cdc42ep2  (Proteintech)

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Septins, CDC42, and CDC42‐effector proteins CDC42EP1/CDC42EP2 in oligodendrocytes and myelin. (A, A′) Representative electron micrographs of optic nerves cross‐sectioned at P75 show that mice lacking Septin8 (A′) but not control (A) mice develop pathological myelin outfoldings in the CNS, in agreement with prior quantitative assessment (Patzig et al. ). Myelin outfolding highlighted in red in (A′); asterisk indicates an axon associated with a myelin outfolding. TEM, transmission electron microscopy. (B) Violin plot of the abundance of the indicated transcripts in mature oligodendrocytes (MOL). Re‐analysis of previously published scRNA‐seq data ( GSE75330 ; Marques et al. ). Each datapoint represents one out of 998 cells designated as mature oligodendrocytes (MOL; clusters MOL1‐MOL6 in Marques et al. ) in C57Bl/6 mice. Note that Cdc42 , Cdc42ep1 , and Cdc42ep2 mRNAs are abundant in MOL when compared to Cdc42ep3 , Cdc42ep4 , and Cdc42ep5 mRNAs. For bulk RNA‐seq data of oligodendrocytes immunopanned from mouse cortices (Zhang et al. ), see Figure . (C) Protocol of myelin purification via sucrose density gradient centrifugation and osmotic shocks. For details see (Erwig et al. ). (D) Immunoblots detecting CDC42, CDC42EP1, CDC42EP2, and all myelin septin filament subunits (SEPTIN2, SEPTIN4, SEPTIN7, SEPTIN8) in myelin purified from brains of C57B/6 N‐mice at postnatal day 20 (P20), P45, and P76. Fast Green serves as loading control. n = 2 mice per age. (E) Immunoblotting reveals reduced abundance of CDC42EP1 and CDC42EP2 and validates diminishment of myelin septins in myelin purified from brains of Septin8 −/− mice at P75 compared to controls (Ctrl). Fast Green as loading control. n = 3 mice per genotype.

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Control, Transmission Assay, Electron Microscopy, RNA Sequencing, Purification, Gradient Centrifugation, Western Blot

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Cdc42 ‐deletion in oligodendrocytes of adult mice impairs myelin structure and alters protein composition. (A–F) Representative electron micrographs (A, A′) and genotype‐dependent quantification (B–F) of cross‐sectioned optic nerves showing myelin pathology in Cdc42 flox/flox ; Plp CreERT2 (icKO) compared to control (Ctrl) mice 4 months post tamoxifen injection (mo PTI). (A, A′) Myelin pathology highlighted in red; asterisks indicate associated axons. (B) Quantitative analysis of electron micrographs of optic nerves 4, 8, and 10 months PTI reveals normal axon density in Cdc42 ‐icKO mice. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (4 months PTI p = 0.441, 8 months PTI p = 0.282, 10 months PTI p = 0.875). C Quantitative analysis shows moderately but significantly reduced percentage of myelinated axons in Cdc42 ‐icKO mice 8 months PTI but not 4 or 10 months PTI. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (4 months PTI p = 0.400, 8 months PTI p = 0.005, 10 months PTI p = 0.537). D Quantitative analysis identifies increased percentage of axon/myelin‐units with myelin outfoldings in Cdc42 ‐icKO mice. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t‐test with Holm‐Šídák correction (4 months PTI p = 0.0135, 8 months PTI p = 0.0163, 10 months PTI p = 0.0005). (E) Quantitative analysis reveals increased percentage of axon/myelin‐units with myelin whorls in Cdc42 ‐icKO mice. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (4 months PTI p = 0.003, 8 months PTI p = 0.015, 10 months PTI p = 0.002). F Quantitative analysis shows increased percentage of axon/myelin‐units that display other pathology 8 and 10 months PTI. Mean ± SEM; datapoints represent individual mice; n = 3–5 mice; multiple unpaired t test with Holm‐Šídák correction (4 months PTI p = 0.263, 8 months PTI p = 0.017, 10 months PTI p = 0.0004). (G, H) Differential proteome analysis comparing the relative abundance of proteins in myelin purified from brains of Cdc42 ‐icKO and control (Crtrl) mice 10 months PTI. (G) Heatmap shows mass spectrometric quantification of known myelin constituents in three biological replicates (M1, M2, and M3) as the average of two technical replicates each, compared to the mean of Ctrl. Note that CDC42, myelin septins (SEPTIN2, SEPTIN4, SEPTIN7, SEPTIN8), and the septin‐associated adaptor protein anillin (ANLN) are diminished in myelin when oligodendrocytes lack Cdc42 . (H, H′) Volcano plots summarizing genotype‐dependent quantitative myelin proteome analysis. Data points represent relative abundance of proteins quantified in myelin of Cdc42 ‐icKO compared to Ctrl mice 10 months PTI. Data points are plotted as log2‐transformed fold‐change on the x‐axis against the −log10‐transformed q value on the y‐axis according to two different data acquisition modes (see Methods section for details) that is, MS E (H; 391 proteins) and UDMS E (B′; 535 proteins). The vertical stippled lines mark a log 2 ‐fold change of 0.5 or −0.5 threshold of changed protein abundance in myelin, and the horizontal stippled line indicates a −log 10 ‐transformed q‐value of 1.3 as significance threshold. Data points representing myelin septin subunits (SEPT2, SEPT4, SEPT7, and SEPT8) and CDC42 are highlighted in red with protein names given; their abundance is strongly reduced in Cdc42 ‐icKO compared to Ctrl myelin. Note that CDC42EP1 and CDC42EP2 are not identified by mass spectrometry in the present dataset. For dataset and exact q values see data Table . (I) Immunoblotting validates reduced abundance of CDC42 and myelin septins and reveals diminishment of CDC42EP1 and CDC42EP2 in myelin purified from Cdc42 ‐icKO mice 10months PTI. Fast Green as loading control. Blots show n = 3 mice per genotype.

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Control, Injection, Purification, Transformation Assay, Quantitative Proteomics, Mass Spectrometry, Western Blot

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Myelin protein composition is altered when Cdc42ep1 and Cdc42ep2 are lacking from oligodendrocytes. (A, B) Differential proteome analysis comparing the relative abundance of proteins in myelin purified from brains of Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox Cnp Cre/wt (dcKO) and control (Ctrl) mice at 20 weeks of age. A heatmap shows mass spectrometric quantification of known myelin constituents in three biological replicates (M1, M2, and M3) as the average of two technical replicates each, compared to the mean of Ctrl. Note that the abundance of myelin septins (SEPTIN2, SEPTIN4, SEPTIN7, and SEPTIN8), the septin‐associated adaptor protein anillin (ANLN), and CDC42 in myelin is reduced when oligodendrocytes lack Cdc42ep1 and Cdc42ep2 . (B) Volcano plots summarizing genotype‐dependent quantitative myelin proteome analysis. Data points represent relative abundance of proteins quantified in myelin of dcKO compared to Ctrl mice. Data points are plotted as log2‐transformed fold‐change on the x‐axis against the −log10‐transformed q value on the y‐axis according to two different data acquisition modes (see Section for details) that is, MS E (B; 459 proteins) and UDMS E (B′; 728 proteins). The vertical stippled lines mark a log 2 ‐fold change of 0.5 or −0.5 threshold of changed protein abundance in myelin, and the horizontal stippled line indicates a −log 10 ‐transformed q‐value of 1.3 as significance threshold. Data points representing myelin septin subunits (SEPTIN2, SEPTIN4, SEPTIN7, SEPTIN8), ANLN, and CDC42 are highlighted in orange with protein names given; their abundance is strongly reduced in dcKO compared to Ctrl myelin. Note that CDC42EP1 is identified by mass spectrometry in EP2 cKO and Ctrl samples in the present UDMS E data set; CDC42EP2 is not identified. For dataset and exact q‐values see data Table . (C) Immunoblot validates reduced abundance of myelin septins and CDC42 and virtual absence of CDC42EP1 and CDC42EP2 in myelin purified from Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO) mice compared to control (Ctrl) mice. Fast Green serves as loading control. (D) qRT‐PCR to determine the abundance of mRNAs encoding myelin septin subunits ( Septin2 , Septin4 , Septin7 , and Septin8 ) and the septin‐associated adaptor anillin ( Anln ) in the white matter (corpora callosa) of dcKO and Ctrl mice at 26 weeks of age. Mean ± SEM; datapoints represent individual mice; n = 5 mice per genotype. Data did not pass Shapiro–Wilk test for normality; unpaired Mann–Whitney test with nonparametric design and 0.05 threshold for p value comparisons ( Septin2 p = 0.547, Septin4 p = 0.690, Septin7 p > 0.999, Septin8 p = 0.134, and Anln p = 0.547).

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Purification, Control, Transformation Assay, Quantitative Proteomics, Mass Spectrometry, Western Blot, Quantitative RT-PCR, MANN-WHITNEY

Journal: Glia

Article Title: CDC42 ‐Effector Proteins Regulate Higher Order Structure of Septins Required for CNS Myelin Integrity

doi: 10.1002/glia.70134

Figure Lengend Snippet: Disorganized higher order structure of myelin septin filaments when oligodendrocytes lack Cdc42ep1 and Cdc42ep2 . (A, B) Representative light micrographs of teased fibers dissected from dorsal spinal cords of Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox (Ctrl, A) and Cdc42ep1 flox/flox ; Cdc42ep2 flox/flox ; Cnp Cre/wt (dcKO, B) mice at 26 weeks of age. All myelin septin subunits (SEPTIN2, SEPTIN4, SEPTIN7, and SEPTIN8) were collectively immunolabeled (shown in red) together with the axonal marker SMI312 (in blue) and imaged using a confocal microscope. Higher order septin structures of up to 12 μm length along the axon are readily detected in Ctrl (arrowheads in A), while septin‐immunopositive structures are more frequent but shorter and fragmented in dcKO mice (arrowheads in B). (C, D) Representative micrographs of selected myelin septins (SEPTIN2, SEPTIN4, and SEPTIN8) collectively immunolabeled on expanded spinal cord, imaged via multiphoton imaging, and 3‐dimensionally reconstructed. Objects are false‐colored in red, green, and blue according to their position along the z‐axis. Objects located in the same z‐plane are assigned the same color. (C, C′) Overview of myelin septin immunolabeling in expanded Ctrl spinal cord (C) and magnified view of the boxed region (C′). (D, D′) Overview of myelin septin immunolabeling in expanded dcKO spinal cord (D) and magnified view of the boxed region (D′). Note that higher order septin structures are readily detected in Ctrl (C′). In dcKO mice, septin‐immunopositive structures are more frequent but shorter and fragmented (D′). (E, G) Representative micrographs and schematics of myelin septins (SEPTIN2, SEPTIN4, SEPTIN8) collectively immunolabeled on cross‐sectioned spinal cord, superresolution imaging at 100× magnification (E), and genotype‐dependent quantification (F, G). Myelin septins are shown in magenta (arrows pointing at puncta in E), the marker for compact myelin PLP is in blue (outlined by stippled lines in E). (F) Genotype‐dependent quantification of the relative frequency of distinct septin puncta per axon/myelin‐profile indicate a shift towards a larger number of myelin septin puncta in control (gray) compared to dcKO (green) spinal cord. Ninety‐two axon/myelin profiles from five control mice and 100 axon/myelin profiles from five dcKO mice; number of myelin septin puncta by Chi‐squared test p = 6.151e−09. (G) Quantification of the mean signal area covered by septin puncta indicates that septin puncta are enlarged in dcKO mice. Mean ± SEM; datapoints represent individual mice; n = 5 mice per genotype; Students t test ( p = 0.0482).

Article Snippet: Primary antibodies were specific for CDC42 (Santa Cruz, 1:500, #sc‐8401), CDC42EP1 (custom‐made rabbit polyclonal antibody against mouse CDC42EP1 epitope VEKHSNRDRDRDPDH, Pineda, 1:1000), CDC42EP2 (Proteintech Group, 1:300, #11824‐1‐AP), SEPTIN2 (Proteintech Group, 1:500, #11397‐1‐AP), SEPTIN4 (IBL, 1:200, #JP18987), SEPTIN7 (IBL, 1:5000, #18991), SEPTIN8 (Proteintech Group, 1:500, #11769‐1‐AP), or ATP1a3 (Abcam, 1:1000, #ab182571).

Techniques: Immunolabeling, Marker, Microscopy, Imaging, Control

Journal: Brain Sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Cell division control 42 effector protein 2 (Cdc42EP2) negatively regulated neurite outgrowth by downregulating βIII-tubulin in E46K α-syn transfectants. ( A ) Quantitative analysis of Cdc42EP2 mRNA expression. SK-N-SH cells were seeded into six-well plates (1 × 10 5 cells/well) and cultured for 16 h to achieve attachment and confluency. After transfection with each pcDNA3.1+-α-syn (WT, A53T, and E46K), the cells were maintained at 37 °C in an atmosphere of 5% CO 2 for 24 h. Then, total RNA was isolated for cDNA synthesis, followed by real-time quantitative PCR using SYBR Green TOPreal TM qPCR PreMIX (Enzynomics) on an Eco Real-Time PCR System (Illumina). Data are presented as means ± SD from four independent experiments. ( B ) Quantitative analysis of βIII-tubulin gene expression using the same techniques and statistical analyses as described in A. ( C ) Western blot analysis of Cdc42EP2 protein levels in α-syn transfectants, including the empty vector group (control). ( D ) Western blot analysis of βIII-tubulin levels in α-syn transfectants, including the empty vector group (control). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of prepared cell lysate samples and transferring the proteins onto membranes, western blotting was performed using specific antibodies for human α-syn, Cdc42EP2, or βIII-tubulin. presents representative blots from three independent experiments. Data are presented as means ± SD for three independent experiments performed in triplicate. Analysis was performed using one-way ANOVA (* p < 0.05, ** p < 0.01) and Student t -test (# p < 0.05, ## p < 0.01).

Article Snippet: The membranes were probed for 16 h at 4 °C with the following primary antibodies: anti-α-syn antibody (BD Biosciences, Franklin Lakes, NJ, USA, 610787, 1:1000 dilution), anti-Cdc42EP2 antibody (Abnova, Taipei, Taiwan, H00010435-M01, 1:1000 dilution), anti-βIII-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-8005, 1:1500 dilution) and anti-GAPDH antibody (Sigma-Aldrich, G8795, 1:2500).

Techniques: Control, Expressing, Cell Culture, Transfection, Isolation, cDNA Synthesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Gene Expression, Western Blot, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Transferring

Journal: Brain Sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: α-Syn-induced Cdc42EP2 expression regulated neurite outgrowth in SK-N-SH cells. Confocal microscopy analysis of SK-N-SH cells transfected with WT, A53T, and E46K α-syn, including empty vector control (vehicle), revealed the correlation of Cdc42EP2 with the mutations of α-syn. Immunofluorescent staining was performed using Alexa Fluor 488-conjugated secondary antibody (green). α-Syn-induced Cdc42EP2 was visualized using Alexa Fluor 594 staining (red). Immunofluorescent images were captured using an LSM510 META confocal microscope with an Axioplan 2 imaging system (Carl Zeiss). Scale bar: 50 μm.

Article Snippet: The membranes were probed for 16 h at 4 °C with the following primary antibodies: anti-α-syn antibody (BD Biosciences, Franklin Lakes, NJ, USA, 610787, 1:1000 dilution), anti-Cdc42EP2 antibody (Abnova, Taipei, Taiwan, H00010435-M01, 1:1000 dilution), anti-βIII-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-8005, 1:1500 dilution) and anti-GAPDH antibody (Sigma-Aldrich, G8795, 1:2500).

Techniques: Expressing, Confocal Microscopy, Transfection, Plasmid Preparation, Control, Staining, Microscopy, Imaging

Journal: Brain Sciences

Article Title: E46K α-Synuclein Mutation Fails to Promote Neurite Outgrowth by Not Inducing Cdc42EP2 Expression, Unlike Wild-Type or A53T α-Synuclein in SK-N-SH Cells

doi: 10.3390/brainsci15010009

Figure Lengend Snippet: Cdc42EP2 knockdown abrogated α-syn-induced neurite outgrowth. Small interfering RNA (siRNA) targeting Cdc42EP2 was transfected into SK-N-SH cells. Unrelated siRNA was used as a negative control. Following a 6-hour interval, we performed cotransfection with secondary α-syn expression cDNA (WT, A53T, or E46K). An empty vector was used as the control. ( A ) At 72 h, the peak point of siRNA action, we assessed morphological changes in the cotransfected SK-N-SH cells using microscopy. Scale bar: 50 μm. ( B ) We performed three independent experiments to evaluate the changes in neurite lengths of the transfectants. Results are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (* p < 0.05 and ** p < 0.01) and Student t -test (## p < 0.01).

Article Snippet: The membranes were probed for 16 h at 4 °C with the following primary antibodies: anti-α-syn antibody (BD Biosciences, Franklin Lakes, NJ, USA, 610787, 1:1000 dilution), anti-Cdc42EP2 antibody (Abnova, Taipei, Taiwan, H00010435-M01, 1:1000 dilution), anti-βIII-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-8005, 1:1500 dilution) and anti-GAPDH antibody (Sigma-Aldrich, G8795, 1:2500).

Techniques: Knockdown, Small Interfering RNA, Transfection, Negative Control, Cotransfection, Expressing, Plasmid Preparation, Control, Microscopy