Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: Increased Parkin levels occur in Fkbp51 KO mice via post-translational regulation. A Parkin expression is significantly increased in the Fkbp51 KO in the hippocampal subregions CA1 and DG, scale bar = 50 μm. B Primary cultured WT and Fkbp51 KO hippocampal neurons labeled at DIV9 with MAP2 (green), Parkin (red), and DAPI (blue), scale bar = 100 μm. WT neurons express more MAP2 but less Parkin than Fkbp51 KO neurons in the soma and dendrites. The magnified panel highlights the distinct labeling patterns in WT and Fkbp51 KO soma and dendrites. C Western blotting confirms significantly higher Parkin protein expression in the Fkbp51 KO hippocampus. Quantification of the western blot density indicates that the Parkin protein is significantly elevated in the Fkbp51 KO hippocampus (over 2.5-fold). D No significant differences were found in Park2 mRNA expression. Data represent the mean ± SEM normalized to GAPDH. Pyr, pyramidal cell layer; Rad, radiatum layer; HR, hilar region; GCL, granule cell layer. Data represent mean ± SEM of 6 mice per genotype. P values were determined by Student’s unpaired t-test. ****P < 0.0001

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: Expressing, Cell Culture, Labeling, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: Ablation of the Fkbp51 gene compromises hippocampal size. A Image acquisition and data processing. Sagittal view with vertical lines indicating the position range where coronal sections were acquired. Stacks of images were analyzed from both 5 male WT and 5 male KO followed by 2D segmentation of areas of interest. Machine learning was applied and landmarks of CA and DG were analyzed. B Representative brain section with hippocampal subregions highlighted. The yellow regions indicate the 2D segmentation outcome, and red lines are the resulting smooth centerlines along the interface. Eight landmarks were measured, annotated in the magnified view at the bottom of the section (1) the cortex plus corpus callosum (CC) length was approximated using the vertical intensity profile of the contrast enhanced grayscale image; (2) the hippocampus height was calculated in the vertical direction, after the “peak point” between CA1 and CA2 was located; (3) the length of the hippocampus was calculated as the arc length of the pyramidal cell layer; (4) the local curve change at the “peak point” between CA1 and CA2 was captured by the radius of the best locally fit circle to the curve (in cyan); (5) similarly, the local curve change of CA3 was captured by the radius of the best locally fit circle; (6) the length of the colored DG segment was calculated as the arc lengths of the corresponding granular cell layer; (7) the DG height was measured vertically, after locating the peak point between the top and bottom granular cell layers; (8) the DG apex was captured by the radius of the best locally fit circle to the DG line. The y-axis in each plot corresponds to measurement in pixels, and the scale varies across the plots for illustration purposes. In blue and red are the measurements for WT and KO, respectively. The plots 2–8 are divided by a black vertical line into left and right hippocampus segment measurements. Bold dots indicate the group averages. CA: cornu ammonis. DG: dentate gyrus; Hippo: hippocampus. P value shown in (1) was determined by Student’s unpaired t-test, shown in (2–8) were determined by One-way ANOVA

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: Morphological differences between Fkbp51 KO and WT primary cultured neurons. A Neurons were labeled with βIII-tubulin for whole neurons (red), MAP2 for soma and dendrites (green), and DAPI for nuclei (blue). Primary cultured neurons were analyzed at DIV3, DIV5, and DIV9. Solid head arrows indicate axon outgrowth, while line head arrows indicate dendrite outgrowth. Inset of each image showed the magnified staining of soma area. Scale bar = 100 μm. B Quantification of MAP2 and βIII-tubulin labelling in primary cultured hippocampal neurons. C, D Statistical analyses of neurite numbers, radius of maximum intersections and intersections at different radii in treated and untreated WT and Fkbp51 KO neurons. DIV: days in vitro. Data represent mean ± SEM of 8–10 neurons from 3 independent experiments. P value shown in B was determined by Student’s unpaired t-test, and P values shown in C and D were determined by two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Immunofluorescence labeling of βIII-tubulin, F-actin, and Tau is included in the Supplementary Information, SI-Fig. 2

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: Cell Culture, Labeling, Staining, In Vitro, Immunofluorescence

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: Fkbp51 KO neuronal polarization sensitive to microtubule stability alteration. A Morphological differences between Fkbp51 KO and WT primary cultured neurons are observable at DIV 9. Neurons are labeled with βIII-tubulin (red) and MAP2 (green). Hippocampal neurons from Fkbp51 KO mice exhibit enhanced neurite outgrowth. Taxol reduced dendrite outgrowth from Fkbp51 KO neurons, while nocodazole reduced minor neurite formation in both WT and Fkbp51 KO, with more obvious neuronal deformation present in Fkbp51 KO. Scale bar = 100 μm. B Quantification of the MAP2 labeling in primary cultured hippocampal neurons. C, D Statistical analyses of neurite numbers, radius of maximum intersections and intersections at different radii in treated and untreated WT and Fkbp51 KO neurons. Data represent mean ± SEM of 8 ~ 10 neurons from 3 independent experiments. P value shown in B was determined by Student’s unpaired t-test, and P values shown in C, D were determined by two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: Cell Culture, Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: In vivo differences in βIII-tubulin, MAP2, and Tau expression in the hippocampus. A No significant differences were found in the expression levels of Tubb3, Map2, or Tau mRNA. B MAP2 and DAPI immunofluorescent labeling of hippocampus and magnified subfields including DG, CA1, and CA3. C In the CA1 and DG hippocampal subfields, MAP2 and βIII-tubulin expression are significantly lower in the Fkbp51 KO than in the WT, particularly in the Rad. Tau protein expression levels are also lower in the Fkbp51 KO than in the WT (scale bar = 50 μm). D Western blotting confirmed the reduced expression of the MAP2, p-MAP2, βIII-tubulin, and Tau proteins. E Quantification of the MAP2, p-MAP2, βIII-tubulin and Tau proteins in the hippocampus. Data represent the mean ± SEM normalized to GAPDH. Pyr, pyramidal cell layer; Rad, radiatum layer; HR, hilar region; GCL, granule cell layer. Data represent mean ± SEM of 6 mice per genotype. P values were determined by Student’s unpaired t-test. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: In Vivo, Expressing, Labeling, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: Increased Parkin levels occur in Fkbp51 KO mice via post-translational regulation. A Parkin expression is significantly increased in the Fkbp51 KO in the hippocampal subregions CA1 and DG, scale bar = 50 μm. B Primary cultured WT and Fkbp51 KO hippocampal neurons labeled at DIV9 with MAP2 (green), Parkin (red), and DAPI (blue), scale bar = 100 μm. WT neurons express more MAP2 but less Parkin than Fkbp51 KO neurons in the soma and dendrites. The magnified panel highlights the distinct labeling patterns in WT and Fkbp51 KO soma and dendrites. C Western blotting confirms significantly higher Parkin protein expression in the Fkbp51 KO hippocampus. Quantification of the western blot density indicates that the Parkin protein is significantly elevated in the Fkbp51 KO hippocampus (over 2.5-fold). D No significant differences were found in Park2 mRNA expression. Data represent the mean ± SEM normalized to GAPDH. Pyr, pyramidal cell layer; Rad, radiatum layer; HR, hilar region; GCL, granule cell layer. Data represent mean ± SEM of 6 mice per genotype. P values were determined by Student’s unpaired t-test. ****P < 0.0001

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: Expressing, Cell Culture, Labeling, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: FKBP51 prevents disrupted SH-SY5Y cell morphology and is associated with ubiquitin-mediated degradation of Parkin. A Double labeling of MAP2/Parkin, Tau/Parkin, and F-actin/Parkin indicates that Fkbp51 siRNA treatment increases the outgrowth of neurites in SH-SY5Y cells, decreases MAP2, Tau, and F-actin expression, and increases Parkin expression. B Western blotting confirms reduced FKBP51 and increased Parkin levels following Fkbp51 siRNA treatment compared to those treated with the scramble siRNA control. C Increased expression of Flag-FKBP51 in SH-5YSY cells corresponds to a dose-dependent decrease in Parkin protein levels. D Parkin expression levels decreased with overexpression of Fkbp51 but not pcDNA3.1 control. E Co-transfection of His-Parkin with ubiquitin HA-UB alone (lane 2), Flag-Fkbp51 and His-Parkin (lane 3), or HA-UB and Flag-Fkbp51 together (lane 4) promotes ubiquitin-dependent degradation of both FKBP51 and Parkin. F Co-IP assays show that FKBP51 binds Parkin, βIII-tubulin, and Tau. Parkin also binds MAP2. G Co-transfection of Flag-Fkbp51 with ParkinS65A reveals S65A is a critical site for FKBP51 and Parkin interaction. Data represent mean ± SEM from 3 independent experiments. P value shown in B was determined by Student’s unpaired t-test, P values shown in C, E and G were determined by One-way ANOVA, and P values shown in D were determined by Two-way ANOVA. *P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: Labeling, Expressing, Western Blot, Over Expression, Cotransfection, Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin

doi: 10.1007/s00018-022-04167-8

Figure Lengend Snippet: Model of morphological and molecular alterations in WT and Fkbp51 KO neuron polarization. A schematic model demonstrating the morphological differences between WT and Fkbp51 KO neurons, with shorter axons and greater dendritic outgrowth in Fkbp51 KO neurons. The underlying mechanism could be due to Fkbp51 inhibition of Parkin activity. In WT neurons, FKBP51 interacts with Parkin and regulates Parkin activity, including its ubiquitination activity, resulting in normal substrate degradation. The loss of FKBP51 interrupts the regular FKBP51 inhibition of Parkin, and may also reduce Parkin self -degradation, resulting in enhanced Parkin accumulation and enhanced Parkin activity, thus enhanced substrate degradation

Article Snippet: The cells were transfected with the Flag- Fkbp51 (HG11487-CF, Sino Biological, Beijing, China), His- Park2 (HG12092-NH, Sino Biological, Beijing, China) or HA-ubiquitin (#18712, Addgene, Cambridge, MA, USA) plasmid for overexpression or with the human Fkbp51 siRNA (sc-35380, Santa Cruz Biotech, Inc., Dallas, TX, USA) for knock-down, using Lipofectamine 3000 (L3000008, Life Technologies, Gaithersburg, MD, USA) according to the manufacturer’s recommendations.

Techniques: Inhibition, Activity Assay