase-positive motor neurons via activation of AMPA/kainate and NMDA receptors. Third, BMAA is found in a free form or bound to proteins—this binding not only leads to protein misfolding and aggregation but also allows a reservoir of the toxin to form inside neuroproteins, from which it leaks out over the years to slowly kill motor neurons (which explains why Chamorro people who emigrated from Guam often developed ALS-PdC years later). Finally, the brains of Chamorro people with ALS-PdC were found to contain BMAA, both in the free and bound forms, whereas those of healthy control Canadians did not.The cyanobacterial hypothesis extended beyond ALS-PdC when researchers discovered that Canadian patients who died from Alzheimer's disease, and who had never eaten a flying fox, also had BMAA in their brain tissues. This finding led to some major questions. Might BMAA be produced by other cyanobacteria and be similarly magnified through the food chain in other ecosystems? Could cyanobacteria trigger ALS, Parkinson's disease, and Alzheimer's disease in particular vulnerable people elsewhere?Evidence for the possibility of widespread exposure to BMAA came from a study of 30 laboratory strains of cyanobacteria, 95% of which tested positive for BMAA. People who live near cyanobacteria-contaminated lakes have been reported to be at increased risk of ALS, as are, perhaps, those who eat contaminated shellfish from Chesapeake Bay, MD, USA. These findings, plus the confirmation by Deborah Mash and colleagues (Miami Brain Endowment Bank, FL, USA) of BMAA bound to proteins in neurons of patients with ALS, but generally not in those of healthy people, are supportive of the theory.However, although neurotoxins are generally included in the list of possible causative mechanisms responsible for neurodegenerative disease, none is known to trigger the type of progressive and selective neuronal loss seen in ALS. “MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), perhaps the best characterised neurotoxin, produces selective damage to substantia nigral dopaminergic neurons and resultant parkinsonism, yet fails to induce further progressive loss of neurons following the initial neurotoxic exposure,” explains Daniel Perl (Uniformed Services University of the Health Sciences, Bethesda, MD, USA). “[Further], Spencer et al exposed macaques to massive daily doses of BMAA and, although weakness was noted clinically, there is no evidence in this report that this was caused by the death of either upper or lower motor neurons, and subsequent in vivo experiments have shown little in the way of selective and/or progressive motor neuron damage.”Cox cites recent Swedish studies that show cognitive deficits and neuropathologies in mice given BMAA but concedes the broader point: “Proof of the BMAA theory would require a non-human primate model which, when dosed with BMAA, exhibits incipient tauopathies or other protein inclusions similar to what is observed in brain tissues from Chamorro ALS-PdC patients or sporadic ALS patients.”Douglas Galasko (Department of Neurosciences, University of California San Diego, CA, USA) is also critical of the BMAA theory. “I am unaware of conclusive evidence that cyanobacteria or BMAA cause ALS or other disorders in human beings. It is unclear why BMAA would selectively affect motor neurons, the cells primarily involved in ALS. Even if BMAA is present in the brain in low abundance, it may not have any adverse consequences.”Cox, however, refers to recent studies that show that BMAA at low concentration (10–30 μM) kills specific subpopulations of motor neurons through direct action at AMPA, NMDA, and metabotropic glutamate receptors. “Further, Doug Lobner and his students at Marquette University (Milwaukee, WI, USA) have shown that BMAA can potentiate insults from other environmental neurotoxins, including methylmercury”, he adds.Galasko also worries how protein-bound BMAA in the cell could be released slowly to cause problems over time. “Cox and collaborators used methods to prepare brain tissue that include boiling in concentrated hydrochloric acid to release protein-bound BMAA. An unanswered question is how a pool of strongly protein-bound BMAA could be released under physiological conditions and cause toxicity.” He adds, “If a substantial protein-associated pool of BMAA is proposed to be critical, it would be important to identify which proteins are involved.” Cox replies that “acid hydrolysis is the most commonly used method for releasing aminoacids from proteins—there are more than 6000 papers that use this technique—but we also get the same results at neutral pH by using proteases. However, Prof Galasko's comment on determining which proteins are involved is spot on, and we are endeavouring to do exactly that with our new Orbital Trap Mass Spectrometer.”Perl has other worries. “Most of the literature related to BMAA cyanobacteria addresses potential biological sources for exposure to BMAA while ignoring the paucity of evidence that BMAA can induce such progressive ALS-related pathology.” Cox agrees, “This is a valid criticism, and a persuasive primate model is needed.”New research might, however, support the idea that BMAA can unleash a neurodegenerative effect. Mash has recently reported that BMAA rapidly crosses the blood–brain barrier in mice where it is “captured” by neuroproteins. “In collaboration with Ken Rodgers and Rachael Dunlop at the University of Technology (Sydney, Australia), we have also found that BMAA mischarges the tRNA for serine in cell cultures, resulting in protein misfolding and aggregation,” says Cox.The BMAA theory has now gathered enough pace to be tested in the clinical sphere. “Todd Levine and David Sapperstein at Phoenix Neurological Associates [Phoenix, AZ, USA] have [FDA] approval for human clinical trials of two different candidate drugs for ALS based on the BMAA theory: a zinc-copper formulation designed to chelate BMAA from patients, and l-serine,” says Cox. l-serine has recently been shown to prevent BMAA misincorporation into proteins and subsequent protein misfolding in cell cultures.Cox's team is also developing tests for exposure to BMAA. “We began by analysing hair”, explains colleague Sandra Banack, “but we now analyse blood serum and cerebrospinal fluid for BMAA.” If the theory holds true, such tests could be important since we are all exposed to cyanobacteria by swimming in lakes and the sea, and, perhaps more importantly, via the consumption of shellfish from contaminated water. Knowledge of whom among us is genetically predisposed to neurodegenerative disease, and who has been exposed to environmental triggers, could help to reduce the burden of neurodegenerative disease.The idea that cyanobacteria could be causing human neurodegeneration might sound like something of a long shot, and until a viable primate model of BMAA-induced neurodegeneration is produced, Cox knows that the BMAA theory will have its detractors. However, he points out that “the scientific method depends on rigorous testing of new ideas. We see the BMAA theory as complementary to current genetic research, and are very open to the possibility that other environmental factors may be involved. Hopefully, through time, better understanding will emerge that will result in better patient outcomes.”Given the absence of curative treatments for neurodegenerative diseases, and increasingly common cyanobacterial blooms, that understanding cannot come quickly enough. Is a cyanobacterial neurotoxin involved in amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease? If so, does that offer any hope of new treatments? And what have flying foxes and Wyoming got to do with it? Adrian Burton investigates. Jackson Hole in northwestern Wyoming, USA, might seem an unlikely place to continue a line of research that started on the tropical island of Guam. Yet, this is exactly where, nearly a decade ago, Paul Cox and colleagues set up the Institute for Ethnomedicine—“cyanobacteria central” in the search for evidence that a toxin produced by the latter organisms can trigger neurodegenerative disease in genetically vulnerable individuals. The Institute, the stated mission of which is to “search for new cures by studying patterns of wellness and disease among indigenous peoples”, boasts an on-site research staff of just four to nine scientists, but has a network of collaborators around the world. Its projects combine intensive laboratory studies with extensive fieldwork. For example, the Institute is helping to develop the anti-HIV drug prostratin, which Cox brought from Samoa in a potion made from mamala tree (Homalanthus nutans) bark given to him by two healers who used it to treat hepatitis. However, the Institute is perhaps best known for its work on cyanobacteria and their possible association with amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, a line of inquiry that started a long time ago, and a long way from Wyoming. US military medical staff stationed on Guam after World War 2 noticed that many of the local Chamorro people developed a rapidly progressive neurodegenerative disorder with symptoms akin to ALS plus parkinsonism dementia (Pd). No infectious or other cause was ever found. However, Cox and colleagues suggest that this disease, known as ALS–PdC (the C for “complex”), is triggered by the ingestion of β-methylamino-l-alanine (BMAA), a toxic, non-protein aminoacid produced by Nostoc cyanobacteria living in the specialised coralloid roots of the cycad tree Cycas micronesica. Based on years of research, their reasoning is as follows. First, Chamorro people make a flour out of the tree's seeds, the kernels and coverings of which contain BMAA; additionally, Chamorro people used to eat flying foxes (Pteropus mariannus), which eat cycad seeds, completing a biomagnification pathway for the toxin (since the Chamorros dropped bats from their diet, ALS-PdC has diminished). Second, in vitro, BMAA selectively kills subpopulations of NADPH-diaphorase-positive motor neurons via activation of AMPA/kainate and NMDA receptors. Third, BMAA is found in a free form or bound to proteins—this binding not only leads to protein misfolding and aggregation but also allows a reservoir of the toxin to form inside neuroproteins, from which it leaks out over the years to slowly kill motor neurons (which explains why Chamorro people who emigrated from Guam often developed ALS-PdC years later). Finally, the brains of Chamorro people with ALS-PdC were found to contain BMAA, both in the free and bound forms, whereas those of healthy control Canadians did not. The cyanobacterial hypothesis extended beyond ALS-PdC when researchers discovered that Canadian patients who died from Alzheimer's disease, and who had never eaten a flying fox, also had BMAA in their brain tissues. This finding led to some major questions. Might BMAA be produced by other cyanobacteria and be similarly magnified through the food chain in other ecosystems? Could cyanobacteria trigger ALS, Parkinson's disease, and Alzheimer's disease in particular vulnerable people elsewhere? Evidence for the possibility of widespread exposure to BMAA came from a study of 30 laboratory strains of cyanobacteria, 95% of which tested positive for BMAA. People who live near cyanobacteria-contaminated lakes have been reported to be at increased risk of ALS, as are, perhaps, those who eat contaminated shellfish from Chesapeake Bay, MD, USA. These findings, plus the confirmation by Deborah Mash and colleagues (Miami Brain Endowment Bank, FL, USA) of BMAA bound to proteins in neurons of patients with ALS, but generally not in those of healthy people, are supportive of the theory. However, although neurotoxins are generally included in the list of possible causative mechanisms responsible for neurodegenerative disease, none is known to trigger the type of progressive and selective neuronal loss seen in ALS. “MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), perhaps the best characterised neurotoxin, produces selective damage to substantia nigral dopaminergic neurons and resultant parkinsonism, yet fails to induce further progressive loss of neurons following the initial neurotoxic exposure,” explains Daniel Perl (Uniformed Services University of the Health Sciences, Bethesda, MD, USA). “[Further], Spencer et al exposed macaques to massive daily doses of BMAA and, although weakness was noted clinically, there is no evidence in this report that this was caused by the death of either upper or lower motor neurons, and subsequent in vivo experiments have shown little in the way of selective and/or progressive motor neuron damage.” Cox cites recent Swedish studies that show cognitive deficits and neuropathologies in mice given BMAA but concedes the broader point: “Proof of the BMAA theory would require a non-human primate model which, when dosed with BMAA, exhibits incipient tauopathies or other protein inclusions similar to what is observed in brain tissues from Chamorro ALS-PdC patients or sporadic ALS patients.” Douglas Galasko (Department of Neurosciences, University of California San Diego, CA, USA) is also critical of the BMAA theory. “I am unaware of conclusive evidence that cyanobacteria or BMAA cause ALS or other disorders in human beings. It is unclear why BMAA would selectively affect motor neurons, the cells primarily involved in ALS. Even if BMAA is present in the brain in low abundance, it may not have any adverse consequences.” Cox, however, refers to recent studies that show that BMAA at low concentration (10–30 μM) kills specific subpopulations of motor neurons through direct action at AMPA, NMDA, and metabotropic glutamate receptors. “Further, Doug Lobner and his students at Marquette University (Milwaukee, WI, USA) have shown that BMAA can potentiate insults from other environmental neurotoxins, including methylmercury”, he adds. Galasko also worries how protein-bound BMAA in the cell could be released slowly to cause problems over time. “Cox and collaborators used methods to prepare brain tissue that include boiling in concentrated hydrochloric acid to release protein-bound BMAA. An unanswered question is how a pool of strongly protein-bound BMAA could be released under physiological conditions and cause toxicity.” He adds, “If a substantial protein-associated pool of BMAA is proposed to be critical, it would be important to identify which proteins are involved.” Cox replies that “acid hydrolysis is the most commonly used method for releasing aminoacids from proteins—there are more than 6000 papers that use this technique—but we also get the same results at neutral pH by using proteases. However, Prof Galasko's comment on determining which proteins are involved is spot on, and we are endeavouring to do exactly that with our new Orbital Trap Mass Spectrometer.” Perl has other worries. “Most of the literature related to BMAA cyanobacteria addresses potential biological sources for exposure to BMAA while ignoring the paucity of evidence that BMAA can induce such progressive ALS-related pathology.” Cox agrees, “This is a valid criticism, and a persuasive primate model is needed.” New research might, however, support the idea that BMAA can unleash a neurodegenerative effect. Mash has recently reported that BMAA rapidly crosses the blood–brain barrier in mice where it is “captured” by neuroproteins. “In collaboration with Ken Rodgers and Rachael Dunlop at the University of Technology (Sydney, Australia), we have also found that BMAA mischarges the tRNA for serine in cell cultures, resulting in protein misfolding and aggregation,” says Cox. The BMAA theory has now gathered enough pace to be tested in the clinical sphere. “Todd Levine and David Sapperstein at Phoenix Neurological Associates [Phoenix, AZ, USA] have [FDA] approval for human clinical trials of two different candidate drugs for ALS based on the BMAA theory: a zinc-copper formulation designed to chelate BMAA from patients, and l-serine,” says Cox. l-serine has recently been shown to prevent BMAA misincorporation into proteins and subsequent protein misfolding in cell cultures. Cox's team is also developing tests for exposure to BMAA. “We began by analysing hair”, explains colleague Sandra Banack, “but we now analyse blood serum and cerebrospinal fluid for BMAA.” If the theory holds true, such tests could be important since we are all exposed to cyanobacteria by swimming in lakes and the sea, and, perhaps more importantly, via the consumption of shellfish from contaminated water. Knowledge of whom among us is genetically predisposed to neurodegenerative disease, and who has been exposed to environmental triggers, could help to reduce the burden of neurodegenerative disease. The idea that cyanobacteria could be causing human neurodegeneration might sound like something of a long shot, and until a viable primate model of BMAA-induced neurodegeneration is produced, Cox knows that the BMAA theory will have its detractors. However, he points out that “the scientific method depends on rigorous testing of new ideas. We see the BMAA theory as complementary to current genetic research, and are very open to the possibility that other environmental factors may be involved. Hopefully, through time, better understanding will emerge that will result in better patient outcomes.” Given the absence of curative treatments for neurodegenerative diseases, and increasingly common cyanobacterial blooms, that understanding cannot come quickly enough.