Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Dietary folic acid prevents peripheral neuropathy in mouse models of neural tube defects and type 2 diabetes

doi: 10.1073/pnas.2528095123

Figure Lengend Snippet: Electrophysiology and in vitro characterization of peripheral neuropathy observed in 6-mo-old Shmt1 +/+ and Shmt1 −/− mice. ( A ) Work flow showing measurement of nerve conduction velocity (NCV) in mice that were randomly weaned to various diet groups. The control group consisted of Shmt1 +/+ mice weaned to AIN93G (2 mg/kg folic acid) only. Shmt1 −/− mice were randomly weaned to AIN93G diet or AIN93G + FA (8 mg/kg folic acid) or AIN93G + 0.1% dU (2 mg/kg folic acid, 0.1% deoxyuridine) for 6 mo followed by electrophysical and biochemical measurements. ( B ) Sciatic motor nerve conduction velocity comparison between female ( Left ) vs. male ( Right ) mice on different diets. Relative NCV (m/s) was plotted on the Y -axis. Each data point represents mean NCV per mouse, calculated from both left and right limbs using the formula: NCV m s = Distance between proximal and distal stimulation Latency of response for proximal stimulus - Latency of response for distal stimulus Latency of response times were recorded for three independent proximal and distal stimulations ( 24 ). On the X -axis, the diet/genotype group corresponding to NCV data are plotted. Data are shown as mean NCV ± SEM (n = 14 to 16 mice/group). Adjusted p-values were represented only where the comparisons were statistically significant ( P < 0.05). ( C ) Additional electrophysiology parameters were extracted for each functional response curve resulting from distal and proximal stimulations. Compound motor action potential (CMAP) is represented by the amplitude (mV) of functional response curve, while the duration of the response curve was also measured. Panel C ( Top and Bottom ) represent data from female and male mice, respectively. No change in CMAP or duration of response were observed for either male or female mice across the diet and genotype groups. ( D ) Sciatic nerve lysates extracted from mice on respective diet and genotype groups were probed with neuron-specific antibodies: neurofascin 155 (NF155, 155kD), myelin associated glycoprotein (MAG, 100kD), myelin basic protein (MBP, 18-21 kD) and internal loading control β3 tubulin (50 kD). Panel D ( Left ) shows semiquantitative immunoblots from N = 3 biological replicates of each diet/genotype group. Panel D ( Right ) shows densitometric analysis of the immunoblots on ImageJ. Relative quantitative expression, normalized to β3 tubulin was plotted on the Y -axis whereas, the diet/genotype groups were plotted on the X -axis. Bar graphs on the Right indicate relative expression ± SEM (n = 3 mice/group). Statistical significance, where applicable, was shown with asterix for *p adj = 0.016 and ***p adj = 0.0001. Changes were seen in MAG but none were observed for MBP or NF155 protein expression across diet/genotype groups for either sex. ( E ) Sciatic nerve mRNAs were extracted from Shmt1 +/+ and Shmt1 −/− female and male mice on AIN93G diet for 6 mo. RT-qPCR was performed to test transcriptional changes in the expression of Schwann cell transcription factors ( Sox10, Sox2, Krox20, Oct6, Mbp , and Mpz ) normalized against endogenous control 18S RNA. Data represent mean normalized mRNA expression ± SEM (n = 3 mice/group for female and n = 4 mice per group for male). Only expression of the Krox20 gene in Shmt1 −/− female was reduced (* P = 0.036).

Article Snippet: The membranes were blocked with blotting grade casein (Biorad) and incubated at 4 °C overnight with primary antibodies against myelin basic protein, MBP (Cell Signaling D8X4Q, 1:1,000), myelin associated glycoprotein, MAG (Cell Signaling D4G3, 1:15,000), neurofascin-155, NF155 (Cell Signaling D7B6O, 1:400), periaxin (SC-515672, 1:250), and loading control β3-tubulin (Cell Signaling D71G9, 1:15,000).

Techniques: In Vitro, Control, Comparison, Functional Assay, Western Blot, Expressing, Quantitative RT-PCR