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Beta-methylamino-alanine (BMAA) injures hippocampal neurons in vivo
NeuroToxicology 28 (2007) 702–704
Brief communication
Beta-methylamino-alanine (BMAA) injures hippocampal neurons in vivo Eric J. Buenz a, Charles L. Howe b,c,d,* b
a BioSciential, LLC, Rochester, MN, USA Translational Immunovirology and Biodefense Program, Mayo Clinic College of Medicine, Rochester, MN, USA c Department of Neurology, Mayo Clinic College of Medicine, Rochester, MN, USA d Department of Neuroscience, Mayo Clinic College of Medicine, Rochester, MN, USA
Received 27 October 2006; accepted 12 February 2007 Available online 24 February 2007
Abstract The unusually high incidence of amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC) among the Chamorro people of Guam has fueled an intense search for the etiologic agent responsible for this neurodegenerative disease. Recently, a biomagnification hypothesis was proposed to account for the role of dietary consumption of b-methylamino-alanine (BMAA) in patients with ALS/PDC. However, this hypothesis is hotly debated and a direct association between BMAA and neuronal injury in vivo has been lacking. We provide evidence that introduction of BMAA into the CNS of mice leads to sporadic death of hippocampal neurons, supporting a direct causal link between BMAA and neuronal injury. # 2007 Elsevier Inc. All rights reserved. Keywords: b-Methylamino-alanine; Neurodegeneration; Chamorro people; Amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC); Biomagnification
1. Introduction The unusually high incidence of amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC) among the Chamorro people of Guam has fueled an intense search for an etiologic agent. Recently it was proposed that individuals receive a dose of b-methylamino-alanine (BMAA) sufficient to induce ALS/PDC after consuming flying foxes that had accumulated this neurotoxin through eating cycad seeds containing cyanobacteria (Miller, 2006). While this biomagnification hypothesis is intriguing, the research methodology used to arrive at this conclusion has been questioned (Duncan and Marini, 2006). Thus, the relationship between BMAA and neurologic injury, and a model by which this injury can occur, has yet to be firmly established. Elevated levels of BMAA have been reported in the brain tissue of individuals with ALS/PDC or Alzheimer disease (AD)
* Corresponding author at: Department of Neurology, Mayo Clinic College of Medicine, Guggenheim 442-C, 200 First St. SW, Rochester, MN 55905, USA. Tel.: +1 507 538 4603; fax: +1 507 284 1086. E-mail address: [email protected] (C.L. Howe). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.02.010
(Cox et al., 2003) and monkeys fed large doses of synthetic BMAA (Duncan and Marini, 2006) develop symptoms similar to ALS/PDC (Spencer et al., 1987). In contrast, mice fed more physiologically appropriate doses of BMAA do not develop neurologic deficits (Cruz-Aguado et al., 2006), and a recent study found no evidence of free BMAA in individuals diagnosed with ALS/PDC or AD (Montine et al., 2005). Such conflicting reports highlight the lack of a direct causal link between BMAA and the pathognomonic features of ALS/PDC and AD. Here we show that introduction of BMAA into the brain results in sporadic neuronal injury to the CA1 region of the hippocampus, an area that is exquisitely sensitive to excitotoxic damage.
2. Methods Male C57BL/6J mice (Jackson Laboratories) between 12 and 14 weeks of age were injected intracranially into the striatum with 10 mL of 100 mM BMAA (Sigma; St. Louis, MO) prepared in PBS. This dose of BMAA was selected based on a previous report of BMAA-induced damage (Santiago et al., 2006). Sham-treated animals were intracranially injected
E.J. Buenz, C.L. Howe / NeuroToxicology 28 (2007) 702–704
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with 10 mL PBS. Handling and testing of all animals conformed to the National Institutes of Health and Mayo Clinic institutional guidelines and was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mice were euthanized 24 h after injection with a lethal dose of pentobarbital and perfused via intracardiac puncture with 50 mL of 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for 24 h in preparation for morphologic analysis. Following postfixation, cuts through the optic chiasm and the infundibulum were made using the ‘‘Atlas of the Mouse Brain and Spinal Cord’’ (sections 220 and 350) as a guide (Buenz et al., 2006; Sidman et al., 1971). The three blocks resulting from this dissection were embedded together in paraffin, and 5 mm-thick sections were cut on a microtome and then mounted on charged slides and stained with hematoxylin and eosin. NSC-34 cells, a spinal motor neuron-like cell line (kindly provided by Neil Cashman, Vancouver Coastal Health Research Institute) were cultured in DMEM (Cellgro, Herndon, VA) containing 10% heat-inactivated fetal calf serum (Cellgro) and 1% penicillin/streptomycin (Cellgro) at 37 8C in 5% CO2. NSC-34 cells were treated with increasing doses of BMAA (50 mM, 100 mM, 500 mM, and 1000 mM) for 18 h. Cell death was assessed by propidium iodide exclusion (Buenz, 2007). 3. Results Vehicle-treated mice (n = 6) had an intact CA1 pyramidal cell layer with healthy pyramidal neurons (Fig. 1A) and abundant apical processes. In contrast, BMAA-treated animals (n = 7) exhibited a wide range of damage to the CA1 region of the hippocampus. While some animals experienced intermittent pyknotic neurons in the pyramidal layer (Fig. 1B), other mice exhibited profound neuronal death (Fig. 1C) and complete regression of the apical processes. It is important to note that analysis of the injection sites showed that the BMAA was not delivered directly to the hippocampus in any of the mice analyzed (data not shown). This observation suggests a systemic level of activity rather than localized cytotoxicity at the site of injection. These findings critically demonstrate the neurotoxic potential of BMAA in vivo. We also examined the effects of BMAA on neurons (NSC34 cells) in vitro to assess direct toxicity. Cells treated with 50 mM BMAA were similar to vehicle treated (P = 0.671; ttest). However, at higher levels we observed a dosedependent induction of NSC-34 neuronal death (Fig. 1D; r2 = 0.846). These results suggest that NSC-34 cells are an appropriate model to examine BMAA-induced neuronal death in vitro.
Fig. 1. Treatment with BMAA injures hippocampal neurons. (A) Vehicletreated mice exhibit normal hippocampal morphology, including readily identifiable apical processes (white streaks below the pyramidal neurons, indicated by the arrow). A healthy neuron in the pyramidal layer of the hippocampus is
indicated by the green arrowhead in panel A. (B and C) Intracranial injection with BMAA results in hippocampal injury ranging from intermittent loss of neurons in the pyramidal layer (B) to the death of essentially all pyramidal neurons and complete regression of the apical processes (C). (D) Treatment of neurons in vitro with BMAA results in dose-dependent death as assessed by a flow cytometric dye exclusion assay. Scale bar equals 20 mm.
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E.J. Buenz, C.L. Howe / NeuroToxicology 28 (2007) 702–704
4. Discussion The finding that BMAA directly induces neuronal death in vivo and in vitro supports the theory that this amino acid may serve as the etiologic agent for ALS/PDC syndrome in the Chamorro people. It is important to note, however, that we did not observe overt cell death in any other neuronal population in the brain, including the cerebellum, brainstem, substantia nigra, or cortex (data not shown). Additionally, we did not see complete destruction of the hippocampal structure, only limited and sporadic injury to CA1 pyramidal neurons. These results may explain BMAA-induced stress markers previously reported in hippocampal neurons (Andersson et al., 1997). Potentially, these hippocampal neurons are a ‘‘canary in a coalmine’’, serving as the most sensitive neuronal population, and perhaps longer incubation times would reveal more robust hippocampal injury and the development of injury in other neuronal populations more relevant to ALS/PDC syndrome. We suggest that our observations provide support for an important, and so far overlooked, model to explain how BMAA, an agent that is not highly neurotoxic (Duncan and Marini, 2006), could ultimately cause ALS/PDC—as with other disorders (Buenz et al., 2006), small repetitive injury events that incite limited and covert neuronal death may accumulate over time and eventually manifest as an overt disease state. Alternately, it may be that BMAA predisposes cells to other types of damage (Lobner et al., 2006), such as that elicited by viral infection (Buenz and Howe, 2006; Buenz et al., 2006). Thus, the biomagnification hypothesis may not only require amplification of the toxin via an intermediate food species, but may also require amplification through time by persistent and repeated dosing that results in accumulation of neuronal injury or it may require a secondary insult to induce neuronal death. If this model is correct, we would predict that a range of neuronal injury would be present in the Chamorro population and that the extent of injury would correlate with age and relative exposure to toxin consumption. Whether higher doses of BMAA or longer incubation times in our experiments would lead to greater neuronal death more closely associated with ALS/PDC and AD remains to be determined. However, our data show that direct introduction of BMAA into the brain does kill neurons, albeit not very
efficiently and in a very limited manner. This observation emphasizes the need for further work to understand the injurious capacity of neurotoxic amino acids, even ones with limited acute potency. Acknowledgements We thank Dr. Emanuel Strehler for critically reading the manuscript. This work was supported by grant RG3636 (CLH) from the National MS Society and by Donald and Frances Herdrich (CLH). We would also like to thank Ruth Stricker and Bruce Dayton for their continued guidance and support. References Andersson H, Lindqvist E, Olson L. Plant-derived amino acids increase hippocampal BDNF, NGF, c-fos and hsp70 mRNAs. Neuroreport 1997;8:1813–7. Buenz EJ. Mitochondrial involvement in Atuna racemosa induced toxicity. J Ethnopharmacol 2007;109:304–11. Buenz EJ, Howe CL. Picornaviruses and cell death. Trends Microbiol 2006;14:28–36. Buenz EJ, Rodriguez M, Howe CL. Disrupted spatial memory is a consequence of picornavirus infection. Neurobiol Dis 2006;24:266–73. Cox PA, Banack SA, Murch SJ. Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci USA 2003;100:13380–3. Cruz-Aguado R, Winkler D, Shaw CA. Lack of behavioral and neuropathological effects of dietary beta-methylamino-l-alanine (BMAA) in mice. Pharmacol Biochem Behav 2006;84:294–9. Duncan MW, Marini AM. Debating the cause of a neurological disorder. Science 2006;313:1737. Lobner D, Piana PM, Salous AK, Peoples RW. Beta-N-methylamino-l-alanine enhances neurotoxicity through multiple mechanisms. Neurobiol Dis 2006. Miller G. Neurodegenerative disease. Guam’s deadly stalker: on the loose worldwide? Science 2006;313:428–31. Montine TJ, Li K, Perl DP, Galasko D. Lack of beta-methylamino-l-alanine in brain from controls, AD, or Chamorros with PDC. Neurology 2005;65:768– 9. Santiago M, Matarredona ER, Machado A, Cano J. Acute perfusion of BMAA in the rat’s striatum by in vivo microdialysis. Toxicol Lett 2006;167:34–9. Sidman RL, Angevine JB, Pierce ET. Atlas of the mouse brain and spinal cord. Cambridge, MA: Harvard University Press; 1971. Spencer PS, Nunn PB, Hugon J, Ludolph AC, Ross SM, Roy DN, et al. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science 1987;237:517–22.
Brief communication
Beta-methylamino-alanine (BMAA) injures hippocampal neurons in vivo Eric J. Buenz a, Charles L. Howe b,c,d,* b
a BioSciential, LLC, Rochester, MN, USA Translational Immunovirology and Biodefense Program, Mayo Clinic College of Medicine, Rochester, MN, USA c Department of Neurology, Mayo Clinic College of Medicine, Rochester, MN, USA d Department of Neuroscience, Mayo Clinic College of Medicine, Rochester, MN, USA
Received 27 October 2006; accepted 12 February 2007 Available online 24 February 2007
Abstract The unusually high incidence of amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC) among the Chamorro people of Guam has fueled an intense search for the etiologic agent responsible for this neurodegenerative disease. Recently, a biomagnification hypothesis was proposed to account for the role of dietary consumption of b-methylamino-alanine (BMAA) in patients with ALS/PDC. However, this hypothesis is hotly debated and a direct association between BMAA and neuronal injury in vivo has been lacking. We provide evidence that introduction of BMAA into the CNS of mice leads to sporadic death of hippocampal neurons, supporting a direct causal link between BMAA and neuronal injury. # 2007 Elsevier Inc. All rights reserved. Keywords: b-Methylamino-alanine; Neurodegeneration; Chamorro people; Amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC); Biomagnification
1. Introduction The unusually high incidence of amyotrophic lateral sclerosis/Parkinson–dementia complex (ALS/PDC) among the Chamorro people of Guam has fueled an intense search for an etiologic agent. Recently it was proposed that individuals receive a dose of b-methylamino-alanine (BMAA) sufficient to induce ALS/PDC after consuming flying foxes that had accumulated this neurotoxin through eating cycad seeds containing cyanobacteria (Miller, 2006). While this biomagnification hypothesis is intriguing, the research methodology used to arrive at this conclusion has been questioned (Duncan and Marini, 2006). Thus, the relationship between BMAA and neurologic injury, and a model by which this injury can occur, has yet to be firmly established. Elevated levels of BMAA have been reported in the brain tissue of individuals with ALS/PDC or Alzheimer disease (AD)
* Corresponding author at: Department of Neurology, Mayo Clinic College of Medicine, Guggenheim 442-C, 200 First St. SW, Rochester, MN 55905, USA. Tel.: +1 507 538 4603; fax: +1 507 284 1086. E-mail address: [email protected] (C.L. Howe). 0161-813X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2007.02.010
(Cox et al., 2003) and monkeys fed large doses of synthetic BMAA (Duncan and Marini, 2006) develop symptoms similar to ALS/PDC (Spencer et al., 1987). In contrast, mice fed more physiologically appropriate doses of BMAA do not develop neurologic deficits (Cruz-Aguado et al., 2006), and a recent study found no evidence of free BMAA in individuals diagnosed with ALS/PDC or AD (Montine et al., 2005). Such conflicting reports highlight the lack of a direct causal link between BMAA and the pathognomonic features of ALS/PDC and AD. Here we show that introduction of BMAA into the brain results in sporadic neuronal injury to the CA1 region of the hippocampus, an area that is exquisitely sensitive to excitotoxic damage.
2. Methods Male C57BL/6J mice (Jackson Laboratories) between 12 and 14 weeks of age were injected intracranially into the striatum with 10 mL of 100 mM BMAA (Sigma; St. Louis, MO) prepared in PBS. This dose of BMAA was selected based on a previous report of BMAA-induced damage (Santiago et al., 2006). Sham-treated animals were intracranially injected
E.J. Buenz, C.L. Howe / NeuroToxicology 28 (2007) 702–704
703
with 10 mL PBS. Handling and testing of all animals conformed to the National Institutes of Health and Mayo Clinic institutional guidelines and was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mice were euthanized 24 h after injection with a lethal dose of pentobarbital and perfused via intracardiac puncture with 50 mL of 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for 24 h in preparation for morphologic analysis. Following postfixation, cuts through the optic chiasm and the infundibulum were made using the ‘‘Atlas of the Mouse Brain and Spinal Cord’’ (sections 220 and 350) as a guide (Buenz et al., 2006; Sidman et al., 1971). The three blocks resulting from this dissection were embedded together in paraffin, and 5 mm-thick sections were cut on a microtome and then mounted on charged slides and stained with hematoxylin and eosin. NSC-34 cells, a spinal motor neuron-like cell line (kindly provided by Neil Cashman, Vancouver Coastal Health Research Institute) were cultured in DMEM (Cellgro, Herndon, VA) containing 10% heat-inactivated fetal calf serum (Cellgro) and 1% penicillin/streptomycin (Cellgro) at 37 8C in 5% CO2. NSC-34 cells were treated with increasing doses of BMAA (50 mM, 100 mM, 500 mM, and 1000 mM) for 18 h. Cell death was assessed by propidium iodide exclusion (Buenz, 2007). 3. Results Vehicle-treated mice (n = 6) had an intact CA1 pyramidal cell layer with healthy pyramidal neurons (Fig. 1A) and abundant apical processes. In contrast, BMAA-treated animals (n = 7) exhibited a wide range of damage to the CA1 region of the hippocampus. While some animals experienced intermittent pyknotic neurons in the pyramidal layer (Fig. 1B), other mice exhibited profound neuronal death (Fig. 1C) and complete regression of the apical processes. It is important to note that analysis of the injection sites showed that the BMAA was not delivered directly to the hippocampus in any of the mice analyzed (data not shown). This observation suggests a systemic level of activity rather than localized cytotoxicity at the site of injection. These findings critically demonstrate the neurotoxic potential of BMAA in vivo. We also examined the effects of BMAA on neurons (NSC34 cells) in vitro to assess direct toxicity. Cells treated with 50 mM BMAA were similar to vehicle treated (P = 0.671; ttest). However, at higher levels we observed a dosedependent induction of NSC-34 neuronal death (Fig. 1D; r2 = 0.846). These results suggest that NSC-34 cells are an appropriate model to examine BMAA-induced neuronal death in vitro.
Fig. 1. Treatment with BMAA injures hippocampal neurons. (A) Vehicletreated mice exhibit normal hippocampal morphology, including readily identifiable apical processes (white streaks below the pyramidal neurons, indicated by the arrow). A healthy neuron in the pyramidal layer of the hippocampus is
indicated by the green arrowhead in panel A. (B and C) Intracranial injection with BMAA results in hippocampal injury ranging from intermittent loss of neurons in the pyramidal layer (B) to the death of essentially all pyramidal neurons and complete regression of the apical processes (C). (D) Treatment of neurons in vitro with BMAA results in dose-dependent death as assessed by a flow cytometric dye exclusion assay. Scale bar equals 20 mm.
704
E.J. Buenz, C.L. Howe / NeuroToxicology 28 (2007) 702–704
4. Discussion The finding that BMAA directly induces neuronal death in vivo and in vitro supports the theory that this amino acid may serve as the etiologic agent for ALS/PDC syndrome in the Chamorro people. It is important to note, however, that we did not observe overt cell death in any other neuronal population in the brain, including the cerebellum, brainstem, substantia nigra, or cortex (data not shown). Additionally, we did not see complete destruction of the hippocampal structure, only limited and sporadic injury to CA1 pyramidal neurons. These results may explain BMAA-induced stress markers previously reported in hippocampal neurons (Andersson et al., 1997). Potentially, these hippocampal neurons are a ‘‘canary in a coalmine’’, serving as the most sensitive neuronal population, and perhaps longer incubation times would reveal more robust hippocampal injury and the development of injury in other neuronal populations more relevant to ALS/PDC syndrome. We suggest that our observations provide support for an important, and so far overlooked, model to explain how BMAA, an agent that is not highly neurotoxic (Duncan and Marini, 2006), could ultimately cause ALS/PDC—as with other disorders (Buenz et al., 2006), small repetitive injury events that incite limited and covert neuronal death may accumulate over time and eventually manifest as an overt disease state. Alternately, it may be that BMAA predisposes cells to other types of damage (Lobner et al., 2006), such as that elicited by viral infection (Buenz and Howe, 2006; Buenz et al., 2006). Thus, the biomagnification hypothesis may not only require amplification of the toxin via an intermediate food species, but may also require amplification through time by persistent and repeated dosing that results in accumulation of neuronal injury or it may require a secondary insult to induce neuronal death. If this model is correct, we would predict that a range of neuronal injury would be present in the Chamorro population and that the extent of injury would correlate with age and relative exposure to toxin consumption. Whether higher doses of BMAA or longer incubation times in our experiments would lead to greater neuronal death more closely associated with ALS/PDC and AD remains to be determined. However, our data show that direct introduction of BMAA into the brain does kill neurons, albeit not very
efficiently and in a very limited manner. This observation emphasizes the need for further work to understand the injurious capacity of neurotoxic amino acids, even ones with limited acute potency. Acknowledgements We thank Dr. Emanuel Strehler for critically reading the manuscript. This work was supported by grant RG3636 (CLH) from the National MS Society and by Donald and Frances Herdrich (CLH). We would also like to thank Ruth Stricker and Bruce Dayton for their continued guidance and support. References Andersson H, Lindqvist E, Olson L. Plant-derived amino acids increase hippocampal BDNF, NGF, c-fos and hsp70 mRNAs. Neuroreport 1997;8:1813–7. Buenz EJ. Mitochondrial involvement in Atuna racemosa induced toxicity. J Ethnopharmacol 2007;109:304–11. Buenz EJ, Howe CL. Picornaviruses and cell death. Trends Microbiol 2006;14:28–36. Buenz EJ, Rodriguez M, Howe CL. Disrupted spatial memory is a consequence of picornavirus infection. Neurobiol Dis 2006;24:266–73. Cox PA, Banack SA, Murch SJ. Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci USA 2003;100:13380–3. Cruz-Aguado R, Winkler D, Shaw CA. Lack of behavioral and neuropathological effects of dietary beta-methylamino-l-alanine (BMAA) in mice. Pharmacol Biochem Behav 2006;84:294–9. Duncan MW, Marini AM. Debating the cause of a neurological disorder. Science 2006;313:1737. Lobner D, Piana PM, Salous AK, Peoples RW. Beta-N-methylamino-l-alanine enhances neurotoxicity through multiple mechanisms. Neurobiol Dis 2006. Miller G. Neurodegenerative disease. Guam’s deadly stalker: on the loose worldwide? Science 2006;313:428–31. Montine TJ, Li K, Perl DP, Galasko D. Lack of beta-methylamino-l-alanine in brain from controls, AD, or Chamorros with PDC. Neurology 2005;65:768– 9. Santiago M, Matarredona ER, Machado A, Cano J. Acute perfusion of BMAA in the rat’s striatum by in vivo microdialysis. Toxicol Lett 2006;167:34–9. Sidman RL, Angevine JB, Pierce ET. Atlas of the mouse brain and spinal cord. Cambridge, MA: Harvard University Press; 1971. Spencer PS, Nunn PB, Hugon J, Ludolph AC, Ross SM, Roy DN, et al. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science 1987;237:517–22.