La cianotoxina β-N-metil-amino-L-alanina (BMAA) es un aminoácido no proteico producido por cianobacterias, capaz de biomagnificarse en las cadenas tróficas de ecosistemas marinos y terrestres. Dada su capacidad de atravesar la barrera hematoencefálica, su ingesta progresiva se asocia con el desarrollo de ciertas retinopatías, así como también de enfermedades neurodegenerativas, tales como la Esclerosis Lateral Amiotrófica (ELA), la Enfermedad de Parkinson (EP) y la Enfermedad de Alzheimer (EA). Los daños causados por la BMAA son múltiples y originados en mecanismos diversos. Así, la BMAA, en presencia de iones bicarbonato (HCO3 - ), puede formar un compuesto denominado carbamato, cuya estructura química es similar al glutamato (Glut), uno de los neurotransmisores más importantes del sistema nervioso. A su vez, el carbamato se une y activa receptores de Glut, ya sean ionotrópicos (como el receptor de N-metil-D- aspartato) o metabotrópicos. La sobreexcitación de estos receptores ocasionada por la BMAA, promueve mecanismos de excitotoxicidad que conducen a alteraciones neuronales. Por otro lado, la BMAA puede ingresar a las células a través del sistema xc, un sistema de transporte sodio-independiente común para cistina y Glut. Una vez en el interior celular, la toxina puede incorporarse erróneamente en las cadenas polipeptídicas en reemplazo de Serina (Ser). Así, unida a componentes proteicos, puede generar un reservorio endógeno de lenta liberación que expone a las neuronas a una baja pero continua dosis de esta toxina. Entre sus varios efectos subcelulares, la BMAA puede afectar la permeabilidad de las membranas mitocondriales comprometiendo su actividad. Además, puede inducir modificaciones en los niveles de Ca 2+, generar estrés oxidativo, promover fallas en la producción de ATP e inducir estrés en el retículo endoplasmático, lo cual conduce a alteraciones en la síntesis y/o distribución de proteínas. Asociado a esto, se originan alteraciones en el transporte axonal y la fragmentación de estas estructuras. Pese a su trascendencia para la salud, aún son desconocidos los efectos directos que genera la exposición a la BMAA de las neuronas y células gliales de la retina (como las células gliales de Müller –CGM-), o del epitelio pigmentario de la retina (EPR). Además, todavía son mayormente desconocidos aquellos factores o moléculas capaces de modular las vías de señalización involucradas en los efectos deletéreos inducidos por la BMAA. Al respecto, recientemente se ha propuesto que la activación de los receptores X retinoides (RXR) protegerían a las neuronas y modularían la respuesta inflamatoria durante las enfermedades neurodegenerativas del sistema nervioso central, y también en retinopatías. Aún se desconoce si estos receptores ejercen un rol protector contra los daños inducidos por la BMAA. En esta Tesis se estudiaron los mecanismos involucrados en los cambios degenerativos inducidos por la BMAA en células de la retina, así como también en células PC12 diferenciadas a neuronas. Asimismo, se evaluó el valor protector de agonistas de los RXRs frente a los efectos deletéreos inducidos por la BMAA en células de la retina. Para estos estudios, se obtuvieron cultivos puros de neuronas amacrinas y fotorreceptores (FRs), de CGM puros, y cultivos neuro-gliales a partir de retinas de ratas neonatas. Además, se utilizaron cultivos de líneas celulares PC12 y epiteliales ARPE- 19. Todos los cuales fueron tratados con la BMAA para evaluar sus efectos sobre estas células y el posible rol protector de los RXRs. Los resultados obtenidos en este trabajo demostraron que aún bajas concentraciones de la BMAA (de 0,4 μM) alteraron la viabilidad no sólo de las neuronas amacrinas y FRs, sino también de las células PC12 diferenciadas a neuronas, de las CGM e incluso de las células del EPR. La BMAA también, indujo alteraciones en la permeabilidad mitocondrial y en la producción de ROS en las células neuronales, gliales y epiteliales, mientras que en las CGM indujo cambios en la morfología nuclear. Por su parte, en neuronas amacrinas, promovió el crecimiento axonal, aunque generando el colapso de sus conos de crecimiento. Estas alteraciones fueron mediadas por la activación de los receptores NMDA en presencia de iones HCO3 - . Además, en estas células, la BMAA se incorporaría erróneamente en las cadenas polipeptídicas en reemplazo de la Ser, dado que la suplementación del medio de cultivo con este aminoácido previno la toxicidad inducida por la BMAA. En cuanto a la acción protectora de los RXRs, nuestros resultados demostraron que su activación bloqueó los efectos tóxicos que produjo la BMAA sobre las neuronas amacrinas y los FRs, así como también sobre las células del EPR. En resumen, en esta Tesis presentamos evidencias de que la BMAA afecta múltiples estructuras subcelulares en las células que conforman la retina, así como también a células PC12 diferenciadas. Estos resultados sugieren que los daños inducidos por la BMAA representan un potencial riesgo para la salud, y podrían contribuir al desarrollo de retinopatías, así como de varias enfermedades neurodegenerativas. Además, este trabajo indicaría que la activación de los RXRs puede presentar un papel protector al ejercer un rol relevante en la supervivencia de las neuronas amacrinas y FRs, así como también de las células del EPR. En su conjunto, estos hallazgos aportan nuevos conocimientos en relación a los mecanismos deletéreos inducidos por la BMAA y podrían ser de utilidad para el desarrollo de futuras estrategias terapéuticas. / The cyanotoxin β–N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by cyanobacteria. It is biomagnified along the food chains in both, marine and terrestrial ecosystems. Due to its ability to cross the brain blood barrier, its ingestion may contribute to the onset of retinopathies, as well as neurodegenerative diseases, like Amyotrophic Lateral Sclerosis, Parkinson (PD) and Alzheimer disease (AD). Damages induced by BMAA are multiple and originated by different mechanisms. In the presence of bicarbonate ions (HCO3 - ), BMAA can produce carbamate, whose chemical structure is similar to that of glutamate (Glut), one of the most important neurotransmitters in the nervous system. In turn, carbamate can bind and activate both ionotropic (like N-Methyl-D-aspartate -NMDA-) and metabotropic Glut receptors. Overactivation of these receptors by BMAA promotes excitotoxicity, which leads to nuclear alterations. On the other hand, BMAA crosses the cell membranes by using the cystine/glutamate antiporter (xc- system), a sodium-independent amino acid transporter. Once inside the cells, the toxin can mistakenly replace the amino acid Serine (Ser) in polypeptide chains, thus generating an endogenous reservoir of BMAA, whose slow- release exposes neurons to a low, but continuous amount of this toxin. Among its various subcellular effects, BMAA can alter mitochondrial membrane permeability compromising mitochondrial activity. Besides, it can alter Ca2+ levels, generate oxidative stress, promote failures in the ATP production and induce endoplasmic reticulum (RE) stress, leading to alterations in the protein synthesis and/or distribution. In this context, BMAA promotes alterations in axonal transport along with fragmentation of these structures. Despite its importance to health, the direct effects of BMAA exposure on retinal neurons and glial cells (such as Müller glial cells –CGM-), or retinal pigment epithelium (RPE) cells, are virtually unknown and the factors or molecules, which could modulate the signaling pathways involved in the deleterious effects induced by BMAA have not been established. In this regard, it has recently been proposed that the activation of Retinoid X Receptors (RXR) can protect neurons and modulate the inflammatory responses during neurodegenerative diseases of the central nervous system, including retinopathies. However, the possible protective roles of RXRs in BMAA-induced damages are still unknown. In this Thesis, we have studied the mechanisms involved in the degenerative changes induced by BMAA into retinal cells, and in neuron-like, differentiated rat pheochromocytoma cells (PC12 cells), as well. We also evaluated the protection of RXR agonists against the deleterious effects of BMAA in retinal cells. For these purposes, we obtained pure neuronal cultures of amacrine neurons and photoreceptors (PHRs); pure MGC cultures, and mixed neuro-glial cultures from newborn rats. In addition, we used PC12 cells and ARPE-19 epithelial cell lines. We treated them with BMAA to evaluate its effects on these cells and the possible protective roles of RXRs. Our results showed that low concentrations of BMAA (0.4 μM) altered, not only the viability of amacrine neurons and PHRs, but also that of neuronally differentiated PC12 cells, MGC and even that of the RPE cells. Also, the cyanotoxin induced alterations in mitochondrial membrane permeability and in ROS production, while in MGC, BMAA induced changes in the nuclear morphology. On the other hand, in amacrine neurons, this toxin promoted axonal growth, although simultaneously generating the collapse of their growth cones. We established that all these alterations were induced by activation of NMDA receptors in the presence of HCO3 - ions. Besides, in all these cell types, BMAA appeared to incorporate into polypeptide chains replacing Ser, since supplementation of the culture media with this amino acid prevented toxicity damages. Regarding the protective roles of RXRs, our results showed that their activation blocked the toxic effects induced by BMAA in amacrine neurons, PHRs, and RPE cells. In summary, in this Thesis we present evidences that BMAA affected multiple subcellular structures in retina cells and in PC12 cells differentiated into neurons. These results suggest that the damaging effects induced by BMAA represent a potential health risk, which could contribute to the development of retinopathies, along with other neurodegenerative diseases. In addition, this work would indicate that RXR activation can promote survival of amacrine PHRs and RPE cells. Taken together, these findings provide new knowledge regarding the deleterious mechanisms induced by BMAA, which could be useful for the development of future effective therapies.

Materiais de Referência (MR) de substâncias químicas têm ampla aplicação, sobretudo na área analítica, servindo de referência para validação de métodos, calibração de instrumentos e controle de qualidade, estabelecendo a comparabilidade de resultados analíticos em escala global e permitindo a transferência da exatidão entre métodos, laboratórios e padrões. Norteado por essas necessidades, o trabalho apresenta uma proposta para certificação de MR baseada nas orientações preconizadas por diretrizes e normas internacionais, principalmente as que seguem o ISO Guia 34, para estabelecer as propriedades certificadas através de técnicas analíticas de Espectrometria de Massas de Alta Resolução, Ressonância Magnética Nuclear, de ¹H e de ¹³C, e de Análise Elementar CHN. A certificação contemplou as caracterizações qualitativa e quantitativa, ensaio de estabilidade e o cálculo da estimativa da incerteza da medição. Como resultado, foi produzido e certificado um lote piloto de MR de β-N-metilamino-alanina (BMAA), uma toxina obtida in-house através de síntese química e purificação, cujos valores de propriedades certificadas foram rastreáveis ao SI e acompanhadas da estimativa da incerteza da medição. / Reference Materials (RM) of chemicals have wide application, particularly in the analyses, providing a reference for validation of methods, instrument calibration and quality control, establishing the comparability of analytical results on a global scale and enabling the transfer of accuracy between methods, laboratories and standards. Guided by these requirements, the paper presents a proposal for certification of MR based on the guidelines recommended by international guidelines and standards, especially those which follow the ISO Guide 34, to establish the certified properties through analytical techniques of mass spectrometry High resolution Nuclear Magnetic Resonance, ¹H and ¹³C, and CHN elemental analysis. The certification included qualitative and quantitative characterization, stability test and the calculation of the estimate of measurement uncertainty. As a result, was produced and certified a pilot batch of RM β-N-methylamino-alanine (BMAA), a toxin obtained in-house via chemical synthesis and purification, whose property values are certified and traceable to the SI accompanied by an estimative of the uncertainty of measurement.

Materiais de Referência (MR) de substâncias químicas têm ampla aplicação, sobretudo na área analítica, servindo de referência para validação de métodos, calibração de instrumentos e controle de qualidade, estabelecendo a comparabilidade de resultados analíticos em escala global e permitindo a transferência da exatidão entre métodos, laboratórios e padrões. Norteado por essas necessidades, o trabalho apresenta uma proposta para certificação de MR baseada nas orientações preconizadas por diretrizes e normas internacionais, principalmente as que seguem o ISO Guia 34, para estabelecer as propriedades certificadas através de técnicas analíticas de Espectrometria de Massas de Alta Resolução, Ressonância Magnética Nuclear, de ¹H e de ¹³C, e de Análise Elementar CHN. A certificação contemplou as caracterizações qualitativa e quantitativa, ensaio de estabilidade e o cálculo da estimativa da incerteza da medição. Como resultado, foi produzido e certificado um lote piloto de MR de β-N-metilamino-alanina (BMAA), uma toxina obtida in-house através de síntese química e purificação, cujos valores de propriedades certificadas foram rastreáveis ao SI e acompanhadas da estimativa da incerteza da medição. / Reference Materials (RM) of chemicals have wide application, particularly in the analyses, providing a reference for validation of methods, instrument calibration and quality control, establishing the comparability of analytical results on a global scale and enabling the transfer of accuracy between methods, laboratories and standards. Guided by these requirements, the paper presents a proposal for certification of MR based on the guidelines recommended by international guidelines and standards, especially those which follow the ISO Guide 34, to establish the certified properties through analytical techniques of mass spectrometry High resolution Nuclear Magnetic Resonance, ¹H and ¹³C, and CHN elemental analysis. The certification included qualitative and quantitative characterization, stability test and the calculation of the estimate of measurement uncertainty. As a result, was produced and certified a pilot batch of RM β-N-methylamino-alanine (BMAA), a toxin obtained in-house via chemical synthesis and purification, whose property values are certified and traceable to the SI accompanied by an estimative of the uncertainty of measurement.

Les suppléments alimentaires à base d’algues sont commercialisés pour leurs bénéfices pour la santé ainsi que pour leur valeur nutritionnelle. Cependant, certains de ces produits peuvent être contaminés par des toxines produites par la cooccurrence d’espèces toxigènes. Afin d’évaluer l’ampleur de la contamination en toxines dans différents suppléments disponibles sur le marché nord-américain, nous avons achetés 37 échantillons de spiruline, d’Aphanizomenon flos-aqua, de Chlorella et de varech et les avons analysés pour 27 toxines par des méthodes de chromatographie liquide et de spectrométrie de masse (LC-MS/MS ou LC-HRMS). Nous avons détecté des microcystines dans les huit échantillons d’Aphanizomenon à des niveaux allant jusqu’à 1000 ng/g et de l’acide 2,4-diaminobutyrique (DAB) dans tous les échantillons de suppléments à des concentrations incluses entre 3 et 1600 ng/g. L’anatoxine A (ANA-a) et le β-amino-N-méthylalanine (BAMA) n’ont pas été détectés tandis que les autres toxines ont été détectées de façon plus aléatoire à des concentrations variables, sans lien clair avec l’espèce de l’algue. En considérant ces résultats, les produits à base d’Aphanizomenon devraient faire l’objet d’un contrôle plus strict pour prévenir leur contamination et restreindre la vente de produits contaminés. Ces découvertes seraient également d’intérêt pour les consommateurs afin qu’ils puissent évaluer les risques associés à la consommation régulière de certains suppléments à base d’algues / Algal dietary supplements are marketed for their health benefits and nutritional value. However, these types of products can be contaminated by toxins produced by co-occurring toxigenic cyanobacteria. In order to evaluate the contamination of different supplements available on the North American market, we purchased 37 samples of spirulina, Aphanizomenon flos-aquae, Chlorella and kelp, and analysed them to identify 27 suspected toxins using liquid chromatography mass spectrometry methods (LC-MS/MS or LC-HRMS). We found microcystins (MC) in the 8 Aphanizomenon samples with levels up to 1000 ng g-1 dw and 2,4-diaminobutyric acid (DAB) in all samples with values ranging from 3 to 1600 ng g-1. Anatoxin-a (ANA) and β-amino-N-methylalanine (BAMA) were not detected, while other toxins were diversely detected with no clear link to the nature of the alga. Considering these results, Aphanizomenon products may require stricter monitoring to prevent further contamination. The findings are also of interest to consumers so that they can properly assess the risks that may be involved in the regular consumption of certain algal dietary supplements.

<p>Phylogeny and morphology were examined for the cyanobionts from the water fern <i>Azolla </i>and the cyanobacterial genus<i> Nostoc</i> originating from symbioses with different host plants (genera <i>Gunnera, Cycas,</i> <i>Dioon,</i> <i>Encephalarthos, Macrozamia, </i>and <i>Anthoceros</i>), the lichen genus<i> Pannaria</i>, and free-living <i>Nostoc</i> isolates from different habitats. <i>Nostoc</i> isolates of <i>Pannaria</i> formed a closely related group, but, in general, no monophyletic nature was attributed to the genus <i>Nostoc</i>, in contrast to the cyanobionts from <i>Azolla </i>which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the <i>Azolla</i> cyanobionts and the <i>Nostoc</i> isolates from the bipartite <i>Pannaria</i> lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic <i>Nostoc </i>species and the other comprising cyanobacteria in association with the water fern <i>Azolla</i>.</p><p>The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-<i>Azolla </i>symbiosis, the cyanobacterium-free <i>Azolla pinnata</i> var <i>imbricata </i>strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus <i>Ochrobactrum </i>in the plant.</p>

Phylogeny and morphology were examined for the cyanobionts from the water fern Azolla and the cyanobacterial genus Nostoc originating from symbioses with different host plants (genera Gunnera, Cycas, Dioon, Encephalarthos, Macrozamia, and Anthoceros), the lichen genus Pannaria, and free-living Nostoc isolates from different habitats. Nostoc isolates of Pannaria formed a closely related group, but, in general, no monophyletic nature was attributed to the genus Nostoc, in contrast to the cyanobionts from Azolla which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the Azolla cyanobionts and the Nostoc isolates from the bipartite Pannaria lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic Nostoc species and the other comprising cyanobacteria in association with the water fern Azolla. The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-Azolla symbiosis, the cyanobacterium-free Azolla pinnata var imbricata strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus Ochrobactrum in the plant.

Neurodegenerative diseases have been shown to correlate positively with an ageing population. The most common neurodegenerative diseases are amyotrophic lateral sclerosis (ALS), Parkinson’s disease and Alzheimer’s disease. The cause of these diseases is believed to be the interaction between genetic and environmental factors, synergistically acting with ageing. BMAA (β-methylamino-L-alanine) is one kind of toxin present in our environment and might play an important role in the development of those diseases. BMAA was initially isolated from cycad seeds in Guam, where the incidence of ALS/Parkinsonism-dementia complex among the indigenous people was 50 – 100 times higher than the rest of the world in the 1950’s. BMAA can induce toxic effects on rodents and primates. Furthermore, it can potentiate neuronal injury on cell cultures at concentrations as low as 10 µM. BMAA was reported to be produced by cyanobacteria, and could bio-magnify through the food chain. In this thesis, work was initially focused on the improvement of an existing analytical method for BMAA identification and quantification using liquid chromatography coupled with tandem mass spectrometry.  Subsequently, the refined method was applied to environmental samples for probing alternative BMAA producer(s) in nature and to seafood samples for estimation of human exposure to this toxin. In Paper I, a systematic screening of the isomers of BMAA in a database was performed and seven potential isomers were suggested. Three of them were detected or suspected in natural samples. In Paper II, a deuterated internal standard was synthesized and used for quantifying BMAA in cyanobacteria. In Paper III, Diatoms were discovered to be a BMAA producer in nature. In Paper IV, ten popular species of seafood sold in Swedish markets were screened for BMAA. Half of them were found to contain BMAA at a level of 0.01 – 0.90 µg/g wet weight. In Future perspectives, the remaining questions important in this field are raised.

The cyanobacterial amino acid β-N-methylamino-L-alanine (BMAA) is a neurotoxin implicated in the etiology of neurodegenerative diseases. Cyanobacteria are cosmopolitan organisms present in various environments. BMAA can cause long-term neurodegenerative alterations in rats exposed during the neonatal period, a period that corresponds to the last trimester and the first few years of life in humans. As BMAA has been reported to be bioaccumulated in the aquatic food chain and detected in mussels, crayfish and fish used for human consumption, the main aim of this thesis has been to investigate the final step in the mammalian food-chain, i.e. the transfer of BMAA into breast milk. Autoradiographic imaging and mass spectrometry analysis showed an enantiomer-selective uptake of BMAA and that the neurotoxin was transferred from lactating mice and rat, via the milk, to the brain of the nursed pups. The results show that transport of BMAA may be disproportional to dose. In addition, BMAA was found present both as free amino acid and tightly associated to proteins in rat brains. Surprisingly, however, no association to milk proteins was found. In vitro studies of murine (HC11) and human (MCF7) mammary epithelial cells suggest that BMAA can pass the human mammary epithelium into milk. Additional transport studies on human intestinal, glioblastoma and neuroblastoma cells showed that L-BMAA was consistently favored over D-BMAA and that the transport was mediated by several amino acid transporters. We also demonstrated that egg-laying quail transfer BMAA to its offspring by deposition in the eggs, particularly in the yolk but also in the albumen. Furthermore, comparative analysis of carboxyl- and methyl-labeled [14C]-BMAA suggested that BMAA was not metabolized to a large degree. Altogether, the results indicate that BMAA can be transferred from mothers, via the milk, to the brain of nursed human infants. Determinations of BMAA in mothers’ milk and cows’ milk are therefore warranted. We also propose that birds’ eggs could be an additional source of BMAA exposure in humans. It might therefore be of concern that mussels are increasingly used as feed in commercial egg production.

No description available.

The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) now appears to be a cause of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). Its production by cyanobacteria throughout the world combined with multiple mechanisms of BMAA neurotoxicity, particularly to vulnerable subpopulations of motor neurons, has significantly increased interest in investigating exposure to this non-protein amino acid as a possible risk factor for other forms of neurodegenerative illness. We here provide a brief overview of BMAA studies and provide an introduction to this collection of scientific manuscripts in this special issue on BMAA.