Mechanisms and modes of β-N-methylamino-lalanine neurotoxicity: the basis for designing therapies
- Authors: Van Onselen, Rianita
- Date: 2019
- Subjects: Cyanobacteria , Amino acids -- Toxicology , Neurotoxic agents
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/32971 , vital:32483
- Description: Since the discovery of the non-canonical amino acid β-N-methylamino-L-alanine (BMAA) and the demonstration of its acute neurotoxicity in chicks and rats, it has been postulated that BMAA might contribute to the development of neurodegenerative diseases worldwide due to its presence in numerous aquatic and terrestrial food webs. This hypothesized link was widely criticized because of the inability to reproduce symptoms in a BMAA-exposed animal model that resembled the symptoms observed in humans, and for the inability to achieve significant levels of toxicity in in vitro models via the postulated mechanisms of toxicity. The most widely described mechanism of BMAA toxicity was excitotoxicity by over-excitation of ionotropic and/or metabotropic glutamate receptors following activation by BMAA. However, the excitotoxic potency of BMAA is much lower than those of other known excitotoxins and it was not known whether BMAA could accumulate in significant concentrations in synapses to cause the said excitotoxicity. Therefore, uptake of BMAA into synaptic vesicles from where it can be released into synapses in high concentrations, was investigated and it was found that, unlike the uptake that was observed for glutamate, BMAA was not taken up into synaptic vesicles. This discovery suggests that BMAA is not released into synapses via synaptic vesicles and that excitotoxicity is an unlikely mechanism of BMAA toxicity in mammalian systems. Misincorporation of BMAA into proteins in the place of L-serine was suggested to be an important mechanism of BMAA toxicity that could lead to protein misfolding and the subsequent protein aggregates that are typically found in the central nervous system (CNS) of neurodegenerative disease patients. However, previous studies in prokaryotes and in a rat pheochromocytoma PC12 cell line showed that misincorporation of BMAA does not occur to any significant extent. However, these studies were criticized for not using human-derived model systems to show that misincorporation does not occur, and it was argued that due to differences in mitochondrial protein synthesis mechanisms, misincorporation of BMAA into human proteins could not be ruled out as a possible mechanism of toxicity. Therefore, misincorporation of BMAA was investigated in a number of human-derived non-neuronal cell lines and directly compared to the misincorporation of other known amino acid analogues. No evidence of misincorporation of BMAA into these cell lines was obtained and therefore it was concluded that misincorporation of BMAA into proteins does not occur in human-derived cell models. Although misincorporation of BMAA into proteins was refuted as a mechanism of toxicity, the strong interactions between BMAA and proteins that require extensive purification procedures to remove the associated BMAA, could not be discounted as a possible contributor to the toxicity of BMAA. Cell-free interactions between BMAA and enzymes, which resulted in reduced activity, were described previously but the nature of these interactions was never determined. Therefore, the direct interactions between BMAA and a range of commercial proteins and melanin (that is known to also have a strong affinity for BMAA) were investigated in an attempt to describe the nature of these interactions. It was discovered that BMAA has a high affinity for hydroxyl groups, and that if these hydroxyl groups in the form of hydroxyl containing amino acid residues occurred in important regulatory or active sites of proteins, BMAA reduced the enzyme activity. Catalase was subsequently selected as an important enzyme required for the maintenance of the delicate reactive oxygen species (ROS) balance in the CNS, to test the effect of BMAA on the activity of the enzyme. BMAA inhibited a human commercial extract of catalase in a cell free system, and this inhibition appeared to be non-competitive in nature. Subsequently, catalase in an extract from a human cell line was also shown to be inhibited by BMAA and it was concluded that this BMAA induced inhibition of catalase could be an important contributor to the toxicity of BMAA in in vivo systems. The affinity of BMAA for hydroxyl groups, especially the reactive L-tyrosine side chain hydroxyl, was recognized as a possible mechanism that can be utilized to protect against the toxicity of BMAA. It was subsequently shown that excess concentrations of L-serine and L-tyrosine could protect against the BMAA-induced enzyme inhibition and improper folding of proteins in a cell-free system. By administering an equimolar concentration of either L-phenylalanine (the soluble precursor of L-tyrosine) or L-serine an hour before administration of BMAA in a rat model, the BMAA-induced neurotoxicity was greatly reduced, especially by treatment with L-phenylalanine, which resulted in a decrease of between 60-70% in the observed neuropathologies. It was recognized that the protection offered by L-phenylalanine was greater than would be expected if protection was by virtue of direct hydroxyl binding alone and it was subsequently hypothesized that the conversion of L-phenylalanine to dopamine could have contributed to the observed protection. Subsequently, the possible protection offered by dopamine, administered as L-DOPA, against BMAA neurotoxicity was investigated in the same neonatal rat model and compared to the protection offered by L-tyrosine. It was discovered that dopamine protected against the BMAAinduced neuronal cell losses in the hippocampus, striatum and spinal cord but it was not as efficient as L-tyrosine in protection against the BMAA-induced proteinopathies, suggesting two distinct mechanisms of BMAA toxicity, one of which is a depletion of dopamine, which had not been previously described. Finally, the nature of the BMAA-induced dopamine depletion was investigated by administering BMAA in combination with other dopaminergic modifiers viz. apomorphine (a D1/D2 receptor agonist), a dopamine transporter inhibitor (GBR12783) and reserpine (a vesicular monoamine transporter -VMAT2- inhibitor) to the neonatal rat model in an attempt to describe how BMAA functions as a dopaminergic toxin. Based on these results it was concluded that BMAA inhibits uptake of dopamine into synaptic vesicles by inhibiting VMAT2-mediated uptake of dopamine, which causes neuronal loss in the hippocampus, striatum and substantia nigra pars compacta, and that the BMAA-induced inhibition of catalase contributes significantly to the toxicity of BMAA by causing an accumulation of hydrogen peroxide in the hippocampus, striatum and spinal cord, which results in extensive neuronal damage in these areas. This work was the first to thoroughly investigate the mechanisms that explain the observed pathologies caused by BMAA in an in vivo model, and was the first to suggest that BMAA can reduce the dopamine in the CNS by inhibiting VMAT2-mediated uptake of dopamine into synaptic vesicles, and increase damage by reactive oxygen species by inhibiting catalase. BMAA is therefore a multimechanistic and multimodal.
- Full Text:
- Date Issued: 2019
- Authors: Van Onselen, Rianita
- Date: 2019
- Subjects: Cyanobacteria , Amino acids -- Toxicology , Neurotoxic agents
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/32971 , vital:32483
- Description: Since the discovery of the non-canonical amino acid β-N-methylamino-L-alanine (BMAA) and the demonstration of its acute neurotoxicity in chicks and rats, it has been postulated that BMAA might contribute to the development of neurodegenerative diseases worldwide due to its presence in numerous aquatic and terrestrial food webs. This hypothesized link was widely criticized because of the inability to reproduce symptoms in a BMAA-exposed animal model that resembled the symptoms observed in humans, and for the inability to achieve significant levels of toxicity in in vitro models via the postulated mechanisms of toxicity. The most widely described mechanism of BMAA toxicity was excitotoxicity by over-excitation of ionotropic and/or metabotropic glutamate receptors following activation by BMAA. However, the excitotoxic potency of BMAA is much lower than those of other known excitotoxins and it was not known whether BMAA could accumulate in significant concentrations in synapses to cause the said excitotoxicity. Therefore, uptake of BMAA into synaptic vesicles from where it can be released into synapses in high concentrations, was investigated and it was found that, unlike the uptake that was observed for glutamate, BMAA was not taken up into synaptic vesicles. This discovery suggests that BMAA is not released into synapses via synaptic vesicles and that excitotoxicity is an unlikely mechanism of BMAA toxicity in mammalian systems. Misincorporation of BMAA into proteins in the place of L-serine was suggested to be an important mechanism of BMAA toxicity that could lead to protein misfolding and the subsequent protein aggregates that are typically found in the central nervous system (CNS) of neurodegenerative disease patients. However, previous studies in prokaryotes and in a rat pheochromocytoma PC12 cell line showed that misincorporation of BMAA does not occur to any significant extent. However, these studies were criticized for not using human-derived model systems to show that misincorporation does not occur, and it was argued that due to differences in mitochondrial protein synthesis mechanisms, misincorporation of BMAA into human proteins could not be ruled out as a possible mechanism of toxicity. Therefore, misincorporation of BMAA was investigated in a number of human-derived non-neuronal cell lines and directly compared to the misincorporation of other known amino acid analogues. No evidence of misincorporation of BMAA into these cell lines was obtained and therefore it was concluded that misincorporation of BMAA into proteins does not occur in human-derived cell models. Although misincorporation of BMAA into proteins was refuted as a mechanism of toxicity, the strong interactions between BMAA and proteins that require extensive purification procedures to remove the associated BMAA, could not be discounted as a possible contributor to the toxicity of BMAA. Cell-free interactions between BMAA and enzymes, which resulted in reduced activity, were described previously but the nature of these interactions was never determined. Therefore, the direct interactions between BMAA and a range of commercial proteins and melanin (that is known to also have a strong affinity for BMAA) were investigated in an attempt to describe the nature of these interactions. It was discovered that BMAA has a high affinity for hydroxyl groups, and that if these hydroxyl groups in the form of hydroxyl containing amino acid residues occurred in important regulatory or active sites of proteins, BMAA reduced the enzyme activity. Catalase was subsequently selected as an important enzyme required for the maintenance of the delicate reactive oxygen species (ROS) balance in the CNS, to test the effect of BMAA on the activity of the enzyme. BMAA inhibited a human commercial extract of catalase in a cell free system, and this inhibition appeared to be non-competitive in nature. Subsequently, catalase in an extract from a human cell line was also shown to be inhibited by BMAA and it was concluded that this BMAA induced inhibition of catalase could be an important contributor to the toxicity of BMAA in in vivo systems. The affinity of BMAA for hydroxyl groups, especially the reactive L-tyrosine side chain hydroxyl, was recognized as a possible mechanism that can be utilized to protect against the toxicity of BMAA. It was subsequently shown that excess concentrations of L-serine and L-tyrosine could protect against the BMAA-induced enzyme inhibition and improper folding of proteins in a cell-free system. By administering an equimolar concentration of either L-phenylalanine (the soluble precursor of L-tyrosine) or L-serine an hour before administration of BMAA in a rat model, the BMAA-induced neurotoxicity was greatly reduced, especially by treatment with L-phenylalanine, which resulted in a decrease of between 60-70% in the observed neuropathologies. It was recognized that the protection offered by L-phenylalanine was greater than would be expected if protection was by virtue of direct hydroxyl binding alone and it was subsequently hypothesized that the conversion of L-phenylalanine to dopamine could have contributed to the observed protection. Subsequently, the possible protection offered by dopamine, administered as L-DOPA, against BMAA neurotoxicity was investigated in the same neonatal rat model and compared to the protection offered by L-tyrosine. It was discovered that dopamine protected against the BMAAinduced neuronal cell losses in the hippocampus, striatum and spinal cord but it was not as efficient as L-tyrosine in protection against the BMAA-induced proteinopathies, suggesting two distinct mechanisms of BMAA toxicity, one of which is a depletion of dopamine, which had not been previously described. Finally, the nature of the BMAA-induced dopamine depletion was investigated by administering BMAA in combination with other dopaminergic modifiers viz. apomorphine (a D1/D2 receptor agonist), a dopamine transporter inhibitor (GBR12783) and reserpine (a vesicular monoamine transporter -VMAT2- inhibitor) to the neonatal rat model in an attempt to describe how BMAA functions as a dopaminergic toxin. Based on these results it was concluded that BMAA inhibits uptake of dopamine into synaptic vesicles by inhibiting VMAT2-mediated uptake of dopamine, which causes neuronal loss in the hippocampus, striatum and substantia nigra pars compacta, and that the BMAA-induced inhibition of catalase contributes significantly to the toxicity of BMAA by causing an accumulation of hydrogen peroxide in the hippocampus, striatum and spinal cord, which results in extensive neuronal damage in these areas. This work was the first to thoroughly investigate the mechanisms that explain the observed pathologies caused by BMAA in an in vivo model, and was the first to suggest that BMAA can reduce the dopamine in the CNS by inhibiting VMAT2-mediated uptake of dopamine into synaptic vesicles, and increase damage by reactive oxygen species by inhibiting catalase. BMAA is therefore a multimechanistic and multimodal.
- Full Text:
- Date Issued: 2019
Ecophysiology and nutrient uptake mechanisms facilitating the prolonged bloom persistence by Cyanothece sp. in Lake St Lucia, South Africa
- Authors: Du Plooy, Schalk Jacobus
- Date: 2017
- Subjects: Cyanobacterial blooms , Cyanobacteria -- Physiology , Cyanobacteria
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/7344 , vital:21324
- Description: Cyanobacterial blooms are becoming more frequent worldwide, with possible negative effects on human health. The effects of climate change and eutrophication have been associated with persistent cyanobacterial blooms becoming more frequent. Altered water characteristics, salinity in particular, influence ecosystem dynamics that may lead to conditions conducive to cyanobacterial blooms. The occurrence of an 18-month long Cyanothece sp. bloom (the longest for any cyanobacterium recorded so far worldwide and the first of the genus) from June 2009 to December 2010 in Africa’s largest estuarine lake, St Lucia, highlighted the susceptibility of ecosystems to anthropogenic alterations. This study investigated the long-term survival and physiological adaptations of Cyanothece sp. to various and dynamic environmental conditions that contributed towards its bloom persistence. The main findings are the high salinities at which Cyanothece sp. could perform important physiological processes such as N uptake, N2 fixation and photosynthesis. Nutrient uptake (both nitrogen and phosphorus) was observed over the full experimental salinity range (0-300) while N2 fixation was only observed up to a salinity of 120. Nutrient uptake rates significantly decreased at this threshold salinity of 120. Interestingly, photosystem II activity was not observed in Cyanothece sp. during this study, but photosystem I activity was robust. Salinity had a minor influence on electron transport rates by photosystem I, high temperature (> 30°C) did however increase electron transport rates. Rapid responses to hypo-osmotic shock (i.e. osmotic downshift during freshening events) by Cyanothece sp. cells also helped minimize cell rupture due to high turgor pressure. Zooplankton abundance within the St Lucia system was negatively correlated with salinity, while grazing experiments indicated that the typical estuarine zooplankton species are able to graze on Cyanothece sp. cells. Therefore, the disappearance of zooplankton at salinities above 60 must have been an important factor in the bloom persistence. Apart from the ecological factors that were at play in St Lucia during the bloom period, the persistence of the Cyanothece sp. bloom can be attributed to the robust nature of their nutrient uptake, nitrogen fixation and photosynthetic systems to maintain activity despite extreme hypersalinity levels.
- Full Text:
- Date Issued: 2017
- Authors: Du Plooy, Schalk Jacobus
- Date: 2017
- Subjects: Cyanobacterial blooms , Cyanobacteria -- Physiology , Cyanobacteria
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: http://hdl.handle.net/10948/7344 , vital:21324
- Description: Cyanobacterial blooms are becoming more frequent worldwide, with possible negative effects on human health. The effects of climate change and eutrophication have been associated with persistent cyanobacterial blooms becoming more frequent. Altered water characteristics, salinity in particular, influence ecosystem dynamics that may lead to conditions conducive to cyanobacterial blooms. The occurrence of an 18-month long Cyanothece sp. bloom (the longest for any cyanobacterium recorded so far worldwide and the first of the genus) from June 2009 to December 2010 in Africa’s largest estuarine lake, St Lucia, highlighted the susceptibility of ecosystems to anthropogenic alterations. This study investigated the long-term survival and physiological adaptations of Cyanothece sp. to various and dynamic environmental conditions that contributed towards its bloom persistence. The main findings are the high salinities at which Cyanothece sp. could perform important physiological processes such as N uptake, N2 fixation and photosynthesis. Nutrient uptake (both nitrogen and phosphorus) was observed over the full experimental salinity range (0-300) while N2 fixation was only observed up to a salinity of 120. Nutrient uptake rates significantly decreased at this threshold salinity of 120. Interestingly, photosystem II activity was not observed in Cyanothece sp. during this study, but photosystem I activity was robust. Salinity had a minor influence on electron transport rates by photosystem I, high temperature (> 30°C) did however increase electron transport rates. Rapid responses to hypo-osmotic shock (i.e. osmotic downshift during freshening events) by Cyanothece sp. cells also helped minimize cell rupture due to high turgor pressure. Zooplankton abundance within the St Lucia system was negatively correlated with salinity, while grazing experiments indicated that the typical estuarine zooplankton species are able to graze on Cyanothece sp. cells. Therefore, the disappearance of zooplankton at salinities above 60 must have been an important factor in the bloom persistence. Apart from the ecological factors that were at play in St Lucia during the bloom period, the persistence of the Cyanothece sp. bloom can be attributed to the robust nature of their nutrient uptake, nitrogen fixation and photosynthetic systems to maintain activity despite extreme hypersalinity levels.
- Full Text:
- Date Issued: 2017
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