Redox and methylation metabolites in aging and autism.

<p>(a) Redox and methylation pathway metabolites in control subjects of 0–20 yrs (n = 12) compared to subjects of 61–80 yrs (n = 10). (b) GSH/GSSG ratio (left) and SAM/SAH ratio (right) in aging. (c) Redox and methylation pathway metabolites in frontal cortex of autistic subjects (n = 9) compared to age-matched controls (n = 9). (d) GSH/GSSG ratio (left) and SAM/SAH ratio (right) in autism. * Indicates a significant difference from 0–20 yrs group (panels a and b) or control group (panels c and d) (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Cobalamin-related redox metabolic pathways in neuronal cells.

<p>Endocytosis brings TC-bound Cbl species to lysosomes where axial ligands are removed by MMACHC and MeCbl or AdoCbl are subsequently formed by SAM and ATP-dependent pathways, respectively. MeCbl is a required cofactor for methionine synthase, whose activity supports a large number of methylation reactions, including DNA methylation, as well as dopamine-stimulated phospholipid methylation, carried out by the D4 dopamine receptor (D4R). AdoCbl supports MMACoA mutase in mitochondria. Cysteine, which is rate-limiting for GSH synthesis, can be provided either by cellular uptake via the cysteine/glutamate transporter EAAT3 (excitatory amino acid transporter 3) or by transsulfuration of HCY via cystathionine. The latter pathway is restricted in human brain, increasing the importance of growth factor-dependent cysteine uptake by EAAT3.</p

Redox and methylation metabolite and cobalamin status in GCLM KO mice.

<p>(a) Redox and methylation metabolite levels in frontal cortex of GCLM KO mice at P40 and P90 (n = 7). Results are expressed as a percentage of the WT level of each metabolite. (b) Levels of five individual Cbl species in frontal cortex of GLCM KO and WT mice at P40 and P90. Inset indicates total Cbl levels. * Indicates a significant difference from control group (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Methionine synthase activity in autism.

<p>Methionine synthase activity in frontal cortex of autistic and age-matched control subjects measured either with only endogenous Cbl or with the addition of OHCbl. * Indicates a significant difference from control group (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Cobalamin status in human frontal cortex.

<p>(a) The general structure of Cbl species in which “X” represents various ligands linked to the cobalt atom, giving rise to the five different Cbl species measured in postmortem frontal cortex. (b) Total Cbl levels in frontal cortex of control subjects divided into four age groups: 0–20 yrs (n = 12), 21–40 yrs (n = 5), 41–60 yrs (n = 10) and 61–80 yrs (n = 12). (c) Levels of five individual Cbl species of control subjects in four age groups. (d) Age-dependent decrease of MeCbl in human frontal cortex (n = 43). Inset: Age trends of serum Cbl, frontal cortex total Cbl and MeCbl. Serum Cbl data is from Ref. 30. (e) Total Cbl levels in placenta (n = 6), frontal cortex of fetal (n = 4) and control (0–20 yrs) subjects (n = 12). (f) Levels of five individual Cbl species in placenta (n = 6), frontal cortex of fetal (n = 4) and control (0–20 yrs) subjects (n = 12). * Indicates a significant difference from 0–20 yrs group (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Redox and methylation-related pathways in neurons.

<p>Neurons can utilize either astrocyte-derived or transsulfuration-derived cysteine for GSH synthesis; however, higher levels of cystathionine in human brain indicate impaired transsulfuration, increasing the importance of the former cellular uptake pathway. GSH levels are a primary determinant of cellular redox status, and oxidative stress can inhibit MS activity by multiple mechanisms, as described herein, including oxidation of Cbl(I), down-regulation of transcription, or alternative mRNA splicing with deletion of the cap domain. Oxidative stress restricts dopamine-stimulated phospholipid methylation, since it is dependent upon MS activity.</p

MS protein status in human cortex.

<p>(<b>A</b>) Western blot analysis of MS protein revealed three primary bands with approximate MW values of 140, 113 and 101 kDa. A similar pattern was observed for young control subjects, age-matched autistic subjects, and elderly subjects. (<b>B</b>) Quantification of western blot data. The density of the three MS variants was not significantly different between the three groups (n = 8).</p

Domain structure and exon composition of cobalamin-dependent MS.

<p>(<b>A</b>) MS is comprised of five domains: HCY-binding (pink), methylfolate-binding (green), cap (yellow), cobalamin-binding (red) and SAM-binding (blue). Structures from <i>E.coli </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056927#pone.0056927-Dixon1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056927#pone.0056927-Bandarian2" target="_blank">[3]</a> and <i>T.maritima </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056927#pone.0056927-Evans1" target="_blank">[4]</a> (PDB codes 1Q8J, 1K98 and 1MSK, respectively were used to construct a composite model). A structurally uncharacterized linker segment between the folate and cap domains is absent. (<b>B</b>) The human MS gene contains 33 exons specifying its five domains in a sequential manner. The location of PCR sequences used in this study is indicated by red and black line segments below the corresponding domains.</p

MS mRNA variants in human cortex.

<p>(<b>A</b>) qRT-PCR was performed using cDNA from the cortex of fetal and 76 year old subjects, with primers spanning exons 13–26, yielding three products (a–c). (<b>B</b>) Exon composition of the full-length upper band (a) and the alternatively spliced middle band lacking exons 16–18 (b) were confirmed by sequencing; the proposed composition of the lower band is also illustrated (c).</p

Control subject clinical details and CAP and COB mRNA values.

<p>Control cortex samples from age 28 weeks to 84.7 years. A.U. = arbitrary units. PMI = postmortem interval.</p