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Do vervets and macaques respond differently to BMAA?
G Model NEUTOX 1989 No. of Pages 2
NeuroToxicology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
NeuroToxicology
Do vervets and macaques respond differently to BMAA? Paul Alan Coxa,* , David A. Davisb , Deborah C. Mashb , James S. Metcalfa , Sandra Anne Banacka a b
Brain Chemistry Labs, Institute for Ethnomedicine, Jackson Hole, WY, USA Department of Neurology, University of Miami, Miller School of Medicine, Miami, FL, USA
A R T I C L E I N F O
Article history: Received 21 April 2016 Received in revised form 26 April 2016 Accepted 26 April 2016 Available online xxx Keywords: BMAA Vervets Macaques Neurofibrillary tangles Beta-amyloid Thioflavin Hyperphosphorylation Neuropathology
A B S T R A C T
Vervets with chronic dietary exposure to BMAA develop neurofibrillary tangles (NFT) and sparse b-amyloid plaque-like deposits in the brain. Macaques dosed via oral gavage with BMAA developed marked neurological signs in the absence of cell death. These differences may result from increased vulnerability of macaques to BMAA, the higher effective dose they received via oral gavage, and the possibility of stable adducts due to the bicarbonate used to neutralize their BMAA dose. Confirmation of chromatolysis and cell death in macaque brains was visualized using toluidine staining. In contrast, immunological staining with AT8 and b-amyloid (1–42) antibodies and thioflavine-S stain in vervet brains suggests early stage labeling of neurites and NFT and plaque-like formation in the absence of neuronal loss. The lack of neurologic deficits reported in vervets is in keeping with early preclinical pathology observed with these immunohistochemical methods. BMAA toxicity in vervet brains causes the early events that occur in the genesis of neurofibrillary pathology. Taken together, these different studies of vervets and macaques demonstrate BMAA toxicity in the brain due to chronic exposures. The use of more sensitive immunohistochemical methods in the vervet study most likely explains the differences in neuropathology reported for vervets and macaques. ã 2016 Elsevier B.V. All rights reserved.
In a pioneering study published nearly 30 years ago, Spencer et al. reported that exposure to BMAA produced neurological signs in macaques (Macaca fascicularis) (Spencer et al., 1987). In response to our recent report that chronic dietary exposure to the cyanobacterial toxin, L-BMAA, triggers the formation of neurofibrillary tangles (NFT) and b-amyloid deposits in the brains of vervets (Chlorocebus sabaeus) (Cox et al., 2016), Spencer et al. (2016) raise several questions about differences between their macaque study and what we reported for vervets. They noted “prominent signs of motor system disease” (Spencer et al., 2016) in macaques and question whether the absence of such clinical signs in our vervets is due to species differences in metabolism or in the purity of the L-BMAA fed to the vervets. They also raised the issue that while we reported “widespread neurofibrillary degeneration and amyloid plaques in L-BMAA-treated vervets, this was not apparent in comparably treated cynomolgus monkeys” (Spencer et al., 2016). They further suggest that we reported that L-serine protects vervets “from the full tauopathic
* Corresponding author. E-mail address: [email protected] (P.A. Cox).
effects of L-BMAA because this essential amino acid competitively blocked the misincorporation of the latter into brain protein” (Spencer et al., 2016). In our recent study (Cox et al., 2016), vervets were fed daily for 140 days a quarter of a banana with a powdered test substance placed inside. Cohorts of eight vervets were given a banana dosed with the dry powder of L-BMAA hydrochloride salt, at either 21 mg/kg/day (low dose cohort) or 210 mg/kg/day (high dose cohorts) while a third cohort of eight received 210 mg/kg/day LBMAA plus 210 mg/kg/day L-serine. A control cohort of eight vervets received 210 mg/kg/day dry rice flour. The L-BMAA hydrochloride salt had 99.08% purity determined by 1H NMR, 13 C NMR, ES-API MS, HESI UPLC–MS/MS, and HPLC. The analysis confirmed that the L-BMAA hydrochloride salt conformed to structure with 154.6 MW and L-optical rotation. Our high dose of 210 mg/kg/day of powdered L-BMAA hydrochloride salt is equivalent to an effective daily dose of 160 mg/kg L-BMAA. The macaques studied by Spencer et al. (1987) received oral gavage doses ranging between 100 and 350 mg/kg/day L-BMAA; thus the highest dose administered by gavage to the macaques was over twice the highest dose we fed to vervets.
http://dx.doi.org/10.1016/j.neuro.2016.04.017 0161-813X/ã 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: P.A. Cox, et al., Do vervets and macaques respond differently to BMAA?, Neurotoxicology (2016), http://dx.doi. org/10.1016/j.neuro.2016.04.017
G Model NEUTOX 1989 No. of Pages 2
2
P.A. Cox et al. / NeuroToxicology xxx (2015) xxx–xxx
The original Spencer et al. (1987) study examined macaque brains for gross chromatolysis visualized in motor neurons with toluidine blue (e.g. necrotic neurons) using standard histological methods available at that time. These studies were not designed to label particular proteins implicated in the disease process using a panel of well-characterized antibodies nor did they use any other method to confirm pathology or loss of neurons in other brain regions studied in our report (Cox et al., 2016). The chromatolysis of neurons is time dependent and usually timed in a way that captures the maximal insult of a toxin exposure. Toluidine blue would not show NFTs or amyloid plaques. Thus, the anatomical approaches are not comparable between the two studies. We used antibodies to label neurofibrillary tangles (NFT) and b-amyloid plaques using three approaches (e.g., tau AT8 and b-amyloid (1–42) with thioflavine-S for confirmation). Thioflavins are dyes used for histology staining and biophysical studies of protein aggregation. We also examined the cytoarchitecture of the vervet brain using a thionin Nissl stain. This analysis revealed preclinical stage NFTs and neurites and plaque-like structures in the absence of neuronal loss. Thus, the anatomical analysis done by Spencer et al. (1987) on macaques is not comparable to the comprehensive immunologic studies we reported for L-BMAA fed vervets (Cox et al., 2016). Spencer subsequently reported that BMAA-fed macaques lack “certain features of the human disease, notably nigral degeneration and plentiful paired helical filaments” (Spencer, 1987, p. 353), which contrasts with the data presented in our vervet study where dense NFT were found in the superficial layers of cortical brain regions consistent with preclinical stages of the Guam disease (Cox et al., 2016). They also stated that “macaques receiving BMAA did not develop a progressive disease, and the most affected animals displayed no evidence of neuronal loss, marked tractal degeneration, or neuromuscular degeneration” (Spencer, 1990, p. 32). These reported differences between macaques and vervets may reflect differential sensitivity of macaques compared to vervets on upper and lower motor neurons or differences in absorption of L-BMAA due to preparation of powdered (vervet protocol) versus oral gavage (macaque dose of liquid solution prepared with sodium bicarbonate). Spencer et al. (1987) used sodium bicarbonate to neutralize their liquid solution prior to oral gavage. Weiss and Choi (1988) found that addition of sodium bicarbonate potentiates the toxicity of L-BMAA in vitro and Nunn and O’Brien (1989) discovered that BMAA forms a stable adduct with bicarbonate. There was no information provided from the Spencer et al. study on blood, brain or CSF concentrations (Spencer et al., 1987). The bicarbonate adduct of L-BMAA has higher affinity for glutamate receptors which may explain the marked excitotoxic effects seen rapidly in macaques using the electrophysiologic and pharmacologic approaches originally described in their report (Spencer et al., 1987). We found that vervets co-administered L-BMAA and L-serine in their diet had a reduced density of NFT. However, we did not claim in this report any specific mechanism for this neuroprotective action (Cox et al., 2016). L-BMAA is toxic to specific subpopulations
of motor neurons through activation of AMPA and NMDA receptors (Rao et al., 2006; Lobner et al., 2007), and causes protein misfolding and aggregation through misincorporation in neural proteins for Lserine (Dunlop et al., 2013; Glover et al., 2014). Arif et al. (2014) hypothesized that binding of BMAA to mGLUr5 results in hyperphosphorylation of tau through deactivation of the phosphatase PP2A, in keeping with our observations that L-BMAA triggers tauopathy. Finally, we agree with Spencer et al. that “neurotoxic substances can trigger neurodegenerative diseases that are often considered to be exclusively of genetic origin” (Spencer et al., 2016). Three decades later, our primate study provides supportive neuropathological evidence of preclinical disease in vervets fed L-BMAA (Cox et al., 2016), that extends and confirms the seminal contribution of L-BMAA toxicity reported by Spencer and coworkers in macaques (Spencer et al., 1987). Conflict of interest The Institute for Ethnomedicine has applied for patents for the use of L-serine to treat neurodegenerative illness (US 13/683,821), and for screening potential drug candidates using BMAA-induced neurodegeneration (US 14/229,624). References Arif, M., Kazim, S.F., Grundke-Iqbal, I., Garruto, R.M., Iqbal, K., 2014. Tau pathology involves protein phosphatase 2A in Parkinsonism-dementia of Guam. Proc. Natl. Acad. Sci. U. S. A. 111, 1144–1149. doi:http://dx.doi.org/10.1073/pnas.1322614111. Cox, P.A., Davis, D.A., Mash, D.C., Metcalf, J.S., Banack, S.A., 2016. Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain. Proc. R. Soc. B 283, 20152397. doi:http://dx.doi.org/10.1098/ rspb.2015.2397. Dunlop, R.A., Cox, P.A., Banack, S.A., Rodgers, K.J., 2013. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation. PLoS One 8, e75376. doi:http://dx.doi.org/ 10.1371/journal.pone.0075376. Glover, W.B., Mash, D.C., Murch, S.J., 2014. The natural non-protein amino acid Nbeta-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis. Amino Acids 46, 2553–2559. Lobner, D., Piana, P.M.T., Salous, A.K., Peoples, R.W., 2007. b-N-methylamino-Lalanine enhances neurotoxicity through multiple mechanisms. Neurobiol. Dis. 25 (2), 360–366. Nunn, P.B., O’Brien, P., 1989. The interaction of b-N-methylamino-L-alanine with bicarbonate: an 1H-NMR study. FEBS Lett. 251 (1–2), 31–35. Rao, S.D., Banack, S.A., Cox, P.A., Weiss, J.H., 2006. BMAA selectively injures motor neurons via AMPA/kainate receptor activation. Exp. Neurol. 201, 244–252. doi: http://dx.doi.org/10.1016/j.expneurol.2006.04.017. Spencer, P.S., Nunn, P.B., Hugon, J., Ludolph, A.C., Ross, S.M., Roy, D.N., Robertson, R. C., 1987. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science 237 (July (4814)), 517–522. Spencer, P.S., Garner, C.E., Palmer, V.S., Kisby, G.E., 2016. Vervets and macaques: similarities and differences in their responses to L-BMAA. Neurotoxicology doi: http://dx.doi.org/10.1016/j.neuro.2016.03.018. Spencer, P.S., 1987. Guam ALS/Parkinsonism-dementia: a long-latency neurotoxic disorder caused by slow toxin(s) in food? Can. J. Neurol. Sci. 14 (August Suppl. (3)), 347–357. Spencer, P.S., 1990. Linking cycad to the etiology of Western Pacific amyotrophic lateral sclerosis. Chapter 6 IN ALS. In: Rose, F. Clifford, Norris, Forbes H. (Eds.), New Advances in Toxicology and Epidemiology. Smith-Gordon. Weiss, J.H., Choi, D.W., 1988. Beta-N-methylamino-L-alanine neurotoxicity: requirement for bicarbonate as a cofactor. Science 241, 973–975.
Please cite this article in press as: P.A. Cox, et al., Do vervets and macaques respond differently to BMAA?, Neurotoxicology (2016), http://dx.doi. org/10.1016/j.neuro.2016.04.017
NeuroToxicology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
NeuroToxicology
Do vervets and macaques respond differently to BMAA? Paul Alan Coxa,* , David A. Davisb , Deborah C. Mashb , James S. Metcalfa , Sandra Anne Banacka a b
Brain Chemistry Labs, Institute for Ethnomedicine, Jackson Hole, WY, USA Department of Neurology, University of Miami, Miller School of Medicine, Miami, FL, USA
A R T I C L E I N F O
Article history: Received 21 April 2016 Received in revised form 26 April 2016 Accepted 26 April 2016 Available online xxx Keywords: BMAA Vervets Macaques Neurofibrillary tangles Beta-amyloid Thioflavin Hyperphosphorylation Neuropathology
A B S T R A C T
Vervets with chronic dietary exposure to BMAA develop neurofibrillary tangles (NFT) and sparse b-amyloid plaque-like deposits in the brain. Macaques dosed via oral gavage with BMAA developed marked neurological signs in the absence of cell death. These differences may result from increased vulnerability of macaques to BMAA, the higher effective dose they received via oral gavage, and the possibility of stable adducts due to the bicarbonate used to neutralize their BMAA dose. Confirmation of chromatolysis and cell death in macaque brains was visualized using toluidine staining. In contrast, immunological staining with AT8 and b-amyloid (1–42) antibodies and thioflavine-S stain in vervet brains suggests early stage labeling of neurites and NFT and plaque-like formation in the absence of neuronal loss. The lack of neurologic deficits reported in vervets is in keeping with early preclinical pathology observed with these immunohistochemical methods. BMAA toxicity in vervet brains causes the early events that occur in the genesis of neurofibrillary pathology. Taken together, these different studies of vervets and macaques demonstrate BMAA toxicity in the brain due to chronic exposures. The use of more sensitive immunohistochemical methods in the vervet study most likely explains the differences in neuropathology reported for vervets and macaques. ã 2016 Elsevier B.V. All rights reserved.
In a pioneering study published nearly 30 years ago, Spencer et al. reported that exposure to BMAA produced neurological signs in macaques (Macaca fascicularis) (Spencer et al., 1987). In response to our recent report that chronic dietary exposure to the cyanobacterial toxin, L-BMAA, triggers the formation of neurofibrillary tangles (NFT) and b-amyloid deposits in the brains of vervets (Chlorocebus sabaeus) (Cox et al., 2016), Spencer et al. (2016) raise several questions about differences between their macaque study and what we reported for vervets. They noted “prominent signs of motor system disease” (Spencer et al., 2016) in macaques and question whether the absence of such clinical signs in our vervets is due to species differences in metabolism or in the purity of the L-BMAA fed to the vervets. They also raised the issue that while we reported “widespread neurofibrillary degeneration and amyloid plaques in L-BMAA-treated vervets, this was not apparent in comparably treated cynomolgus monkeys” (Spencer et al., 2016). They further suggest that we reported that L-serine protects vervets “from the full tauopathic
* Corresponding author. E-mail address: [email protected] (P.A. Cox).
effects of L-BMAA because this essential amino acid competitively blocked the misincorporation of the latter into brain protein” (Spencer et al., 2016). In our recent study (Cox et al., 2016), vervets were fed daily for 140 days a quarter of a banana with a powdered test substance placed inside. Cohorts of eight vervets were given a banana dosed with the dry powder of L-BMAA hydrochloride salt, at either 21 mg/kg/day (low dose cohort) or 210 mg/kg/day (high dose cohorts) while a third cohort of eight received 210 mg/kg/day LBMAA plus 210 mg/kg/day L-serine. A control cohort of eight vervets received 210 mg/kg/day dry rice flour. The L-BMAA hydrochloride salt had 99.08% purity determined by 1H NMR, 13 C NMR, ES-API MS, HESI UPLC–MS/MS, and HPLC. The analysis confirmed that the L-BMAA hydrochloride salt conformed to structure with 154.6 MW and L-optical rotation. Our high dose of 210 mg/kg/day of powdered L-BMAA hydrochloride salt is equivalent to an effective daily dose of 160 mg/kg L-BMAA. The macaques studied by Spencer et al. (1987) received oral gavage doses ranging between 100 and 350 mg/kg/day L-BMAA; thus the highest dose administered by gavage to the macaques was over twice the highest dose we fed to vervets.
http://dx.doi.org/10.1016/j.neuro.2016.04.017 0161-813X/ã 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: P.A. Cox, et al., Do vervets and macaques respond differently to BMAA?, Neurotoxicology (2016), http://dx.doi. org/10.1016/j.neuro.2016.04.017
G Model NEUTOX 1989 No. of Pages 2
2
P.A. Cox et al. / NeuroToxicology xxx (2015) xxx–xxx
The original Spencer et al. (1987) study examined macaque brains for gross chromatolysis visualized in motor neurons with toluidine blue (e.g. necrotic neurons) using standard histological methods available at that time. These studies were not designed to label particular proteins implicated in the disease process using a panel of well-characterized antibodies nor did they use any other method to confirm pathology or loss of neurons in other brain regions studied in our report (Cox et al., 2016). The chromatolysis of neurons is time dependent and usually timed in a way that captures the maximal insult of a toxin exposure. Toluidine blue would not show NFTs or amyloid plaques. Thus, the anatomical approaches are not comparable between the two studies. We used antibodies to label neurofibrillary tangles (NFT) and b-amyloid plaques using three approaches (e.g., tau AT8 and b-amyloid (1–42) with thioflavine-S for confirmation). Thioflavins are dyes used for histology staining and biophysical studies of protein aggregation. We also examined the cytoarchitecture of the vervet brain using a thionin Nissl stain. This analysis revealed preclinical stage NFTs and neurites and plaque-like structures in the absence of neuronal loss. Thus, the anatomical analysis done by Spencer et al. (1987) on macaques is not comparable to the comprehensive immunologic studies we reported for L-BMAA fed vervets (Cox et al., 2016). Spencer subsequently reported that BMAA-fed macaques lack “certain features of the human disease, notably nigral degeneration and plentiful paired helical filaments” (Spencer, 1987, p. 353), which contrasts with the data presented in our vervet study where dense NFT were found in the superficial layers of cortical brain regions consistent with preclinical stages of the Guam disease (Cox et al., 2016). They also stated that “macaques receiving BMAA did not develop a progressive disease, and the most affected animals displayed no evidence of neuronal loss, marked tractal degeneration, or neuromuscular degeneration” (Spencer, 1990, p. 32). These reported differences between macaques and vervets may reflect differential sensitivity of macaques compared to vervets on upper and lower motor neurons or differences in absorption of L-BMAA due to preparation of powdered (vervet protocol) versus oral gavage (macaque dose of liquid solution prepared with sodium bicarbonate). Spencer et al. (1987) used sodium bicarbonate to neutralize their liquid solution prior to oral gavage. Weiss and Choi (1988) found that addition of sodium bicarbonate potentiates the toxicity of L-BMAA in vitro and Nunn and O’Brien (1989) discovered that BMAA forms a stable adduct with bicarbonate. There was no information provided from the Spencer et al. study on blood, brain or CSF concentrations (Spencer et al., 1987). The bicarbonate adduct of L-BMAA has higher affinity for glutamate receptors which may explain the marked excitotoxic effects seen rapidly in macaques using the electrophysiologic and pharmacologic approaches originally described in their report (Spencer et al., 1987). We found that vervets co-administered L-BMAA and L-serine in their diet had a reduced density of NFT. However, we did not claim in this report any specific mechanism for this neuroprotective action (Cox et al., 2016). L-BMAA is toxic to specific subpopulations
of motor neurons through activation of AMPA and NMDA receptors (Rao et al., 2006; Lobner et al., 2007), and causes protein misfolding and aggregation through misincorporation in neural proteins for Lserine (Dunlop et al., 2013; Glover et al., 2014). Arif et al. (2014) hypothesized that binding of BMAA to mGLUr5 results in hyperphosphorylation of tau through deactivation of the phosphatase PP2A, in keeping with our observations that L-BMAA triggers tauopathy. Finally, we agree with Spencer et al. that “neurotoxic substances can trigger neurodegenerative diseases that are often considered to be exclusively of genetic origin” (Spencer et al., 2016). Three decades later, our primate study provides supportive neuropathological evidence of preclinical disease in vervets fed L-BMAA (Cox et al., 2016), that extends and confirms the seminal contribution of L-BMAA toxicity reported by Spencer and coworkers in macaques (Spencer et al., 1987). Conflict of interest The Institute for Ethnomedicine has applied for patents for the use of L-serine to treat neurodegenerative illness (US 13/683,821), and for screening potential drug candidates using BMAA-induced neurodegeneration (US 14/229,624). References Arif, M., Kazim, S.F., Grundke-Iqbal, I., Garruto, R.M., Iqbal, K., 2014. Tau pathology involves protein phosphatase 2A in Parkinsonism-dementia of Guam. Proc. Natl. Acad. Sci. U. S. A. 111, 1144–1149. doi:http://dx.doi.org/10.1073/pnas.1322614111. Cox, P.A., Davis, D.A., Mash, D.C., Metcalf, J.S., Banack, S.A., 2016. Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain. Proc. R. Soc. B 283, 20152397. doi:http://dx.doi.org/10.1098/ rspb.2015.2397. Dunlop, R.A., Cox, P.A., Banack, S.A., Rodgers, K.J., 2013. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation. PLoS One 8, e75376. doi:http://dx.doi.org/ 10.1371/journal.pone.0075376. Glover, W.B., Mash, D.C., Murch, S.J., 2014. The natural non-protein amino acid Nbeta-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis. Amino Acids 46, 2553–2559. Lobner, D., Piana, P.M.T., Salous, A.K., Peoples, R.W., 2007. b-N-methylamino-Lalanine enhances neurotoxicity through multiple mechanisms. Neurobiol. Dis. 25 (2), 360–366. Nunn, P.B., O’Brien, P., 1989. The interaction of b-N-methylamino-L-alanine with bicarbonate: an 1H-NMR study. FEBS Lett. 251 (1–2), 31–35. Rao, S.D., Banack, S.A., Cox, P.A., Weiss, J.H., 2006. BMAA selectively injures motor neurons via AMPA/kainate receptor activation. Exp. Neurol. 201, 244–252. doi: http://dx.doi.org/10.1016/j.expneurol.2006.04.017. Spencer, P.S., Nunn, P.B., Hugon, J., Ludolph, A.C., Ross, S.M., Roy, D.N., Robertson, R. C., 1987. Guam amyotrophic lateral sclerosis-parkinsonism-dementia linked to a plant excitant neurotoxin. Science 237 (July (4814)), 517–522. Spencer, P.S., Garner, C.E., Palmer, V.S., Kisby, G.E., 2016. Vervets and macaques: similarities and differences in their responses to L-BMAA. Neurotoxicology doi: http://dx.doi.org/10.1016/j.neuro.2016.03.018. Spencer, P.S., 1987. Guam ALS/Parkinsonism-dementia: a long-latency neurotoxic disorder caused by slow toxin(s) in food? Can. J. Neurol. Sci. 14 (August Suppl. (3)), 347–357. Spencer, P.S., 1990. Linking cycad to the etiology of Western Pacific amyotrophic lateral sclerosis. Chapter 6 IN ALS. In: Rose, F. Clifford, Norris, Forbes H. (Eds.), New Advances in Toxicology and Epidemiology. Smith-Gordon. Weiss, J.H., Choi, D.W., 1988. Beta-N-methylamino-L-alanine neurotoxicity: requirement for bicarbonate as a cofactor. Science 241, 973–975.
Please cite this article in press as: P.A. Cox, et al., Do vervets and macaques respond differently to BMAA?, Neurotoxicology (2016), http://dx.doi. org/10.1016/j.neuro.2016.04.017