Pharmacological HIF2α inhibition improves VHL disease-associated phenotypes in zebrafish model.

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl(-/-) mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl(-/-) embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

Early hippocampal cell death, and late learning and memory deficits in rats exposed to the environmental toxin BMAA (ß-N-methylamino-L-alanine) during the neonatal period.

We have reported previously that exposure to the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) during the neonatal period causes cognitive impairments in adult rats. The aim of this study was to investigate the long-term effects of neonatal BMAA exposure on learning and memory mechanisms and to identify early morphological changes in the neonatal brain. BMAA was injected subcutaneously in rat pups on postnatal days 9-10. BMAA (50 and 200 mg/kg) caused distinct deficits in spatial learning and memory in adult animals but no morphological changes. No impairment of recognition memory was detected, suggesting that neonatal exposure to BMAA preferentially affects neuronal systems that are important for spatial tasks. Histopathological examination revealed early neuronal cell death as determined by TUNEL staining in the hippocampus 24 h after a high dose (600 mg/kg) of BMAA whereas no changes were observed at lower doses (50 and 200 mg/kg). In addition, there was a low degree of neuronal cell death in the retrosplenial and cingulate cortices, areas that are also important for cognitive function. Taken together, these results indicate that BMAA is a developmental neurotoxin inducing long-term changes in cognitive function. The risk posed by BMAA as a potential human neurotoxin merits further consideration, particularly if the proposed biomagnifications in the food chain are confirmed.

The discovery of BMAA, and examples of biomagnification and protein incorporation involving other non-protein amino acids.

Because of the similarity of ALS/PDC symptoms to those of the paralytic disease lathyrism, cycad seeds from Guam were analyzed for the presence of the non-protein amino acid b-ODAP, which is known to cause lathyrism. Although b-ODAP was not detected, a novel non-protein amino acid, now known as BMAA, was isolated. Primates are more sensitive to BMAA than rodents. It is possible that BMAA when ingested at a low concentration over a long period might be responsible for ALS/PDC. Some non-protein amino acids, including domoic acid and indospicine, are known to be biomagnified. Other non-protein amino acids including azetidine-2-carboxylic acid, canavanine, and selenium containing analogues of cystine and methionine have been shown to be misincorporated into proteins. Perhaps BMAA will not be the only non-protein amino acid that will be found in the brain tissues of those who died of a neurological disease.

Previous studies underestimate BMAA concentrations in cycad flour.

The traditional diet of the Chamorro people of Guam has high concentrations of the neurotoxin BMAA, beta-methyl-amino-L-alanine, in cycad tortillas and from animals that feed on cycad seeds. We measured BMAA concentration in washed cycad flour and compared different extraction methods used by previous researchers in order to determine how much BMAA may have been unaccounted for in prior research. Samples were analyzed with AQC precolumn derivatization using HPLC-FD detection and verified with UPLC-UV, UPLC-MS, and triple quadrupole LC/MS/MS. Although previous workers had studied only the free amino acid component of BMAA in washed cycad flour, we detected significant levels of protein-associated BMAA in washed cycad flour. These data support a link between ALS/PDC and exposure to BMAA.

BMAA--an unusual cyanobacterial neurotoxin.

Abstract The toxin ss-N-methylamino-L-alanine (BMAA) was proposed to contribute to the ALS/Parkinsonism-dementia complex of Guam (ALS/PDC) based on its presence in cycad seeds, which constituted a dietary item in afflicted populations, and its ability to induce a similar disease phenotype in primates. Although the role of BMAA in human neurodegenerative disease is still highly debated, it appears to injure cultured neurons via mechanisms involving overactivation of neuroexcitatory glutamate receptors. However, BMAA lacks the side-chain acidic group of glutamate and other excitatory amino acids, and in its place has an amino group. In past studies we found that toxic and excitatory effects of BMAA on cultured neurons were dependent upon the presence of bicarbonate in the medium, and suggested that formation of a carbamate adduct of the side-chain amino group might produce structures capable of activating glutamate receptors. Also, while BMAA is a weal agonist at NMDA-type glutamate receptors, we found low levels of BMAA to selectively damage vulnerable sub-populations of neurons, including motor neurons, via activation of AMPA/kainate receptors. Recent reports that BMAA is produced by cyanobacteria in diverse ecosystems and is present in brain and spinal cord tissues from sporadic ALS and Alzheimer's patients as well as brains of ALS/PDC patients provide strong motivation for further investigations of its toxic mechanisms and contributions to human disease.

Cyanobacteria and BMAA: possible linkage with avian vacuolar myelinopathy (AVM) in the south-eastern United States.

Avian vacuolar myelinopathy (AVM) is a neurological disease that produces uncoordinated behavior in affected birds in wetland ecosystems of the south-eastern United States. Feeding and sentinel trials, field surveys, and genetic studies have implicated the introduced flowering plant species Hydrilla verticillata (Hydrocharitaceae) and an associated epiphytic cyanobacterial species (Order Stigonematales) as a causal link to AVM. All five morphotypes of cyanobacteria have been shown to produce the neurotoxic amino acid BMAA, including cyanobacteria of the Stigonematales that are epiphytic on Hydrilla verticillata. If biomagnification of BMAA occurs in these wetland ecosystems, as has been observed in the Guam ecosystem, then the consumption of fish (e.g. shad and herring) and waterfowl (e.g. Canada geese and mallards) from AVM-confirmed reservoirs in Arkansas, Texas, Georgia, North Carolina and South Carolina could represent a significant human health risk.

Human exposure to cyanobacteria and BMAA.

Cyanobacteria are found worldwide, primarily in aquatic habitats. They are increasing in abundance as a result of increasing nutrient inputs from various human activities. Recent data indicate that most cyanobacteria produce the neurotoxin beta-N-methylamino-L-alanine (BMAA), and this toxin can biomagnify UP some food chains to rather high concentrations in animals used as food by humans. BMAA may pose an increasing human health risk.

Cyanobacteria and BMAA exposure from desert dust: a possible link to sporadic ALS among Gulf War veterans.

Veterans of the 1990-1991 Gulf War have been reported to have an increased incidence of amyotrophic lateral sclerosis (ALS) compared to personnel who were not deployed. An excess of ALS cases was diagnosed in Gulf War veterans younger than 45 years of age. Increased ALS among Gulf War veterans appears to be an outbreak time-limited to the decade following the Gulf War. Seeking to identify biologically plausible environmental exposures, we have focused on inhalation of cyanobacteria and cyanotoxins carried by dust in the Gulf region, particularly Qatar. Cyanobacterial crusts and mats are widespread in the deserts of Qatar, occupying up to 56% of the available area in some microhabitats. These cyanobacterial crusts, which help bind the desert sands, are dormant throughout most of the year, but during brief spring rains actively photosynthesize. When disturbed by vehicular traffic or other military activities, the dried crusts and mats can produce significant dust. Using HPLC/FD, an amino acid analyzer, UPLC/MS, and triple quadrupole LC/MS/MS we find that the dried crusts and mats contain neurotoxic cyanobacterial toxins, including beta-N-methylamino-L-alanine (BMAA) and 2,4 diaminobutyric acid (DAB). If dust containing cyanobacteria is inhaled, significant exposure to BMAA and other cyanotoxins may occur. We suggest that inhalation of BMAA, DAB, and other aerosolized cyanotoxins may constitute a significant risk factor for the development of ALS and other neurodegenerative diseases.

Possible therapy for ALS based on the cyanobacteria/BMAA hypothesis.

Although the cyanobacteria/BMAA hypothesis of the cause of ALS and other age-related neurodegenerative diseases remains to be proven, it is not too early to ask whether treatment would be possible if the hypothesis were correct. This paper reviews the possible ways that chronic BMAA neurotoxicity could be prevented or treated.

Beta-N-methylaminoalanine (BMAA): metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro.

The non-protein amino acid, beta-N-methylaminoalanine (BMAA), is neurotoxic and has been implicated in the amyotrophic lateral sclerosis-Parkinsonism-dementia (ALS-PD) complex of Guam. This concept remains controversial, in part because of the lack of a convincing animal model. The neuropharmacology of BMAA is well established, but little is known of its metabolism. This paper reports aspects of the metabolism, and metabolic effects, of BMAA in rat tissues. BMAA changed the distribution of taurine, glycine and serine between rat brain slices and their incubation medium; the glutamate/glutamine cycle between neurones and glia was also compromised. In model experiments BMAA reacted non-enzymatically with pyridoxal-5'-phosphate, releasing methylamine. Rat liver and kidney homogenates, but not brain homogenates, also formed methylamine and 2,3-diaminopropanoic acid when incubated with BMAA. These results provide evidence that several biochemical mechanisms are involved in the neurotoxicity of BMAA. The novel discovery that methylamine is formed from BMAA in rat liver and kidney preparations may be significant since chronic administration of methylamine to rats causes oxidative stress. The extent to which this reaction occurs in different animal species might be a decisive factor in selecting an animal model.

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells.

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1alpha in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1alpha accumulation in different tumor cell lines. Hypoxia-induced HIF-1alpha accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F(0)F(1)-ATPase, prevented hypoxia-induced HIF-1alpha protein accumulation and had no effect on HIF-1alpha induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1alpha content was pronounced in cells exposed to hypoxia (1.5% O(2)) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1alpha regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1alpha accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function.

Aluminum L-glutamate complex in rat brain cortex: in vivo prevention of aluminum deposit by magnesium D-aspartate.

Our previous experiments in the rat showed that aluminum L-glutamate complex (Al L-Glu) crosses the blood-brain barrier and accumulates in selective brain areas and that Al salts may increase D-aspartic acid forms in living brain proteins, probably by inducing more thermodynamically stable structures than L isomers. As magnesium blocks NMDA receptors, D-aspartic acid was used in the present study in the form of magnesium salt to prevent the excitotoxicity of dicarboxylic amino acids. Effects on brain amino acids and Al cortex levels in mature rats were studied after chronic treatment with Al L-Glu or Na L-Glu alone or in association with magnesium D-aspartate (Mg D-Asp). Results demonstrate that treatment with Mg D-Asp induces a decrease in the Al concentration in brain cortex of Al L-Glu-treated rats. In aluminum-free treated controls, treatment with Mg D-Asp in association with Na L-Glu also induces a decrease in Al concentration in brain cortex. These data indicate that Mg D-Asp administration protects rat brain cortex from Al accumulation and suggest that this treatment may be useful in preventing brain Al intoxication.

Neuron damage induced by some potent kainoids and neuroprotective action of new agonists for metabotropic glutamate receptors.

Selective and characteristic neuron damage induced by acromelic acid, a potent kainate analogue, was investigated in comparison to a kainate-induced one. A single systemic injection of acromelic acid A caused behavioral and pathological effects distinct from those seen after systemic kainate. There was an initial marked tonic extension of the rat hindlimb, often followed by convulsions and, in surviving rats, by a transient flaccid paralysis and ultimately, a persistent spastic paraplegia. Pathological examination suggested specific lesions of interneurons in the lower spinal cord with little or no damage to the hippocampal neurons preferentially affected by systemic kainate. Another agonist for kainate-type receptors, which is not a kainoid, demonstrated neurological symptoms and neuron damage quite similar to those of kainate. Pharmacological actions of our newly developed agonists for metabotropic glutamate receptors were described with special reference to kainate excitotoxicity. Intraventricular DCG-IV, a new agonist, caused selective neuron damage in the cingulate cortex and the hippocampal subiculum at relatively high doses, but other agonists did not cause neuron damage in the rat. DCG-IV considerably alleviated the kainate-induced limbic seizures. At relatively low doses, DCG-IV protected some kinds of neurons in the hippocampal CA3 and the amygdala against kainate neurotoxicity, when intraventricularly injected to the rat. These new agonists would provide useful probe for elucidating the mechanism underlying neuron damage induced by kainate-type agonists.

2-(Carboxycyclopropyl)glycines: binding, neurotoxicity and induction of intracellular free Ca2+ increase.

The excitatory actions of the eight stereoisomers of 2-(carboxycyclopropyl)glycine (CCG), conformationally rigid glutamate analogues, were analyzed for the glutamate receptor subtypes by means of binding assays with rat brain membranes. All CCG isomers inhibited the binding of [3H]3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid ([3H]CPP) to N-methyl-D-aspartate (NMDA) receptors. The (2S,3R,4S) isomer (L-CCG-IV) was the most potent agonist for the NMDA receptor and its binding potency was 17- and 790-fold higher than that of L-glutamate and NMDA, respectively. The (2S,3S,4R) isomer (L-CCG-III) showed a potent inhibitory activity for [3H]D-aspartate uptake. Further, L-CCG-IV caused a marked increase of intracellular free Ca2+ concentration [( Ca2+]i) and potent neurotoxicity in the single rat cerebral cortical neurons in vitro, and both were blocked effectively by the NMDA antagonists. Significant correlations were observed between neurotoxicity and the increase of [Ca2+]i and [3H]CPP binding affinity to the NMDA receptor.

Astroglial ablation prevents MPTP-induced nigrostriatal neuronal death.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin which destroys nigrostriatal dopamine neurons, resulting in irreversible idiopathic parkinsonism. MPTP displays dopaminergic neurotoxicity to humans, monkeys, cats and rodents. The oxidative conversion of MPTP to 1-methyl-4-phenylpyridine (MPP+) is responsible for the generation of its neurotoxicity. This metabolism is mediated by the action of monoamine oxidase B, which in the substantia nigra pars compacta (SNc) is localized specifically in astroglia. Employing various combinations of intra-SNc injections of MPTP and the astroglia-specific toxin, L-alpha-aminoadipic acid (L-alpha-AA), we examined the effects of selective astroglial ablation on MPTP-induced nigrostriatal neuronal death in the rat. Varying nigrostriatal cell loss was assessed primarily by the aid of fluorescent retrograde axonal tracing. Treatment with MPTP alone caused tremendous nigrostriatal cell loss, while intra-SNc co-injections of MPTP and L-alpha-AA produced protection against MPTP neurotoxicity in a dose-dependent fashion. Similar effects of L-alpha-AA occurred in the SNc pretreated with the gliotoxin just prior to or 1 day before MPTP administration. However, this preventive action by L-alpha-AA was considerably reduced 3 days after its intra-SNc injection. Interestingly, 7 days following L-alpha-AA pretreatment, nigrostriatal cell loss was even enhanced rather than attenuated by MPTP administered into the SNc. Thus, our data provide clear morphological evidence for the critical importance of the presence of astroglia in the onset of MPTP neurotoxicity.

Gliotoxic effects of alpha-aminoadipic acid on monolayer cultures of dissociated postnatal mouse cerebellum.

The cytotoxic effects of DL-, D- and L-alpha-aminoadipic acid, a six-carbon homologue of glutamate, were investigated in cell cultures of dissociated postnatal mouse cerebellum. Treatment with alpha-aminoadipic acid resulted in rapid nuclear and cytoplasmic swelling and, after longer periods of exposure, karyopyknosis of astrocytes, identified by indirect immunofluorescence labelling with anti-human glial fibrillary acidic protein antiserum. The number of astrocytes with pyknotic nuclei depended on the concentration of alpha-aminoadipic acid as well as on the duration of drug action. The presence of 0.21 mM DL-alpha-aminoadipic acid or 0.10 mM L-alpha-aminoadipic acid for 40 h caused karyopyknosis in 50% of the astrocytes. In contrast, D-alpha-aminoadipic acid, had little gliotoxic activity. None of the cytotoxic effects of DL-alpha-aminoadipic acid or L-alpha-aminoadipic acid observed for astrocytes were seen for the neurons present in the cultures when the drug was added after 4 days in vitro. Neurotoxic effects were evident, however, when alpha-aminoadipic acid was included in the culture medium at plating. These results indicate that alpha-adminoadpic acid can be used to substantially reduce the number of astroglia in cerebellar cultures and that dissociated cell cultures will provide a useful model with which to study the mechanisms of alpha-aminoadipic acid induced glial toxicity.