Journal: Brain

Article Title: In-depth analysis reveals complex molecular aetiology in a cohort of idiopathic cerebral palsy

doi: 10.1093/brain/awab209

Figure Lengend Snippet: Functional mechanism of defective TYW1. ( A ) Brain-section immunostaining of wild-type mice at postnatal 8 weeks, where Tyw1 (green) and NeuN (red) are colabelled, showing Tyw1 expression in neurons within the cortex, hippocampus and cerebellum. Blue: DAPI. Scale bar = 75 μm. ( B ) Immunostaining of normal human brain tissue microarrays, where TYW1 (green) and NeuN (red) are colabelled, revealing TYW1 expression in neurons within a variety of brain regions. Blue: DAPI. Scale bar = 50 μm. ( C ) EdU was intraperitoneally injected at 2 h into pregnant mice before euthanizing them to harvest embryos at E13.5. Cells incorporated with EdU were determined in the embryonic brain sections under fluorescent microscopy and were analysed with ImageJ software. Compared with those of WT littermates, significantly reduced percentages of EdU + areas were observed in the neocortex (NCX) of Tyw1 −/− embryonic brains, as well as in the medial ganglionic eminence (MGE) and lateral ganglionic eminence (LGE). Red: EdU. Blue: DAPI. n = 3 per genotype. For a specific sample, each measurement was performed twice in separate fields. ** P < 0.01, unpaired t -test. Scale bar = 100 μm. ( D ) EdU was injected into the pregnant mice carrying embryos at E15.5, which were observed after 72 h. The embryonic cortex was divided into 10 equally spaced bins along the vertical axis. The percentage of EdU + area in each bin was measured. In Tyw1 −/− mouse cortices, there were less EdU + cells in superficial layers (layers II–III) than those in WT mice, while the number of EdU + cells in the deep layer (layer VI) was greater than that in WT mice. Light blue: EdU. Blue: DAPI. n = 3 per genotype. Scale bar = 100 μm. ( E ) Brain-section immunostaining of mice at postnatal 8 weeks, where Cux1 + and Foxp2 + cells were labelled for neurons in superficial layers (layers II–III) and the deep layer (layer VI), respectively. Compared with those of WT mice, Tyw1 −/− mouse brains showed a significantly reduced thickness of superficial layers at both frontal and motor cortices, while in the deep layers, the reverse phenomenon occurred, albeit without statistical significance. WM: white matter. Green: Cux1. Red: Foxp2. Blue: DAPI. n = 3 per genotype. For a specific sample, each measurement was performed twice in separate fields. *** P < 0.001, n.s.= no Figure 4: Continued significant difference, unpaired t -test. Scale bar = 200 μm. ( F–G ) Proliferation, apoptosis and migration analysis of primary neurons from the cortices of E13.5 mice. ( F ) Quantification of the proliferative and apoptotic primary neurons from E13.5 mouse brain cortices. The primary neurons from the Tyw1 −/− mice showed significantly decreased proliferation (as indicated by EdU staining), while the intensity of apoptosis did not change significantly (as determined by TUNEL staining). *** P < 0.001, n.s.: no significant difference, unpaired t -test. Scale bar = 100 μm. ( G ) Quantification of migrating primary neurons from the E13.5 mouse brain cortex. In the transwell assay, the primary neurons from the Tyw1 −/− mice showed significantly decreased migration. Red arrows indicate cells that already migrated through pores, while arrowheads indicate cells that were migrating in pores. *** P < 0.001, n.s. = no significant difference, unpaired t -test. Scale bar = 250 μm. ( H ) Schematics of the mechanism of ribosomal frameshift under the conditions of wild-type and mutant alleles of TYW1 . GAA is the anticodon of tRNA Phe for phenylalanine, and UUU is the codon of mRNA for phenylalanine. In the four mRNAs, with normalized lengths, of SASS6 , NCAPD2 , CENPE and STIL , the positions of UUU codons are indicated by red bars. The introduction of mutated TYW1 led to promotion of a ribosomal frameshift at the interaction between tRNA Phe and mRNAs, resulting in the reduced production of a subset of proteins involved in cell cycling. ( I ) Measurements of mRNA and protein levels in the E13.5 mouse brain cortex. In the Tyw1 −/− group, the mRNA levels of Cenpe , Stil , Ncapd2 and Sass6 did not change significantly compared with those in the WT group. However, the protein levels of Cenpe, Stil, Ncapd2 and Sass6 showed significant reductions compared with those in the WT group, with remaining levels of 0.38, 0.63, 0.36 and 0.14, respectively. β-actin was used as the internal control for both RNA and protein measurements. Neither RNA nor protein of Tyw1 could be detected in the Tyw1 −/− group. MW (Tyw1) = 84 kDa. MW (Cenpe) =312 kDa. MW (Stil) =143 kDa. MW (Ncapd2) =157 kDa. MW (Sass6) =74 kDa and 65 kDa. MW (β-actin) = 43 kDa. Quantitative PCR with reverse transcription and western blotting data were from three independent experiments. * P < 0.05, *** P < 0.001, unpaired t -test.

Article Snippet: To detect expression of TYW1 and GPAM in human brain tissue, the normal brain tissue microarrays (BNC17011, Biomax, MD, USA) were prepared.

Techniques: Functional Assay, Immunostaining, Expressing, Injection, Microscopy, Software, Migration, Staining, TUNEL Assay, Transwell Assay, Mutagenesis, Control, Real-time Polymerase Chain Reaction, Reverse Transcription, Western Blot

Journal: Brain

Article Title: In-depth analysis reveals complex molecular aetiology in a cohort of idiopathic cerebral palsy

doi: 10.1093/brain/awab209

Figure Lengend Snippet: Functional mechanism of defective GPAM. ( A ) Brain-section immunostaining of wild-type mice at postnatal 8 weeks, where Gpam (green) and Gfap (red) are colabelled, showing Gpam expression in astrocytes within many brain regions. Blue: DAPI. Scale bar = 100 μm. ( B ) Immunostaining of the normal human brain tissue microarrays, where GPAM (green) and GFAP (red) are colabelled, revealing GPAM expression in astrocytes within a variety of brain regions. Blue: DAPI. Scale bar = 50 μm. ( C ) Brain-section immunostaining of the Gpam −/− mice at postnatal 8 weeks revealing a significantly reduced number of Gfap + cells in the medulla, as compared with that in WT mice. The same tendency was observed in other brain regions albeit without statistical significance. Green: Gfap. Red: NeuN. Blue: DAPI. Scale bar = 100 μm. n = 3 per genotype. For a specific sample, each measurement was performed twice in separate fields. ** P < 0.01, n.s.: no significant difference, unpaired t -test. ( D ) Flow cytometry of whole-brain cell suspension from P1 mice stained with Gfap, Ki67 and Caspase-3. Percentages of the Gfap + Ki67 + cells were significantly lower in the Gpam −/− mice brain compared with those in WT mice, indicating inhibited proliferation of astrocytes. Percentages of Gfap + Caspase-3 + cells showed no difference between the two groups, indicating no activated apoptosis. n = 8 per genotype with equal numbers of male and female mice. ** P < 0.01, n.s.: no significant difference, unpaired t -test. ( E ) Nissl staining of sagittal brain sections of mice at postnatal 8 weeks showing a significantly thinner white matter in the motor cortex of the Gpam −/− group, as compared with that of the WT group. The same tendency was observed in the frontal cortex, Figure 6: Continued albeit without statistical significance. Scale bar = 200 μm. n = 3 per genotype. For a specific sample, each measurement was performed four times in separate fields. ** P < 0.01, n.s.: no significant difference, unpaired t -test. ( F ) Brain-section immunostaining of mice at postnatal 8 weeks showing a lower density of myelin binding protein (Mbp) in the medulla of the Gpam −/− mice, compared with that of WT mice. Green: Mbp. Blue: DAPI. Scale bar = 100 μm. ( G ) Western blot of the mouse brain medulla showing significant reductions of Gfap and Mbp in the Gpam −/− group compared with those in the WT group. Gapdh and β-actin were used as controls. MW (Gfap) = 50 kDa; MW (Mbp) = 19 and 26 kDa, respectively. For a specific sample, each measurement was performed three times independently. * P < 0.05, unpaired t -test. ( H ) Schematics of the mechanism of GPAM involvement in lipid metabolism and its influence on astrocytic proliferation and oligodendrocytic myelination, by regulating the production of PA, PI, PC, PE and PS. GPAT4: glycerol-3-phosphate acyltransferase 4. G3P: glycerol-3-phosphate. LPA: lysophosphatidic acid. PA: phosphatidic acid. DAG: diacylglycerol. TAG: triacylglycerol. CDP-DAG: cytidine diphosphate diacylglycerol. PI: phosphatidylinositol. PC: phosphatidylcholine. PE: phosphatidylethanolamine. PS: phosphatidylserine. MBP: myelin binding protein. PLP: proteolipid protein. ( I ) Brain-medulla-section immunostaining showing significant reductions of Filipin, as well as Mbp and Gfap, in Gpam −/− mice compared with those in WT mice. The percentage of the Filipin + area per imaging field was proportional to the cholesterol abundance. Scale bar = 100 μm. n = 3 per genotype. For a specific sample, each measurement was performed four times in separate fields. * P < 0.05, unpaired t -test. ( J ) Quantification of PA, PC and PE levels in the brain medullas of mice at postnatal 8 weeks revealing a significant reduction in the Gpam −/− group compared with those in the WT group. n = 3 per genotype. For a specific sample, each measurement was performed twice or thrice. * P < 0.05, ** P < 0.01, unpaired t -test.

Article Snippet: To detect expression of TYW1 and GPAM in human brain tissue, the normal brain tissue microarrays (BNC17011, Biomax, MD, USA) were prepared.

Techniques: Functional Assay, Immunostaining, Expressing, Flow Cytometry, Suspension, Staining, Binding Assay, Western Blot, Imaging