Figure 1.
Uptake and incorporation of 3H-BMAA into proteins by MRC-5 cells.
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).
Figure 2.
Inhibition of incorporation of radiolabel into cell protein by cycloheximide.
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 (***P<0.001 ****P<0.0001).
Figure 3.
Recovery of radiolabel from cell proteins following incubation of cells with 3H-BMAA.
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, P<0.0001 using Student’s two-tailed t-test, three independent tests (n = 3).
Figure 4.
Inhibition of incorporation or radiolabel into cell proteins by l-serine.
Panel A, Incorporation of radiolabeled BMAA was inhibited by l-serine in a concentration dependent manner. Panel B, d-serine (D-SER) had no significant impact on the incorporation of BMAA (NS, P = 0.4419). l-serine significantly inhibited the incorporation of BMAA compared to control cells (CTRL, ***P = 0.0002) and d-serine (D-SER, **P<0.01). Panel C, there was a significantly (****P<0.001) greater incorporation of BMAA when all protein amino acids were omitted from the culture medium (ALL) compared to when none were (NONE). When only l-serine was omitted (l-SERINE) incorporation was restored to approximately 80% (****P = 0.0009). Student’s two-tailed T-test, values are mean +/− SD for three independent experiments (n = 3).
Figure 5.
Identification of BMAA in cell proteins using tandem mass spectrometry.
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 (m/z 258), and ratios of m/z transitions during collision-induced dissociation of the m/z 459 parent ion matched those of an authenticated BMAA standard. Panel B, linear (r2 = 0.99012) recovery of BMAA from the hydrolyzed proteins as determined by triple quad LC/MS/MS. Values are mean +/− SD from three independent incubations (n = 3).
Figure 6.
Incubation with BMAA results in the formation of autofluorescent bodies and apoptotic changes in cells.
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 l-serine. Cells were incubated in 6 well plates and showed consistent perinuclear and cytosolic autofluorescence of varying degrees of intensity in BMAA treated cultures. The overall intensity of the signal was reduced when cells were co-incubated with l-serine. Images are representative of at least 12 fields of view from triplicate cultures. Scale bar is 100 um. Panel C, incubation with BMAA induced significant (**P = 0.0044) apoptosis in SH-SY5Y cells compared to control cells, which could be abrogated when co-incubated with l-serine (***P = 0.0005) or CHX (**P = 0.002). Student’s two-tailed T-test, values are mean + SD from three independent incubations (n = 3).