Journal: Glia

Article Title: Prostaglandin D2 Synthase Controls Schwann Cells Metabolism and Peripheral Myelin Homeostasis

doi: 10.1002/glia.70137

Figure Lengend Snippet: In the absence of L‐PGDS, myelin protein expression is maintained. (a, b) qRT–PCR analyses on mRNA prepared from (a) 1 month old wild type littermate controls (WT) and l‐pgds −/− (KO) sciatic nerves and (b) 8 months old wild type littermate controls (WT) and l‐pgds −/− (KO) sciatic nerves. All samples were tested for Mbp or Mpz expression. In ( a ) Mbp expression is slightly increased in null mutants, whereas Mpz expression is comparable. Data have been normalized to 36B4 expression level and analyzed with the CFX Manager Software from Biorad. N = pool of 2 nerves/mice, 3 different mice/genotype. Error bars represent mean ± s.e.m. ( Mbp Unpaired t ‐test. p = 0.0502, t = 2.773, df = 4; MPZ : Unpaired t test analysis, p = 0.5796; t = 0.6020 df = 4). In (b) no differences in the expression levels were observed between the genotypes. Data have been normalized to 36B4 expression level and analyzed with the CFX Manager Software from Biorad. N = pool of 2 nerves/mice, 3 different mice/genotype. Error bars represent mean ± s.e.m. ( Mbp Unpaired t ‐test. p = 0.346, t = 1.067, df = 4; MPZ : Unpaired t test analysis, p = 0.1172; t = 1.992 df = 4). (c) Representative western blotting analyses on sciatic nerves prepared from 1 month old wild type littermate controls (WT) and l‐pgds −/− (KO) sciatic nerves. Lysates were tested for MBP, MPZ, and Tubulin (Tub) as a loading control. The graphs showed the quantification of the represented western blotting. Error bars represent mean ± s.e.m. N = 3 different mice/genotype. (d) Representative western blotting analyses on sciatic nerves prepared from 8 months old wild type littermate controls (WT) and l‐pgds −/− (KO) sciatic nerves. Lysates were tested for MBP, MPZ, and Tubulin (Tub) as a loading control. The graphs showed the quantification of the represented western blotting. Error bars represent mean ± s.e.m. N = 3 different mice/genotype. (e) Left panels: Representative immunofluorescence of organotypic mouse Schwann cell—neuronal cocultures myelinated for 14 days and then treated for an additional 7 days with 25 μM A‐T56. Middle panels: Representative immunofluorescence of mouse Schwann cells prepared from P2 sciatic nerves of l‐pgds −/− and wild type control littermate pups, seeded onto purified wild type DRG neurons. Cultures were allowed to myelinate for 35 days. Right panels: Representative immunofluorescence of primary rat Schwann cell cocultured with purified DRG neurons prepared E14.5 l‐pgds −/− and wild type control littermate allowed to myelinate for 7 days. At the end of the myelination, cultures were fixed and stained for MBP (fluorescein) and beta 3 tubulin (middle panels) or neurofilament (right and left panels) (rhodamine). Myelin degeneration was observed in AT‐56 treated cultures and in l‐pgds −/− mouse Schwann cells cocultured with wild type DRG neurons. Bar: 100 μm.

Article Snippet: Results were analyzed using the CFX Manager Software from Biorad according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Software, Western Blot, Control, Immunofluorescence, Purification, Staining

Journal: Glia

Article Title: Prostaglandin D2 Synthase Controls Schwann Cells Metabolism and Peripheral Myelin Homeostasis

doi: 10.1002/glia.70137

Figure Lengend Snippet: L‐PGDS regulates genes involved in lipid homeostasis and Schwann cell energetic metabolism. (a) Heat–map showing the main upregulated genes identified in RNAseq analyses of rat primary Schwann cells—mouse neuronal myelinating cocultures. After 14 days in myelinating conditions, cultures were treated for an additional 7 days with 25 μM AT‐56 or with DMSO as a control vehicle before processing (Alignments on rat genome with p < 0.001). (b) qRT–PCR analyses on mRNA prepared from rat Schwann cell–mouse neuronal cocultures myelinated for 14 days and then treated for an additional 7 days with 25 μM AT‐56 or with DMSO as vehicle control. The expression of Pdk4 , Hmgcs2 , Acsl3 , and Angptl4 is increased in AT‐56 treated cocultures. Data have been normalized to gapdh expression level and analyzed with the StepOne Software v2.3 for Pdk4 , Acsl3 , and Angptl4 (Applied Biosystems). The expression of Hmgcs2 has been normalized to TBP expression level and analyzed with the CFX Manager Software from Biorad. N = pool of at least 10 coverslips/condition, from 3 different rat Schwann cell–mouse neuronal cocultures. Error bars represent mean ± s.e.m. (Two–way ANOVA; pdk4 **** p < 0.0001 F (2, 12) = 256.5; Hmgcs2 : Unpaired t ‐test. * p = 0.0268, t = 3.418, df = 4; Acsl3 *** p = 0.0004 F (2, 12) = 15.92; Angptl4 **** p < 0.0001 F (2, 12) = 315.1). (c) qRT–PCR analyses on mRNA prepared from 8 months old wild type littermate controls (WT) and l‐pgds −/− (KO) sciatic nerves tested for Hmgcs2 and Bdh1 expression. Data have been normalized to 36B4 expression level and analyzed with the CFX Manager Software from Biorad. The expression of both genes is increased in MPZ‐Cre//l‐pgds flx/flx sciatic nerves. N = pool of 2 nerves/mice, 3 different mice/genotype. Error bars represent mean ± s.e.m. (Unpaired t ‐test. Bdh1 : ** p = 0.0055, t = 5.456, df = 4). (d) qRT–PCR analyses on mRNA prepared from 10 months old wild type littermate controls (WT) and MPZ‐Cre//l‐pgds flx/flx sciatic nerves tested for P dk4 and Hmgcs2 expression. Data have been normalized to 36B4 expression level and analyzed with the CFX Manager Software from Biorad. The expression of both genes is increased in MPZ‐Cre//l‐pgds flx/flx sciatic nerves. N = pool of 2 nerves/mice, 4 ( Pdk4 ) N = 6 ( Hmgcs2 ) different mice/genotype. Error bars represent mean ± s.e.m. (Unpaired t ‐test. Pdk4 * p = 0.0308, t = 2.808, df = 6; Hmgcs2 : * p = 0.0213, t = 2.854, df = 8). (e) STRING pathway analyses and functional enrichments identified genes involved in “cellular response to fatty acid” (GO:0071398) and in “regulation of lipid metabolic process” (GO:0019216) with a false discovery rate of 2.13e‐05. (f) STRING pathway analyses and functional enrichments identified genes involved in “mitochondrial part” (GO:0044429) with false discovery rate of 0.00094. (g) KEGG pathway analyses identify genes enriched in the PPARγ signaling pathway (false discovery rate 1.20e‐05). (h) Representative immunofluorescence of rat Schwann cell—mouse neuronal myelinated cocultures treated with 100 μM or 300 μM Arachidonic acid (complexed to BSA) for an additional 7 days after 14 days in myelinating conditions. At the end of the treatment, cultures were fixed and stained for MBP (rhodamine) and Neurofilament (fluorescein). Myelin degeneration was observed upon 300 μM Arachidonic acid treatment with no signs of axonal swelling. The same amount of BSA was added to control cocultures. Bar: 100 μm. (i, j) qRT‐PCR analyses on mRNA prepared from rat Schwann cell—mouse neuronal cocultures treated for additional 7 days with 100 μM or 300 μM Arachidonic acid after 14 days in myelinating conditions. Arachidonic acid induces the expression of Pdk4 (i ) and Hmgcs2 (j (KO)) mRNA. Data have been normalized to 36B4 ( Pdk4 ) and TBP ( Hmgcs2 ) expression level and analyzed with the CFX Manager Software from Biorad. N = pool of at least 10 coverslips/condition, from 3 different rat Schwann cell—mouse neuronal cocultures. Error bars represent mean ± s.e.m. (Unpaired t ‐test. Pdk4 : **** p < 0.0001, t = 38.16, df = 4; Hmgcs2 : * p = 0.0429, t = 2.92, df = 4).

Article Snippet: Results were analyzed using the CFX Manager Software from Biorad according to the manufacturer's instructions.

Techniques: Control, Quantitative RT-PCR, Expressing, Software, Functional Assay, Immunofluorescence, Staining