A patent application relating to the potential of L-serine to be used as a therapeutic agent for the treatment of neurodegenerative diseases has been submitted (PCT/US2012/066373). This patent does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials and there are no consultancies or additional products in development associated with it.
Conceived and designed the experiments: RAD KJR. Performed the experiments: RAD KJR. Analyzed the data: SAB. Contributed reagents/materials/analysis tools: KJR PAC. Wrote the paper: RAD PAC SAB KJR. Supervised the project: KJR PAC.
Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation
Protein translation is a highly efficient and accurate process for assembling the 20 standard amino acids into proteins. Error rates in translation are relatively rare (1 in 103 to 104) and rely on the ability of the system to discriminate between the 20 protein (or canonical) amino acids
During protein translation the genetic code is interpreted from information contained in the nucleic acid sequence in messenger RNA (mRNA) into the primary amino acid sequence of a polypeptide chain. Fidelity of protein synthesis, at the translational level, relies on the specificity of protein amino acid and cognate tRNA recognition by tRNA synthetases. In certain cases when two protein amino acids have a similar structure, a proofreading step checks the structural fidelity of an amino acid to the catalytic site of the tRNA synthetase and the bond is hydrolyzed if the wrong amino acid is attached
β-N-methylamino-
MRC-5 cells, a human lung fibroblast cell line, were from American Tissue and Cell Culture (ATCC, Virginia, USA). SH-SY5Y cells, a human neuroblastoma cell line, were from the European Collection of Cell Cultures (ECACC, Public Health England, UK). Human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cords as described previously
MRC-5 cells, (passage number 14–19), and SH-SY5Y cells, (passage number 30–32), were maintained in DMEM, or DMEM/Hams F12 respectively containing 10 % fetal bovine serum (FBS), 4 mM
MRC-5 cells were incubated with 3H-BMAA (31.25 nM) in Hank’s buffered salt solution (HBSS) containing 10% FBS. After 2, 4, and 16 hours, cells were washed three times in phosphate buffered saline (PBS) supplemented with 10% FBS and lysed by freeze thawing three times in Triton X-100 (0.1%). The protein concentration in the lysate was determined using the BCA assay
To examine the ability of individual amino acids to reduce incorporation or radiolabel into proteins, MRC-5 cells were incubated with 3H-BMAA (31.25 nM) for 16 hours in the presence of individual amino acids (250 µM) and the radiolabel present in the cell proteins assessed as before. All of the 20 protein amino acids (
SH-SY5Y cells were incubated with 3H-BMAA (31.25 nM) for 24 hours and cell proteins isolated by TCA (10%) precipitation. Proteins were washed three times in TCA (10%), rinsed in ice-cold acetone and re-dissolved in PBS. The amount of radiolabel released from the proteins (i.e. not TCA precipitable) after incubation at 37°C with DTT (1 mM) and SDS (2%) with DTT (DTT/SDS) was determined by LSC. Cell proteins were also incubated with pronase (2 mg/mL) for 48 hours in 100 µM Tris hydrochloric acid (HCl) buffer pH 8 containing 20 mM CaCl2 at 37°C or in HCl (12 M) for 12 hours and the release of radiolabel quantified relative to that of the buffer alone (for pronase) or water (for HCl). All protein samples were processed in triplicate.
After incubation with 3H-BMAA or non-labeled BMAA, cells were washed three times with PBS, lysed by freeze thawing in Triton X-100 (0.1%) and cell proteins precipitated in TCA (10%). Protein pellets were washed three times with TCA (10%) and hydrolyzed in boiling 6 M HCl at 110°C for 16 hours as previously described
Hydrolyzed samples as above were resuspended in 20 mM HCl. All samples were appropriately diluted for a balanced reaction before being derivatized with AQC and checked by dilution series and by evaluating double derivatized lysine relative to single derivatized lysine. Samples were analyzed using a triple quadrupole, heated electrospray ionization tandem mass spectrometer (HESI-MS/MS) instrument (Thermo Scientific Finnigan TSQ Quantum UltraAM, San Jose, CA) after separation with an Ultra High Pressure Liquid Chromatography (Waters Acquity-UHPLC) system with a Binary Solvent Manager, Sample Manager and a Waters AccQTag Ultra C18 column (part# 186003837, 2.1×100 mm) at 55°C.
Separation was achieved using gradient elution at 0.65 ml/min in aqueous 0.1% (v/v) formic acid (Eluent A) and 0.1% (v/v) formic acid in acetonitrile (Eluent B): 0.0 min = 99.1% A; 0.5 min = 99.1% A curve 6; 2 min = 95% A curve 6; 3 min = 95% A curve 6; 5.5 min = 90% A curve 8; 6 min = 15% A curve 6; 6.5 min = 15% A curve 6; 6.6 min = 99.1% A curve 6; 8 min = 99.1% A curve 6. Nitrogen gas was supplied to the HESI probe with a nebulizing pressure of 40 psi and a vaporization temperature of 400°C. The tandem mass spectrometer was operated under the following conditions: the capillary temperature was set at 270°C, capillary offset of 35, tube lens offset of 110, auxiliary gas pressure of 35, spray voltage 3500, source collision energy of 0, and multiplier voltage of -1654. A divert valve was used to deflect flow during the beginning and end of the gradient. The second quadrupole was pressurized to 1.0 mTorr with 100% argon. Product-ion analysis of BMAA used
Detection limits (LOD) and limits of quantification (LOQ) of BMAA on the LC-MS/MS were determined experimentally by injecting a dilution series of authenticated stock solutions at 4 concentrations (0.015, 0.15, 1.5, 15 µg/l). The EPA Method Detection Level (MDL) was used which defines the LOD as the minimum concentration of substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. The MDL (48 femtomoles) was calculated using the standard deviation of replicates multiplied by the t statistic with α = 0.01 and n = 1 degrees of freedom. The LOQ (0.48 picomoles) was calculated by multiplying the MDL by 10. For validation purposes, the standard curve for BMAA had the following equation: f(x) = 31887463155463x + 19212, r2 = 0.99). The intra-day precisions for the transition
MRC-5 cells were incubated in DMEM supplemented with 300 µM BMAA in the presence or absence of 300 µM
LDH release from cells was determined as described previously
Apoptosis or necrosis was measured by simultaneous staining with propidium iodide (PI) and Annexin V using the BD Pharminigen™ Annexin V–FITC apoptosis detection kit and flow cytometry performed as described previously
Statistical comparisons were made using Student’s unpaired two tailed T-tests in GraphPad Prism, version 4.0c.
Incubation of human MRC-5 fibroblasts with 3H-BMAA in culture medium depleted in amino acids resulted in a time-dependent increase in radiolabel in cell lysates (
Panel A, uptake of radiolabel by cells was expressed as disintegrations per minute (DPM) per µg of cell protein. Panel B, radiolabel in the cell protein fraction was expressed as DPM per µg of total cell protein. Values are mean +/− SD for three independent experiments (n = 3).
MRC-5, HUVEC and SH-SY5Y cells were incubated with 3H-BMAA with or without CHX (2 µg/ml). Cells proteins were isolated and the amount of radiolabel present in proteins (DPM per cell protein) determined and expressed as a percentage of control where control treatments were designated 100%. Parallel cultures of MRC-5 cells were incubated with 3H-leucine (41 nM) with or without CHX and the effects of CHX similarly determined (open bars). Values are mean +/− SD for three independent experiments (n = 3). Radiolabel incorporation in each cell type was compared with and without CHX using Student’s two-tailed t-test (***
To further examine the association between BMAA and cell proteins, we examined the ability of a range of treatments to release radiolabel from cell proteins. Radiolabeled cell proteins were generated by incubating SH-SY5Y cells with 3H-BMAA for 24 hours. The radiolabel could not be removed from the isolated cell proteins by incubation with a 100 fold molar excess of the reducing agent DTT or by heating with the detergent SDS in the presence of DTT (
SH-SY5Y cells were incubated with 3H-BMAA, cell proteins precipitated by TCA (10%) precipitation and the amount of radiolabel released from proteins (i.e. not TCA precipitable) after incubation with DTT or DTT and SDS quantified. Percentage of radiolabel remaining in proteins following treatment is expressed as a % of buffer alone. Cell proteins were also incubated with pronase or HCl and the release of radiolabel quantified relative to that of buffer alone (for Pronase) or water (for Acid). Values are mean +/− SD,
To determine if a specific protein amino acid was being replaced by BMAA, we examined competition between all 20 protein amino acids and 3H-BMAA for incorporation into cell proteins. We found that incorporation of 3H-BMAA into cell proteins was inhibited in the presence of
Panel A, Incorporation of radiolabeled BMAA was inhibited by
To further confirm that BMAA was present in cell proteins, we incubated MRC-5 cells with a range of concentrations of BMAA and analyzed the hydrolyzed cell proteins by tandem mass spectroscopy on a triple quadrupole LC/MS/MS. Retention times, unique daughter ion (
Panel A, a BMAA standard and a hydrolyzed protein sample (from MRC-5 cells treated with 250 µM BMAA for 24 hours) were run on triple quadrupole LC/MS/MS. Retention times, unique daughter ion (
Autofluorescence, indicative of protein aggregation, developed in cells incubated with 300 µM BMAA for 96 hours (
Panel A, Autofluorescence was observed in the cytosol and perinuclear regions of MRC-5 cells incubated with 300 µM BMAA. Panel B, autofluorescence was reduced when cells were co-incubated with 300 µM
Taken together these four different lines of evidence suggest that incorporation of BMAA into cell proteins is a protein synthesis-dependent process, which can be inhibited by
Protein misfolding, evidenced by the presence of aggregated and tangled proteins manifested by neuroanatomical abnormalities such as Bunina bodies, Lewy body inclusions, tauopathies, senile plaques, and neurofibrillary tangles, is increasingly viewed as an engine for disparate progressive neurodegenerative illnesses. Few environmental agents have been identified that can induce protein misfolding, subsequent protein aggregation, and apoptosis with the notable exception of prions which are believed to be responsible for Kuru, Creutzfeldt-Jakob Disease, or Bovine Spongiform Encephalopathy. We here report that unusual amino acids can, in a similar manner, induce protein misfolding through misincorporation for one of the regular 20 canonical amino acids.
Motor neurons, as a special class of post-mitotic cells, are particularly vulnerable to terminally aggregated proteins since they are unable to reduce the burden of protein aggregates by distributing them amongst daughter cells. This was evident in a study in which neurodegeneration was the primary pathology in a mouse with a minor translational proofreading defect
Our finding that BMAA can be misincorporated for serine in human proteins raises the possibility that such misincorporation results in neurodegenerative illness. Phosphorylation of proteins typically occurs on serine, threonine, tyrosine, and histidine residues. Some serine sites such as tau serine residue 422 or TAR DNA-binding protein 43 (TDP-43) serine residues 409/410 have been identified as being key to neuropathologies
Exposure of human beings to BMAA has been documented on Guam where the presence of protein-associated BMAA in cycad flour was first observed by Polsky
We found the misincorporation of BMAA into human proteins to be a protein-synthesis dependent process in primary human endothelial cells, and in human fibroblast and neuroblastoma cell lines since it was significantly reduced when protein synthesis was partially inhibited by the addition of CHX (
BMAA and other non-protein amino acids can mimic one of the 20 canonical amino acids and become mistakenly incorporated into proteins
Aggregated proteins are characteristic of neurodegenerative disease, hence they are sometimes known as “proteinopathies” owing to the deposition of aggregates of tau, α-synuclein, huntingtin, superoxide dismutase-1, or TDP-43, which characterize such human neurodegenerative disorders as frontotemporal degeneration, Parkinson’s disease, Lewy body disease, Huntington’s disease, and ALS. Our treatment of fibroblast cells with BMAA resulted in the development of cellular autofluorescence characteristic of protein aggregation (
BMAA incorporation into proteins was found to induce apoptosis in neuroblastoma cells
Following a single i.v. bolus injection of 14C-labelled BMAA into the femoral vein of rats, Xie
BMAA induces peptide changes in neonatal mice, with demonstrable cognitive deficits in the adult mice
Whilst the acute effects of BMAA on neuronal cells
The finding that
We thank D. M. Kinney, the Deerbrook Charitable Trust, and the University of Technology, Sydney.