Tissue localization of [¹⁴C]-labeled L-BMAA in a lactating mouse dam with discontinued nursing.

<p>Autoradiogram showing very high levels of radioactivity in the mammary glands (MG) and other tissues of a lactating dam that nursed for 2 h and was killed 24 h after injection of [¹⁴C]L-BMAA. Compare the level of radioactivity of the dam nursing for 24 h (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078133#pone-0078133-g001" target="_blank"><b>Figure 1A</b></a>). The high retention of labeled substance in the mammary glands and all other tissues in the dam with discontinued nursing is due to interruption of milk excretion. White areas correspond to high levels of radioactivity.</p

Influx of [¹⁴C]-labeled L- and D-BMAA in mammary gland HC11 cells.

<p>Levels of radioactivity in cultured undifferentiated and differentiated mammary gland cells exposed to [¹⁴C]-labeled BMAA (1 µM) during 15 min. [¹⁴C]L-BMAA is taken up at a significantly higher rate than [¹⁴C]D-BMAA in both undifferentiated and differentiated cells. Differentiation of the mammary gland cells to a secretory state significantly increases the uptake of [¹⁴C]L- but not [¹⁴C]D-BMAA. Mean values (cpm/mg protein) from seven experiments ± SD are plotted. ***<i>p</i><0.001 compared to undifferentiated cells, and ^###<i>p</i><0.001 compared to D-BMAA within same cell phenotype.</p

Levels of [¹⁴C]-labeled L- and D-BMAA in pup stomach milk and brain following administration to the nursing dams.

<p><b>A</b> Levels of radioactivity in coagulated stomach milk in suckling mouse pups nursed by dams given a single injection of [¹⁴C]L- and [¹⁴C]D-BMAA. The level of L-BMAA in the milk is significantly higher than D-BMAA suggesting a high secretion of the L-enantiomer into milk. <b>B</b> Levels of radioactivity in the brain of suckling mouse pups nursed by dams given a single injection of [¹⁴C]L- and [¹⁴C]D-BMAA. The level of L-BMAA in the brain of the pups is significantly higher compared with D-BMAA. At 24 h there were significantly higher levels of L-BMAA in the brains of suckling pups compared to the levels in the maternal brains. At the end of the experiment the levels of L-BMAA in the maternal brains were significantly higher than the levels of D-BMAA. Mean values (cpm/g coagulated milk or tissue) ± SD are plotted. *<i>p</i><0.05 compared to D-BMAA within each time point, #<i>p</i><0.05 compared to their pups, and φ φ <i>p</i><0.01 compared to dams treated with L-BMAA (Mann-Whitney <i>U</i>-test). The data analysis is based on the dam as a statistical unit (2 pups/dam/time point).</p

Tissue localization of [¹⁴C]-labeled L- and D-BMAA in nursing mouse dams.

<p>Autoradiograms showing high levels of radioactivity in the mammary glands (MG) of nursing dams 24 h after injection of [¹⁴C]L-BMAA (<b>A</b>, <b>B</b>) or [¹⁴C]D-BMAA (<b>C</b>, <b>D</b>). Enlargements of the posterior mammary glands are shown in <b>B</b> and <b>D</b>. White areas correspond to high levels of radioactivity. BM  =  bone marrow, St  =  stomach, *  =  glandular part of stomach, In  =  lower gastrointestinal tract, Ki  =  kidney, Li  =  liver.</p

Tissue localization of [¹⁴C]-labeled L- and D-BMAA in suckling pups following a single administration to the nursing dams.

<p>Autoradiograms showing very high levels of [¹⁴C]L- and [¹⁴C]D-BMAA enantiomers in coagulated milk filling the stomach (St) of pups nursed for 8 or 24 h. White areas correspond to high levels of radioactivity. Th  =  thymus, He  =  heart, St  =  stomach, In  =  lower gastrointestinal tract, Ki  =  kidney, Li  =  liver, Sp  =  spleen.</p

Uncovering Effects of Ex Vivo Protease Activity during Proteomics and Peptidomics Sample Extraction in Rat Brain Tissue by Oxygen-18 Labeling

In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen-18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations

High Resolution Metabolite Imaging in the Hippocampus Following Neonatal Exposure to the Environmental Toxin BMAA Using ToF-SIMS

The environmental neurotoxin β-<i>N</i>-methylamino-l-alanine (BMAA) is suggested to be linked with neurodegenerative disease. In a rat model, neonatal exposure to BMAA induced selective uptake in the hippocampus and caused cell loss, mineralization and astrogliosis as well as learning and memory impairments in adulthood. Moreover, neonatal exposure resulted in increased protein ubiquitination in the cornus ammonis 1 (CA1) region of the adult hippocampus indicating that BMAA may induce protein aggregation. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) based imaging is a powerful technology for spatial profiling of small molecular weight compounds in biological tissues with high chemical specificity and high spatial resolution. The aim of this study was to characterize neurochemical changes in the hippocampus of six month-old rats treated neonatally (postnatal days 9–10) with BMAA. Multivariate data analysis of whole section ToF-SIMS scans was performed to delineate anatomical regions of interest based on their chemical distribution pattern. Further analysis of spectral data obtained from the outlined anatomical regions, including CA1 and dentate gyrus (DG) revealed BMAA-induced long-term changes. Increased levels of phospholipids and protein fragments in the histopathologically altered CA1 region as well as phosphate depletion in the DG were observed. Moreover, high resolution SIMS imaging revealed a specific localization of phosphatidylcholine lipids, protein signals and potassium in the histopathologically altered CA1. These findings demonstrate that ToF-SIMS based imaging is a powerful approach for probing biochemical changes in situ and might serve as promising technique for investigating neurotoxin-induced brain pathology

Neurotoxin-Induced Neuropeptide Perturbations in Striatum of Neonatal Rats

The cyanobacterial toxin β-<i>N</i>-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9–10) with BMAA (40–460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain

Neurotoxin-Induced Neuropeptide Perturbations in Striatum of Neonatal Rats

The cyanobacterial toxin β-<i>N</i>-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9–10) with BMAA (40–460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain

Neurotoxin-Induced Neuropeptide Perturbations in Striatum of Neonatal Rats

The cyanobacterial toxin β-<i>N</i>-methylamino-l-alanine (BMAA) is suggested to play a role in neurodegenerative disease. We have previously shown that although the selective uptake of BMAA in the rodent neonatal striatum does not cause neuronal cell death, exposure during the neonatal development leads to cognitive impairments in adult rats. The aim of the present study was to characterize the changes in the striatal neuropeptide systems of male and female rat pups treated neonatally (postnatal days 9–10) with BMAA (40–460 mg/kg). The label-free quantification of the relative levels of endogenous neuropeptides using mass spectrometry revealed that 25 peptides from 13 neuropeptide precursors were significantly changed in the rat neonatal striatum. The exposure to noncytotoxic doses of BMAA induced a dose-dependent increase of neurosecretory protein VGF-derived peptides, and changes in the relative levels of cholecystokinin, chromogranin, secretogranin, MCH, somatostatin and cortistatin-derived peptides were observed at the highest dose. In addition, the results revealed a sex-dependent increase in the relative level of peptides derived from the proenkephalin-A and protachykinin-1 precursors, including substance P and neurokinin A, in female pups. Because several of these peptides play a critical role in the development and survival of neurons, the observed neuropeptide changes might be possible mediators of BMAA-induced behavioral changes. Moreover, some neuropeptide changes suggest potential sex-related differences in susceptibility toward this neurotoxin. The present study also suggests that neuropeptide profiling might provide a sensitive characterization of the BMAA-induced noncytotoxic effects on the developing brain