Journal: Traffic (Copenhagen, Denmark)

Article Title: Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

doi: 10.1111/tra.12581

Figure Lengend Snippet: FIGURE 3 VMP1 marks ER subdomains enriched in phospholipid synthesizing enzymes. (A) HeLa cells were co-transfected with VMP1-GFP and different ER proteins (Sec61, Atl3, PIS1, CEPT1). VMP1 puncta are usually associated with ER subdomains enriched for phospholipid synthetizing enzymes. (B) Percentage of co-localization for cells expressing VMP1-GFP with Sec61, mCh-Atl3, mCh-PIS1 or mCh-CEPT1, as measured by Mander's co-localization coefficient. Regions of 100 μm2 were used for Mander's coefficient analysis (20 regions from 3 different experiments). Significance was analyzed using the student t test (C) for better visualization of tubules, Cos-7 cells were co-transfected with VMP1-GFP and Sec61 or CEPT1. VMP1 is more associated with CEPT1 than with Sec61 as Pearson´s coefficient demonstrates. (D) SR-SIM showing that VMP1 and CEPT1 are present in the same ER subdomains. Regions of 25 μm2 from 2 different cells are shown. Scale bars: 5 and 0.2 μm for detailed regions

Article Snippet: Primary antibodies used for cell fractionation and western blot were: VMP1 (Cell Signaling #12929), Calnexin (BD Biosciences#610523), Rab7 (Cell Signaling #D95F2), Syntaxin6 (BD Biosciences #610635), EEA1 (BD Biosciences #610456), PIS1 (Santa Cruz #sc-514255) and CEPT1 (Thermo Fisher Scientific #PA5-23876).

Techniques: Transfection, Expressing

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

doi: 10.1111/tra.12581

Figure Lengend Snippet: FIGURE 4 Colocalization of VMP1/PIS1 domains with endosomes and autophagosomes. HeLa cells were transfected with VMP1-GFP and m-cherry-PIS1 plasmids. Then, cells were exposed to both EGF treatment and 2 hours starvation previous fixation and immunostaining against EGFR or LC3 as indicated in the figure. PIS1-VMP1 subdomains are more associated with autophagy than with endocytosis. The mean and SD were obtained from 3 independent transfections (approximately 20-30 cells per transfection)

Article Snippet: Primary antibodies used for cell fractionation and western blot were: VMP1 (Cell Signaling #12929), Calnexin (BD Biosciences#610523), Rab7 (Cell Signaling #D95F2), Syntaxin6 (BD Biosciences #610635), EEA1 (BD Biosciences #610456), PIS1 (Santa Cruz #sc-514255) and CEPT1 (Thermo Fisher Scientific #PA5-23876).

Techniques: Transfection, Immunostaining

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

doi: 10.1111/tra.12581

Figure Lengend Snippet: FIGURE 5 VMP1 depletion affects dynamics of phospholipid synthesizing enzymes enriched ER subdomains. (A) Control and VMP1 silenced cells expressing mCherry-CEPT1 and mCherry-PIS1. Puncta formation of phospholipid biosynthetic enzymes along the ER decreases in VMP1 silenced cells. (B) Western-blot quantification of the protein level of CEPT1 and PIS1 in both control and VMP1-silenced cells. Protein extracts from 3 (COS-7) and 4 (HeLa) independent inhibitions were analyzed by western blot with specific antibodies and the levels quantified by densitometry. The mean and the SD (SD) are shown. Significance was analyzed using the student t test. (C) Control and VMP1-silenced cells expressing PIS1-GFP were visualized by confocal microscopy 24 hours after transfection. In the details, same threshold was applied to all images to analyze the differences in the size of PIS1 puncta. (D) Control and VMP1 depleted HeLa and Cos-7 cells were transfected with the PIS1-GFP plasmid and visualized by live cell imaging 24 hours after transfection. Images show the same region of the cell in an interval of 60 seconds to show the dynamics of punta. Green color has been assigned to time 0 sec and red color to 60 sec for better visualization of the movements. Dynamics of PIS1 puncta in the ER is impaired in the absence of VMP1

Article Snippet: Primary antibodies used for cell fractionation and western blot were: VMP1 (Cell Signaling #12929), Calnexin (BD Biosciences#610523), Rab7 (Cell Signaling #D95F2), Syntaxin6 (BD Biosciences #610635), EEA1 (BD Biosciences #610456), PIS1 (Santa Cruz #sc-514255) and CEPT1 (Thermo Fisher Scientific #PA5-23876).

Techniques: Control, Expressing, Western Blot, Confocal Microscopy, Transfection, Plasmid Preparation, Live Cell Imaging

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

doi: 10.1111/tra.12581

Figure Lengend Snippet: FIGURE 10 Model of VMP1 localization and function at MCS. VMP1 is an ER transmembrane protein that colocalizes with the phospholipid synthesizing enzymes PIS1 and CEPT at MCSs between the ER and different organelles, forming a distinct ER subdomain. The presence of VMP1 is required for correct lipid transfer between organelles. VMP1 depleted cells show abnormal distribution of phospholipids leading to defects in mitochondria morphology, autophagosome formation and endosome maturation. ER is depicted in blue and Atlastin (Atl-3) is depicted in red

Article Snippet: Primary antibodies used for cell fractionation and western blot were: VMP1 (Cell Signaling #12929), Calnexin (BD Biosciences#610523), Rab7 (Cell Signaling #D95F2), Syntaxin6 (BD Biosciences #610635), EEA1 (BD Biosciences #610456), PIS1 (Santa Cruz #sc-514255) and CEPT1 (Thermo Fisher Scientific #PA5-23876).

Techniques:

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: MiRNA-based short-hairpins (miR5 and miR7) targeting distinct regions of ATP10B mRNA, and a scrambled non-targeting sequence (SCR) packaged in AAV2/7 vector under a neuronal promoter (CMVenhanced-synapsin) were stereotactically injected in the right SNpc of rats. (A) Overview of the protocol followed in this study and vector constructs. (B, C) The percentage of double TH+ RFP+ cells within the total TH+ population in the SNpc was determined for the three vectors 2 weeks post-injection. (B) Each data point represents an individual animal, and the number of TH+ and TH+ RFP+ cells from 8 sections was quantified using stereological methods. (C) Representative image of the SNpc of a rat injected with the miR5 vector 2 weeks post-injection, fluorescently stained with TH and RFP antibodies, shows efficient transduction of dopaminergic neurons.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Sequencing, Plasmid Preparation, Injection, Construct, Staining, Transduction

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A, C, G) Indicative of decreased ipsilateral dopaminergic neurotransmission, rats with ATP10B KD showed decreased preference in the use of the contralateral paw in the cylinder test (A), increased bias to ipsilateral swing in the elevated body swing test (C), and spontaneous ipsilateral turning in the open field test (G). (B) Motor coordination and balance was assessed using an accelerated rotarod test (4-40 rpm), with the average performance post-lesion normalized to the individual rat performance prior to surgery. (D, E, F) Spontaneous motor behavior was recorded during a 5-min open field test and analyzed for total distance traveled (D), velocity (E) and rearing (F). Data are mean + s.e.m. *** p < 0.001 (two-way ANOVA, treatment factor), # p < 0.05, ## p < 0.01, ### p < 0.001 (Dunnett’s post hoc test miR5 vs. SCR at corresponding time point), $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 (Dunnett’s post hoc test miR7 vs. SCR at corresponding time point).

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques:

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: ATP10B KD in SNpc neurons leads to Parkinsonian motor deficits at 12 months post injection . (A) Motor coordination and balance was assessed using an accelerated rotarod test (4-40 rpm), with the average performance post-lesion normalized to the individual rat performance prior to surgery (p = 0.0083; SCR vs. miR5 p = 0.0043, SCR vs. miR7 p = 0.1111). (B, C, H) Motor asymmetry was examined by quantifying the preference in the use of the contralateral paw in the cylinder test (p = 0.0110; SCR vs. miR5 p = 0. 00068, SCR vs. miR7 p = 0. 0799), the bias to ipsilateral swing in the EBST (p = 0.0016; SCR vs. miR5 p = 0.0321, SCR vs. miR7 p = 0.0009), and the spontaneous ipsilateral turning behavior in the open field test (p = 0.0123; SCR vs. miR5 p = 0.0388, SCR vs. miR7 p = 0.0112). (E, F, G) Spontaneous motor behavior was recorded during a 5-min open field test and analyzed for total distance traveled (p = 0.0380; SCR vs. miR5 p = 0.1035, SCR vs. miR7 p = 0.0312), velocity (p = 0.0249; SCR vs. miR5 p = 0.0722, SCR vs. miR7 p = 0.0212), and frequency of rearing (p = 0.0064; SCR vs. miR5 p = 0.0083, SCR vs. miR7 p = 0.0166). (D) The catalepsy bar test was performed to assess muscular rigidity. Time spent by the rat grabbing the elevated bar before correcting its posture on the floor was measured (p = 0.0174; SCR vs. miR5 p = 0.0181, SCR vs. miR7 p = 0.0481). In (A, C-H) data are mean ± s.e.m, and analyzed using non-parametric one-way ANOVA (Kruskall Wallis) and Dunn’s post-hoc test versus SCR. SCR (n=15), miR5 (n=15), miR7 (n=17). In (B) data are mean ± s.e.m and analyzed using one-way ANOVA and Dunnet’s post-hoc test versus SCR. SCR (n=12), miR5 (n=10), miR7 (n=11). Each dot represents the value of one animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: Striatal DAT binding potential (BPnd), measured by 18F-FE-PE2I microPET imaging, in the ipsilateral (right) and the contralateral (left) striatum observed in miR5-injected animals (n=5), SCR-injected animals (n=3) and miR7-injected animals at 7-9 months (n=2) and 12 months (n=3).

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Binding Assay, Imaging, Injection

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A) Striatal DAT binding potential (BPnd), measured by 18 F-FE-PE2I microPET imaging, in the ipsilateral (R) vs. the contralateral (L) striatum in miR5-injected animals (n=5) and SCR-injected animals (n=3) (treatment factor p = 0.0007; SCR vs. miR5 2-4 months p = 0.0134, SCR vs. miR5 4-6 months p = 0.0448, SCR vs. miR5 7-9 months p = 0.0448, SCR vs. miR5 12 months p = 0.0024). Data are mean ± s.e.m. and analyzed using two-way ANOVA and Sidak’s post hoc test miR5 vs. SCR at corresponding time point. The effect could be reproduced in miR7-injected animals at 7-9 months (n=2) and 12 months (n=3). (B) Parametric images of 18 F-FE-PE2I BPnd in representative SCR (upper) and miR5 (middle) rats over time; lower row represents representative miR7 images at the final two time points. Transverse images are shown in neurological orientation and are overlaid onto a reference CT skull template with the miR5/miR7 lesioned striatum on the right-hand side.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Binding Assay, Imaging, Injection

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: ATP10B KD in SNpc neurons leads to time-dependent loss of dopaminergic terminals in the dSTR . TH positive area was quantified using QuPath in 8 different sections that cover the STR. (A) TH positive area in the contralateral (Contra) and ipsilateral (Ipsi) dorsal STR of 1 month post-injection rats (SCR Contra vs. SCR Ipsi p = 0.5704, miR Contra vs. miR5 Ipsi p = 0.0046, miR7 Contra vs. miR7 Ipsi p = 0.6951). SCR (n=6), miR5 (n=6), miR7 (n=6). (B) Representative image of TH immunohistochemical staining on 4 different sections from one SCR, miR5 and miR7 rat 1 month post-injection. (C) TH positive area in the contralateral and ipsilateral dorsal STR of 1 year post-injection rats(SCR Contra vs. SCR Ipsi p = 0.5397, miR Contra vs. miR5 Ipsi p = 0.0025, miR7 Contra vs. miR7 Ipsi p = 0.0026). SCR (n=8), miR5 (n=8), miR7 (n=8). (D) Representative image of TH immunohistochemical staining on 4 different sections from one SCR, miR5 and miR7 rat 1 year post-injection. Data are mean ± s.e.m, and anayzed using a paired t-test. Each dot represents the average of the 8 sections per animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: TH positive area was quantified using ImageJ in 6 different sections that cover the STR. (A) Percentage of TH positive area ipsilateral (R) versus contralateral (L) dorsal STR of 1 year post-injection rats, SCR (n=5) and miR5 (n=7). Data are mean ± s.e.m and analyzed using t test, ** p = 0.0025. Each dot represents the average of the 6 sections per animal. (B) Representative image of TH immunohistochemical staining on 6 different sections from one SCR and miR5 rat 1 year post-injection.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: TH positive cells were counted using stereology in 7 different sections that cover the SNpc. (A) Number of TH+ cells in the contralateral (Contra) and ipsilateral (Ipsi) SNpc of 1 month post-injection rats(SCR Contra vs. SCR Ipsi p = 0.98, miR Contra vs. miR5 Ipsi p = 0.2227, miR7 Contra vs. miR7 Ipsi p = 0.0160). SCR (n=6), miR5 (n=6), miR7 (n=5). (B) Representative image of TH immunofluorescence staining on 1 SNpc section from one SCR, miR5 and miR7 rat 1 month post-injection. (C) Number of TH+ cells in the contralateral (Non Inj) and ipsilateral (Inj) SNpc of 1 year post-injection rats(SCR Contra vs. SCR Ipsi p = 0.7125, miR Contra vs. miR5 Ipsi p = 0.0071, miR7 Contra vs. miR7 Ipsi p < 0.0001). SCR (n=8), miR5 (n=8), miR7 (n=8). (D) Representative image of TH immunofluorescence staining on 1 SNpc section from one SCR, miR5 and miR7 rat 1 year post-injection. Data are mean ± s.e.m, and analyzed using a paired t-test. Each dot represents the average of the 7 sections per animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Injection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A-E) Protein signal values were normalized to the endogenous protein (β-actin) levels, and the ipsilateral (Ipsi) versus contralateral (Contra) percentage of the protein signal is represented. Data are presented as mean ± s.e.m., with each dot representing an individual animal. (F) Representative western blot images of α-synuclein and lysosomal proteins analyzed in whole SN extracts from SCR, miR5, and miR7 rats.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

doi: 10.1101/2024.07.09.602700

Figure Lengend Snippet: (A) Representative immunofluorescent images of LAMP1, LAMP2a, cathepsin B and Gcase co-stained with TH marker in the SNpc of SCR, miR5 and miR7 rats at 1 year post-injection. (B-E) Density of LAMP1+, LAMP2a+, cathepsin B+, or Gcase+ spots (number of spots per total TH+ surface) and average spot volume in TH+ cells quantified in the injected side versus non-injected side. SCR (n=8), miR5 (n=8), miR7 (n=8). (B) LAMP1+ number of spots (SCR vs. miR5 p = 0.9089, SCR vs. miR7 p < 0.0001) and LAMP1+ average spot volume (SCR vs. miR5 p > 0.9999, SCR vs. miR7 p = 0.0060). (C) LAMP2a+ number of spots (SCR vs. miR5 p = 0.0017, SCR vs. miR7 p < 0.0001) and LAMP2a+ average spot volume (SCR vs. miR5 p = 0.9435, SCR vs. miR7 p = 0.0009). (D) Cathepsin B+ number of spots (SCR vs. miR5 p = 0.0016, SCR vs. miR7 p < 0.0001) and cathepsin B+ average spot volume (SCR vs. miR5 p = 0.5022, SCR vs. miR7 p < 0.0001). (E) Gcase+ number of spots (SCR vs. miR5 p = 0.9987, SCR vs. miR7 p = 0.568) and Gcase+ average spot volume (SCR vs. miR5 p = 0.0012, SCR vs. miR7 p = 0.0020). Data are mean ± s.e.m and analyzed using one-way ANOVA followed Šídák’s multiple comparisons test, with the exception of LAMP1 average spot volume which was analyzed using Kruskal-Wallis followed Dunn’s multiple comparisons test. Each dot represents the average of 3 sections per animal.

Article Snippet: The AAV2/7 viral vectors were produced by the Leuven Viral Vector Core using the following transfer plasmids for viral vector production: SCR (Addgene plasmid RRID: Addgene_216393), miR5 (Addgene plasmid RRID: Addgene _216391), miR7 (Addgene plasmid RRID: Addgene_216392) ( ).

Techniques: Staining, Marker, Injection