Figure 1.
Increased cell death in Ung−/− neurons to methotrexate and homocysteine: reversal by folic acid supplementation. Neocortical (CTX) and hippocampal (HIP) neurons were exposed to the dihydrofolate-reductase inhibitor methotrexate (MTX) or vehicle.
A
, Increase in LDH activity relative to vehicle-treated sister cultures. Two-way ANOVA (72 h) for genotype: F
(1,150) = 9.8, p < 0.001 in CTX and F
(1,70) = 292.3, p < 0.001 in HIP; treatment: F
(1,150) = 27.3, p < 0.001 in CTX and F
(1,70) = 69.1, p < 0.001 in HIP; genotype × treatment interaction: F
(1,150) = 5.2, p < 0.001 in CTX and F
(1,70) = 28.2, p < 0.001 in HIP.
B
, MTT was measured as a metabolic activity marker and presented as loss of MTT metabolism relative to vehicle-treated sister cultures. ANOVA for genotype: F
(1,70) = 0.6, p = 0.5 in CTX and F
(1,70) = 305.5, p < 0.001 in HIP; treatment: F
(1,70) = 11.3, p < 0.001 in CTX and F
(1,70) = 101.4, p < 0.001 in HIP; interaction: F
(1,70) = 4.0, p = 0.006 in CTX and F
(1,70) = 42.2, p < 0.001 in HIP.
C
,
D
, Viable versus damaged neurons were identified by phase-contrast microscopy and propidium iodide counterstaining. Viable neurons are presented as a percentage of all neurons/high-power field. ANOVA for genotype: F
(1,40) = 33.1, p < 0.001 in CTX and F
(1,40) = 232, p < 0.001 in HIP; treatment: F
(1,40) = 35.7, p < 0.001 in CTX and F
(1,40) = 56.9, p < 0.001 in HIP. Interaction: F
(1,40) = 9.8, p < 0.001 in CTX and F
(1,40) = 27, p < 0.001 in HIP.
E–G
, CTX and HIP neurons were exposed to either HC plus vehicle or HC plus FA. Basal FA concentration in Neurobasal medium is 4 mg/L (∼10 μm).
E
, Increase in LDH activity relative to vehicle-treated sister cultures. Two-way ANOVA (72 h) for factor genotype: F
(1,28) = 8.2, p = 0.008 in CTX and F
(1,28) = 29.3, p < 0.001 in HIP; treatment: F
(1,28) = 3.3, p = 0.017 in CTX and F
(1,28) = 13.4, p < 0.001 in HIP; genotype × treatment interaction: F
(1,28) = 0.711, p = 0.7 in CTX and F
(1,28) = 3.1, p = 0.026 in HIP.
F
,
G
, Viable versus damaged neurons were identified by phase-contrast microscopy and propidium iodide counterstaining. Viable neurons are presented as a percentage of all neurons/high-power field. ANOVA for genotype: F
(1,32) = 0.7, p = 0.40 in CTX and F
(1,32) = 39, p < 0.001 in HIP; treatment: F
(1,32) = 1.5, p = 0.25 in CTX and F
(1,32) = 38.1, p < 0.001 in HIP. Interaction: F
(1,32) = 0.5, p = 0.7 in CTX and F
(1,32) = 10.4, p < 0.001 in HIP. Scale bars:
D
,
G
, 50 μm.
A–F
, #
p < 0.05 for neocortical and *p < 0.05 for hippocampal neurons, Ung−/− versus Ung+/+ within the same MTX, HC, or HC plus FA treatment condition.
E
,
F
, +
p < 0.05 versus corresponding HC without FA. Baseline LDH release and MTT metabolism are given in supplemental Tables 2A (for
A
,
B
) and 2B (for
E
) (available at www.jneurosci.org as supplemental material). All experiments were performed at least in triplicate.