Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 1. Cbln1 is specifically expressed by CSN residing in medial sensorimotor cortex. A, Schematic of the mouse brain and spinal cord, with inset delineating the three spatially, segmen- tally, and molecularly distinct CSN subpopulations: CSNBC-lat (blue) reside in rostro-lateral sensorimotor cortex and extend axons only to bulbar-cervical segments; CSNTL (red) reside in medial sensorimotor cortex and extend axons to thoraco-lumbar spinal segments; and CSNBC-med (purple) also reside in medial sensorimotor cortex and extend axons only to bulbar-cervical segments. CSNTL and CSNBC-med are both located in medial sensorimotor cortex, cannot be spatially distinguished, and are collectively referred to as CSNmedial. Klhl14 expression delineates Klhl14-positive CSNBC-lat from Klhl14-negative CSNmedial. Nearly all CSNTL express Crim1 while CSNBC-lat are Crim1-negative. B, Prior transcriptomic analysis comparing CSNBC-lat and CSNmedial gene expression identified Cbln1 as a gene that is not expressed by CSNBC-lat (blue) but whose expression increases from P1 to P7 in CSNmedial (red; Sahni et al., 2021a). C–E, In situ hybridization confirms that Cbln1 is expressed in Layer V, where CSN reside. Cbln1 expression increases from P1 to P7 and is restricted to medial Layer V throughout the rostro-caudal extent of sensorimotor cortex. Scale bars are 1 mm.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Expressing, Gene Expression, In Situ Hybridization

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 2. Cbln1 is expressed by CSN in Layer V. In situ hybridization at P7 shows that Cbln1 is expressed in medial Layer V in Fezf2 WT (A, C) but not in Fezf2 null (B, D) mice. Fezf2 null mice completely lack CSN (Chen et al., 2005; Molyneaux et al., 2005). This indicates that Cbln1 is expressed by CSN in Layer V in medial sensorimotor cortex. Scale bars are 100 mm for insets and 500 mm for other images.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: In Situ Hybridization

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 3. Time course of Cbln1 expression. Cbln1 is expressed throughout the rostro-caudal extent of sensorimotor cortex in medial but not lateral Layer V at P1 (A, B), P4 (C, D), P10 (E, F), and P14 (G, H). Cbln1 expression in Layer V is absent in P21 (I), P28 (J), and in more than three-month-old (K) mice. Scale bars are 100 mm for insets and 500 mm for all other images.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 4. Cbln1 is expressed by CSNTL and not by CSNBC-med. Dual fluorescence in situ hybridization for Cbln1 (white) and Crim1 (green; Crim1 expression marks CSNTL) and immunocytochem- istry for Ctip2 (red; high Ctip21 marks all CSN) were performed on coronal WT brain sections at P4. Sections were also stained for DAPI (blue, nuclei). Representative sections across the ros- tral-caudal extent of sensorimotor cortex are shown (A, B, D). In confocal insets (A*, B*, B#, D*), yellow arrowheads indicate examples of Cbln11, Crim11, high Ctip21 cells (CSNTL), while blue arrowheads indicate examples of Cbln11, Crim1, Ctip2 cells. Finally, pink arrowheads indicate examples of Cbln1, Crim1, and high Ctip21 neurons in either medial Layer V (A*, B*, D*), which correspond to CSNBC-med, or in lateral Layer V (B#), which correspond to CSNBC-lat. The overlap of Cbln1, Crim1, and Ctip2 expression was quantified in four mice in medial Layer V (C). We find that within CSNmedial (high Ctip21), Cbln11 neurons are almost exclusively Crim11, indicating that Cbln11 CSN are CSNTL. Scale bars are 500 mm.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Fluorescence, In Situ Hybridization, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 5. Cbln1 protein is present in CSN. (A) Cbln1 protein is detected in medial (A9) but not lateral (A0') Layer V, and co-localizes with high Ctip2+ neurons (CSN). (B) As a positive con- trol, we observe Cbln1 protein in the parafascicular nucleus of the thalamus, consistent with prior reports using the Cbln1 E3 antibody (Wei et al., 2007; Kusnoor et al., 2010). In confocal insets (A*, A#, B*), yellow arrowheads indicate examples of Cbln11, Ctip21 cells (CSNTL), blue arrowheads indicate examples of Cbln11, Ctip2 cells, and pink arrowheads indicate examples of Cbln1, Ctip21 cells (CSNBC-med). Apparent Cbln1 antibody staining in the upper layers of the somatosensory cortex is not specific when compared with secondary antibody only negative con- trol staining. Sections were also stained for DAPI (blue, nuclei). Scale bars are 500 mm.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 6. Other Cbln family members are not expressed by developing CSN. Prior differential gene expression analysis (Sahni et al., 2021a) identified Cbln1 (inset in I) as expressed by CSNmedial. In that dataset, Cbln2 (A), Cbln3 (E), and Cbln4 (I) exhibit no expression by CSNmedial (blue) or CSNBC-lat (red) at P1, P4, and P7. Chromogenic in situ hybridization at P1 (B, F, J), P4 (C, G, K), and P7 (D, H, L) confirms that Cbln3 and Cbln4 are not expressed in Layer V of the developing neocortex and that Cbln2 is mostly excluded from Layer V. Neurons expressing Cbln2 in Layer V are likely callosal projection neurons (Arlotta et al., 2005). Scale bars are 100 mm for insets and 500 mm for other images.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Gene Expression, Expressing, Chromogenic In Situ Hybridization

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 7. Cbln1 is not required for axon extension to lumbar L1–L2. A, Experimental outline: Retrograde labeling with injection of CTB-555 at lumbar L1–L2 was performed in Cbln1 WT, Cbln1 Het, and Cbln1 null mice at P5, and the number of retrogradely labeled neurons throughout the rostro-caudal extent of medial sensorimotor cortex was analyzed at P7. B, There is no qualitative difference in the proportion of retrogradely labeled CSN in medial sensorimotor cortex at rostral, middle, or caudal levels between Cbln1 WT or Het (n ¼ 3) and Cbln1 null (n ¼ 2) mice. C–H, Representative rostral, middle, and caudal sections from a Cbln1 Het and a Cbln1 null mouse. Scale bars are 100 mm for insets and are 500 mm for all other images. I, Experimental outline. Anterograde labeling via biotinylated dextran amine (BDA) iontophoresis into caudomedial sensorimotor cortex was performed in Cbln1 Het and Cbln1 null mice at P28. The number of BDA-labeled axons at cervical C1–C2, thoracic T1–T2, and lumbar L1–L2 was counted at P35 for three axial sections per spinal level, and the average number of counts is plotted. J, There is no qualitative difference in the proportion of axons that reach thoracic T1–T2 or lumbar L1–L2 from cervical C1–C2 between Cbln1 Het and Cbln1 null mice. K–P, Representative axial sections from cervical C1–C2, thoracic T1–T2, and lumbar L1–L2 in Cbln1 Het and Cbln1 null mice. Scale bars are 100 mm.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Labeling, Injection

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 8. Cbln1 overexpression in CSNBC-lat is sufficient to redirect axon extension to distal thoracic spinal segments. A, Experimental outline: In one set of experiments, plasmids were designed to express either EGFP alone, or both EGFP and Cbln1 (Cbln1 OE). The constructs were delivered to developing CSNBC-lat in lateral cortex using in utero electroporation at E12.5, and tis- sue was collected at P4 for analysis. CSNBC-lat axons were visualized using immunocytochemistry for EGFP. B, C, Electroporation location and distribution in the brain are well-matched between EGFP (control) and Cbln1 OE mice. D–G, The number of CSNBC-lat axons at cervical C1–C2 and thoracic T1–T2 were counted in axial sections of the spinal cord in EGFP and Cbln1 OE mice. H, The proportion of individual axons that reach thoracic T1–T2 from cervical C1–C2 is significantly higher in Cbln1 OE (red, n ¼ 4) compared with EGFP (control; blue, n ¼ 6) mice (p ¼ 0:01 by two-tailed Student’s t test). I, CST fluorescence intensity was quantified along the rostro-caudal extent of the thoracic cord and normalized to the fluorescence intensity at thoracic T2. Significantly more Cbln1-expressing CSNBC-lat axons extend into the distal thoracic cord when compared with EGFP controls (p ¼ 0:04 by two-way ANOVA with repeated measures followed by Fisher’s least significant difference post hoc test). J, K, CSNBC-lat axons extend into the distal thoracic cord in thoracic sagittal sections in Cbln1 OE (arrowheads in K’, K”) but not in EGFP (control) mice. L, Schematic indicating anatomic location of sections displayed in B–K. M, Experimental outline: In a second set of experiments, AAV particles engineered to express either EGFP alone (AAV-EGFP) or both EGFP and Cbln1 (AAV-Cbln1) were injected into rostrolateral sensorimotor cortex at P0 to test whether Cbln1 is sufficient to redirect axon extension by postmitotic CSNBC-lat. AAV-injected mice were then analyzed at P14. N–Q, The number of axons that reach cervical C1–C2 and thoracic T1–T2 were counted in axial sections of the spinal cord in AAV-EGFP and AAV-Cbln1 mice. R, There are significantly more axons that reach thoracic T1–T2 from cervical C1–C2 in AAV-Cbln1 (red, n ¼ 3) compared with AAV-EGFP (blue, n ¼ 3; p ¼ 0:02 by one-tailed Student’s t test). Scale bars are 100 mm for D–G and N–Q and 500 mm for B, C, J, and K. Each data point in H and R is the axon count averaged over three axial sections per spinal level per mouse.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Over Expression, Construct, In Utero, Electroporation, Immunocytochemistry, Control, Two Tailed Test, Fluorescence, Expressing, Injection, One-tailed Test

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 9. Cbln1 overexpression in CSNmedial is sufficient to increase the number of axons extending past the cervical spinal cord. A, Experimental outline: AAV particles engineered to express either EGFP alone (AAV-EGFP, control) or both EGFP and Cbln1 (AAV-Cbln1) were injected into medial sensorimotor cortex at P0. AAV-injected mice were analyzed at P14. B, CST intensity was quantified in three axial sections per spinal level (cervical C1–C2, thoracic T1–T2, and lumbar L1–L2) per mouse, and the mean value across three axial sections is plotted. The proportion of axons that reach thoracic T1–T2 from cervical C1–C2 (T1/C1) is significantly higher in AAV-Cbln1 (red, n ¼ 13) compared with AAV-EGFP (blue, n ¼ 15) (p ¼ 0:03 by one-tailed Student’s t test). In contrast, the proportion of axons that reach lumbar L1–L2 from cervical C1–C2 is not significantly different between AAV-Cbln1 and AAV-EGFP mice (p ¼ 0:11 by one-tailed Student’s t test). C, We modeled the distribution of T1/C1 in control AAV-EGFP or AAV-Cbln1-injected mice as a mixture of two Gaussians. The distribution of T1/C1 in control AAV-EGFP-injected mice appears bimodal with one Gaussian centered at 0:32 6 0:08 and the other at 0:57 6 0:08, likely reflecting variability in the proportion of CSNBC-med or CSNTL labeled by each injection. In con- trast, the distribution of T1/C1 in AAV-Cbln1-injected mice appears unimodal with one Gaussian centered at 0:54 6 0:27 and the other centered at 0:62 6 0:03. These Gaussians are similar to the Gaussian with control AAV-EGFP injections centered at 0.57, which is likely comprised of a higher proportion of CSNTL compared with CSNBC-med. This suggests that Cbln1 overexpression might specifically shift the segmental targeting of CSNBC-med past the bulbar-cervical cord into the thoraco-lumbar cord, but not overtly affect the segmental targeting of CSNTL. D–I, Representative axial sections from cervical C1–C2, thoracic T1–T2, and lumbar L1–L2 from control AAV-EGFP and AAV-Cbln1-injected mice. Scale bars are 100 mm.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Over Expression, Control, Injection, One-tailed Test, Labeling

Journal: The Journal of Neuroscience

Article Title: Cbln1 Directs Axon Targeting by Corticospinal Neurons Specifically toward Thoraco-Lumbar Spinal Cord

doi: 10.1523/jneurosci.0710-22.2023

Figure Lengend Snippet: Figure 10. Cbln1 expression is regulated by Klhl14 but not by Crim1. A, B, Coronal section of a P4 brain that was electroporated in utero at E12.5 with Klhl14 shRNA. A, EGFP fluorescence (green) shows the site of electroporation in lateral cortex. B, In situ hybridization image of the same section in (A) shows Cbln1 expression. Cbln1 is normally restricted to medial cortex. However, Klhl14 knock-down by shRNA causes ectopic Cbln1 expression in lateral cortex (arrowheads in B”) in the electroporated cortical hemisphere (compare B” with contralateral B’). C, D, Coronal section of a P4 brain that was electroporated in utero at E12.5 with a plasmid containing Cbln1 and EGFP (Cbln1-EGFP). C, EGFP fluorescence (green) shows the site of electroporation in lateral cortex. D, In situ hybridization image of the same section in C showing Klhl14 expression in Cbln1-misexpressing CSNBC-lat remains unchanged, indicating that Klhl14 expression in lat- eral Layer V is unaffected by Cbln1 misexpression. E, F, Coronal section of a P4 brain that was electroporated in utero at E12.5 with Cbln1-EGFP. E, EGFP fluorescence (green) shows the site of electroporation in lateral cortex. F, In situ hybridization image of the same section in E showing that there is no ectopic Crim1 expression in Cbln1-misexpressing CSNBC-lat. G, H, Coronal section of a P4 brain that was electroporated in utero at E12.5 with Crim1-EGFP. Crim1 misexpression in CSNBC-lat can redirect axons toward caudal thoracic spinal segments (Sahni et al., 2021b). G, EGFP fluorescence (green) shows the site of electroporation in lateral cortex. H, In situ hybridization image of the same section in (G) showing that there is no ectopic Cbln1 expression in Crim1-misexpressing CSNBC-lat. I, J, Crim1 expression in medial Layer V does not differ between Cbln1 WT and Cbln1 null mice. K, L, Cbln1 expression in medial Layer V does not differ between Crim1 WT and Crim1 null mice. M, Summary schematic displaying molecular controls over CSN axon extension both at, and beyond, the transition between cervical and thoracic spinal seg- ments. Together with the previous investigations identifying Crim1 and Klhl14 function (Sahni et al., 2021b), our data suggest a model whereby Cbln1 directs CSN axon extension from the cer- vical into the thoracic cord, whereas Crim1 directs those CSN axons that cross this transition zone to extend further toward caudal thoracic and lumbar spinal segments. This indicates that CSN segmental axon targeting toward thoracic and lumbar segments involves multiple, distinct molecular regulators acting at distinct spinal levels. Klhl14, which is specifically expressed in CSNBC-lat and restricts CSNBC-lat axon extension to the bulbar-cervical segments, represses the expression of both Cbln1 and Crim1 in CSNBC-lat. This indicates that Klhl14 represses a broad program of thor- aco-lumbar directed axon extension in CSNBC-lat. This program, mediated by multiple independent molecular controls, would otherwise direct CSN axons past the cervical cord toward caudal thoracic and lumbar segments. Scale bars are 100 mm for insets and 500 mm for all other images.

Article Snippet: The following day, we followed the RNAscope protocol for detecting Crim1 (C1, Opal 520, FP1487001KT; Akoya Biosciences) and Cbln1 (C2, Opal 650, FP1496001KT; Akoya Biosciences) gene expression followed by incubation with anti-rat Alexa Fluor 546 (Invitrogen; catalog #A-11081) for 3 h at room temperature.

Techniques: Expressing, In Utero, shRNA, Fluorescence, Electroporation, In Situ Hybridization, Knockdown, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: mRNA expression of CYP11A1 in human brain tissues . A , schematic diagram describing the cholesterol side-chain cleavage reaction by CYP11A1. qRT-PCR analyses of CYP11A1 in ( B ) total brain, cerebellum, cortex, parietal lobe, occipital pole, temporal lobe, and spinal cord and ( C ) human testes, adrenal glands, brain, and spinal cord. Data for brain and spinal cord in ( C ) are the same as the total brain and spinal cord data in ( B ), respectively. Gene expression is shown as relative expression to α-tubulin. Data are presented as mean ± SD, N = 3. Each data point represents a replicate of the same RNA sample. ( D – G ) RNAscope in situ hybridization analyses of human cerebellum and cerebral cortex tissue (male, 63-years-old). A chromogenic assay was performed with two targets: CYP11A1 ( red ) and myelin basic protein (MBP; teal ). Each punctate dot represents one molecule of mRNA. CYP11A1-positive cells can be found in the granule layer ( D ), molecular layer and Purkinje layer ( E ) of the cerebellum, as well as gray matter of the cortex ( F ), indicated by black arrows . No CYP11A1-positive cells were observed in white matter ( G ). Little colocalization between MBP and CYP11A1 can be observed. qRT-PCR, quantitative RT-PCR.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, RNAscope, In Situ Hybridization, Chromogenic Assay

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: RNA and protein expression of CYP11A1 and its cofactors in glial cells. A – C qRT-PCR analyses of CYP11A1, FDX1, and FDXR in human glial cells, with H295R-S1 as a positive control. Gene expression is shown as relative expression to α-tubulin. Data are presented as mean ± SD, N = 3. Each data point represents total RNA extracted from cells of a different passage for each cell line. D and E representative immunoblots of CYP11A1, FDX1, and FDXR in human glial cells, with MA-10 and H295R-S1 as positive controls. GAPDH was used as a loading control. The 15 μg of total cell lysate was loaded in each lane. No specific bands for CYP11A1 can be observed in glial cells. qRT-PCR, quantitative RT-PCR.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Gene Expression, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: Confocal images of H295R-S1, MGM-1, MGM-3, NHA, and HMC3 cells for CYP11A1 expression . Cells stained with anti-CYP11A1 antibody ( green ), with mitochondria labeled with Mitotracker Red and nucleus stained with DAPI ( blue ). Images were taken at 63× magnification. H295R-S1 cells stain very strongly for CYP11A1, which overlaps entirely with mitochondrial staining. Very faint CYP11A1 staining colocalizing with mitochondria can be seen in MGM-1, MGM-3, NHA, and HMC3 cells, with the strongest signal in MGM-3 cells. DAPI, 4′,6-diamidino-2-phenylindole; NHA, normal human astrocyte.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Expressing, Staining, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: Expression of CYP11A1, FDXR, and POR in MGM-1 cells after treatments that increase pregnenolone synthesis . A – C , qRT-PCR analysis of CYP11A1 expression ( A ) in addition to expression of CYP450 cofactors, FDXR ( B ) and POR ( C ), following treatment of 0.76 mM AMG, 50 μM ketoconazole (KC), or 50 μM 22(R)-hydroxycholesterol (22(R)-HC) for 2 h in MGM-1 cells. Gene expression is shown as relative expression to β-actin. Data are presented as mean ± SD, N = 3. D , representative immunoblot showing protein expression of CYP11A1, FDXR, and POR following the aforementioned treatments. β-Actin was used as a loading control. No significant changes were observed for either RNA or protein expression of CYP11A1, FDXR, and POR following AMG, KC, and 22(R)-HC treatments. AMG, aminoglutethimide; qRT-PCR, quantitative RT-PCR.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: RNA Expression of CYP11A1 variants in the human CNS and peripheral tissues and cells . qRT-PCR analyses of total CYP11A1 ( gray ), CYP11A1 variant 1 ( blue ), and CYP11A1 variant 2 ( red ) expression in human peripheral steroidogenic tissues ( A ), human CNS tissues ( B ), and cell lines ( C ). Gene expression is shown as relative expression to β-actin. Data are presented as mean ± SD. CNS, central nervous system; qRT-PCR, quantitative RT-PCR.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: RNA Expression, Quantitative RT-PCR, Variant Assay, Expressing, Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: Localization of CYP11A1 isoforms. Representative confocal images of MGM-1 cells transfected with CYP11A1a-myc-DDK or CYP11A1b-myc-DDK expression vector. Cells transfected with empty plasmid were used as a negative control. Cells were stained with anti-myc-tag antibody ( green ), with mitochondria labeled with Mitotracker Red and nuclei stained with DAPI ( blue ). Images were taken at 63× magnification. CYP11A1a is strictly localized to the mitochondria. CYP11A1b appears more dispersed throughout the cell and has limited colocalization with mitochondria. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, Staining, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: CYP11A1a but not CYP11A1b overexpression leads to increased pregnenolone production that can be inhibited by AMG . A and B , qRT-PCR analysis of CYP11A1 expression in MGM-1 cells transfected with empty plasmid (negative control) or expression vectors for CYP11A1a or CYP11A1b with Myc-DDK tag. Gene expression is shown as fold change versus negative control, normalized to the expression of β-actin. Data are presented as mean ± SD, N = 3. Each data point represents total RNA extracted from pooled cells from different passages. A , transfected MGM-1 cells showed more than 200-fold increase in total CYP11A1 mRNA expression. B , expression changes of CYP11A1 variant 1 exon 1 indicate that the correct CYP11A1 isoform had been expressed. C , representative immunoblots for myc-tag in transfected MGM-1 cells. β-Actin was used as a loading control. Thirty micrograms of protein were loaded into each lane. A band corresponding to CYP11A1a was observed at around 50 kDa and a band corresponding to CYP11A1b was observed at around 42 kDa. No specific bands could be observed in WT or transfection control MGM-1 cells. D and E , ELISA measurements of pregnenolone secreted by transfected MGM-1 cells, with or without 22(R)-hydroxycholesterol stimulation. F – I , ELISA measurements of pregnenolone secreted by transfected MGM-1 cells treated with different doses of AMG for 2 h under basal ( F and H ) and 22(R)-hydroxycholesterol stimulated ( G and I ) conditions. Each data point represents the average of one experiment, where each treatment was performed in triplicate within each experiment. Data are presented as mean ± SD, N = 3. Statistics performed compared to control ( D and E ) or to 0 mM AMG group ( F – I ). CYP11A1a+ cells synthesized significantly more pregnenolone than controls and CYP11A1b+ cells, which were significantly inhibited by AMG. CYP11A1b+ cells behaved similarly to WT MGM-1 cells for pregnenolone synthesis. (∗∗ p < 0.01, ∗∗∗ p < 0.001). AMG, aminoglutethimide; qRT-PCR, quantitative RT-PCR.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Negative Control, Gene Expression, Variant Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Synthesized

Journal: The Journal of Biological Chemistry

Article Title: The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

doi: 10.1016/j.jbc.2022.102110

Figure Lengend Snippet: Effect of iron chelation on pregnenolone secretion in MGM-1 cells . ELISA measurements of secreted pregnenolone when MGM-1 WT ( A , B , E , and F ) and CYP11A1a+ ( C and D ) cells were pretreated with different doses of deferoxamine ( A – D ) or deferiprone ( E – F ) for 24 h. Each data point represents the average of one experiment, where each treatment was performed in triplicate within each experiment. Data are presented as mean ± SD, N = 3. Statistics performed compared to the no treatment group within each panel. Deferoxamine significantly inhibited pregnenolone production in CYP11A1+ cells and 22(R)-HC stimulated WT cells but had no statistically significant effect on WT cells under basal conditions. Deferiprone significantly inhibited pregnenolone production at high doses in basal and 22(R)-HC stimulated WT cells. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). 22(R)-HC, 22(R)-hydroxycholesterol.

Article Snippet: The following primary antibodies were used: anti-CYP11A1 rabbit mAb (Cell Signaling Technology; #14217, 1:1000 dilution, antigen: human CYP11A1 N terminus), anti-CYP11A1 rabbit pAb (Proteintech; #13363-1-AP, 1:1000 dilution, antigen: CYP11A1 aa 1–300), anti-CYP11A1 rabbit pAb (Abcam; #ab75497, 1:500 dilution, antigen: human CYP11A1 aa 288–337), anti-CYP11A1 rabbit pAb (Abcam; #ab232763, 1:1000 dilution, antigen: CYP11A1 aa 350–521), anti-GAPDH rabbit mAb (Cell Signaling Technology; #2118, 1:2000 dilution), anti-FDXR rabbit pAb (Proteintech; #15584-1-AP, 1:1000 dilution), anti-ADX rabbit mAb (Abcam; #ab108257, 1:1000 dilution), antibeta actin mouse mAb (Abcam; #ab8226, 1:3000 dilution), anti-CYP reductase rabbit pAb (Abcam; #ab13513, 1:1000 dilution), and anti-Myc-tag rabbit mAb (Cell Signaling Technology; #2278S, 1:1000 dilution).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Cd59 and inflammation regulate Schwann cell development

doi: 10.7554/eLife.76640

Figure Lengend Snippet: ( A ) Bright-field images of whole-mount chromogenic in situ hybridization (CISH) showing cd59 expression (purple) in the floorplate (FP) and hypochord at 24 hours post fertilization (hpf). Schematic (right panel) indicates location of cd59 expression (purple) in FP, hypochord, and neural tube in transverse section. ( B ) Fluorescent in situ hybridization (FISH) (RNAscope; ACD) showing cd59 expression (cyan) in the FP (outlined in white dashed lines) and lack of expression in sox10:megfp -positive neural crest cells (NCCs) (orange) in transverse sections (z projection of 20 µm). DAPI-positive nuclei indicated in blue. ( C ) FISH (RNAscope; ACD) showing cd59 expression (cyan) in sox10:megfp -positive (orange) SC precursors (SCPs) at 36 hpf, immature SCs (iSCs) at 48 hpf, and SCs at 72 hpf on the posterior lateral line nerve (pLLN). Representative images each display a transverse section (z projection of 20 µm) of single SC on the pLLN. ( D ) Imaris renderings show cd59 puncta that are localized within the SC at each stage. Two viewpoints are provided to better visualize the location of the puncta. All florescent images were acquired with confocal imaging. Scale bars: ( A ) lateral view, 100 µm; transverse section, 25 µm; ( B ) 10 µm; ( C ) 5 µm. Artwork created by Ashtyn T. Wiltbank with Illustrator (Adobe).

Article Snippet: After staining, all slides were mounted in DAPI fluoromount-G (Southern Biotech) and were coverslipped (VWR).

Techniques: Chromogenic In Situ Hybridization, Expressing, In Situ Hybridization, RNAscope, Imaging

Journal: eLife

Article Title: Cd59 and inflammation regulate Schwann cell development

doi: 10.7554/eLife.76640

Figure Lengend Snippet:

Article Snippet: After staining, all slides were mounted in DAPI fluoromount-G (Southern Biotech) and were coverslipped (VWR).

Techniques: Expressing, Membrane, Transgenic Assay, Mutagenesis, Recombinant, Purification, Gel Extraction, Cloning, TA Cloning, Imaging, RNAscope, Multiplex Assay, In Situ, Reverse Transcription, Electron Microscopy, Labeling, Sequencing, In Situ Hybridization, Software