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AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

In JCI Insight on 22 August 2023 by Meena, N. K., Randazzo, D., et al.

Fig.2.D

Systemic gene transfer rescues muscle pathology after short-term treatment at a high vector dose.(A) Experimental design: 3.5-month-old KO mice received a single injection of systemic vector (SYS; n = 3) at a dose of 5 × 1013 vg/kg. Age-matched wild-type (WT) and untreated Gaa–/– (KO) mice were u... more

Systemic gene transfer rescues muscle pathology after short-term treatment at a high vector dose.(A) Experimental design: 3.5-month-old KO mice received a single injection of systemic vector (SYS; n = 3) at a dose of 5 × 1013 vg/kg. Age-matched wild-type (WT) and untreated Gaa–/– (KO) mice were used as controls. Muscle samples were collected 1 month (mo) after dosing. (B) Western blot analyses of whole muscle lysates with anti-human GAA antibody. Gapdh was used as a loading control. Graph shows GAA activity in muscle tissues from WT, KO, and SYS-treated KO mice. (C) Glycogen content in muscle tissues across the groups. (D and E) Western blot analyses of whole muscle lysates with the indicated antibodies. Gapdh was used as a loading control. The treatment normalized the levels of lysosomal/autophagosomal markers (D) and AMPK activity (E). A significant decrease in both nonphosphorylated 4EBP1 (non-p4EBP1) and total 4EBP1 indicates an improvement in the mTORC1 signaling. (F) Top panel: Immunostaining of single fibers with markers for lysosomes (LAMP1; green), autophagosomes (LC3; red), and nuclei (Hoechst dye; blue); enlarged lysosomes and autophagic buildup (multicolored area in the core of the fiber) are seen in virtually all fibers from KO mice; lysosomes and autophagosomes appear as abundant dot-like LAMP1/LC3-positive structures (often located adjacent to each other; inset) in fibers from WT and SYS-treated KO mice. Bottom panel: Differential interference contrast images of the fibers shown on the top panel; ordered sarcomeric organization is fully restored after treatment. Statistical significance was determined by 1-way ANOVA. Graphs represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Bars: 20 μm. 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1. less

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AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

In JCI Insight on 22 August 2023 by Meena, N. K., Randazzo, D., et al.

Fig.5.D

Muscle glycogen, autophagosomal-lysosomal markers, and mTORC1/AMPK signaling return to near normal after short-term systemic gene transfer at an intermediate vector dose.(A) Experimental design: 3-month-old KO mice received a single injection of systemic (SYS n = 4) or liver-secreted (LS n = 4) v... more

Muscle glycogen, autophagosomal-lysosomal markers, and mTORC1/AMPK signaling return to near normal after short-term systemic gene transfer at an intermediate vector dose.(A) Experimental design: 3-month-old KO mice received a single injection of systemic (SYS n = 4) or liver-secreted (LS n = 4) vector at a dose of 2.5 × 1013 vg/kg. Age-matched wild-type (WT) and untreated Gaa–/– (KO) mice were used as controls. Muscle samples were collected 1 month (mo) after dosing. (B) Western blot analyses of whole muscle lysates with anti-human GAA antibody. The 110 kDa GAA precursor protein is clearly detectable after LS treatment. Gapdh was used as a loading control. Graph shows GAA activity in muscle tissues from WT, untreated KO, and KO treated with SYS or LS vector. (C) Glycogen content in muscle tissues across the groups. (D and E) Western blot analyses of whole muscle lysates with the indicated antibodies. Statistical significance was determined by 1-way ANOVA. Graphs represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. less

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AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

In JCI Insight on 22 August 2023 by Meena, N. K., Randazzo, D., et al.

Fig.9.F

Systemic gene transfer reverses muscle pathology in old KO mice.(A) Experimental design: 9-month-old KO mice received a single injection of SYS (n = 8) vector at a dose of 2.5 × 1013 vg/kg. Age-matched (15.5-month-old) wild-type (WT) and 13.5-month-old untreated Gaa–/– (KO) mice were used as cont... more

Systemic gene transfer reverses muscle pathology in old KO mice.(A) Experimental design: 9-month-old KO mice received a single injection of SYS (n = 8) vector at a dose of 2.5 × 1013 vg/kg. Age-matched (15.5-month-old) wild-type (WT) and 13.5-month-old untreated Gaa–/– (KO) mice were used as controls. Muscle samples were collected 7 months after dosing. (B) SYS-treated KO mice appear healthy for their age group; a 13.5-month-old KO mouse shows profound muscle weakness and wasting; see Supplemental Videos 1 and 2. (C) Western blot analyses of whole muscle lysates with anti-human GAA antibody. Gapdh was used as a loading control. Graph shows GAA activity in muscle tissues from WT, untreated KO, and SYS-treated KO mice. (D) Glycogen content in muscle tissues across the groups. (E) PAS-stained sections of gastrocnemius muscle from WT, KO, and SYS-treated mice; muscle fibers from SYS-treated mice appear normal. Bars: 50 μm. (F) Western blot analyses of whole muscle lysates with the indicated antibodies. (G) Immunostaining of single fibers with markers for lysosomes (LAMP1; green), autophagosomes (LC3; red), and nuclei (Hoechst dye; blue); muscle fibers from SYS-treated KO mice are free from autophagic buildup. Bright autofluorescent particles consist of lipofuscin, a typical feature in patients and in a KO model of the disease (87); autophagic buildup in 13.5-month-old untreated KO occupies large portions of the fibers (the image shows projection view produced from 6 consecutive optical sections (Z-stack). Bars: 20 μm. (H) Muscle strength was assessed using grip strength test after treatment. Statistical significance was determined by 1-way ANOVA and unpaired 2-tailed Student’s t test. Data presented as mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. less

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CD38/ADP-ribose/TRPM2-mediated nuclear Ca2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes.

In Exp Mol Med on 1 July 2023 by Rah, S. Y., Joe, Y., et al.

Fig.1.D

Nuclear CD38 is involved in gluconeogenesis by glucagon.a After isolating nuclei with sucrose ultracentrifugation, the outer membrane of the nuclei was separated by treatment with sodium citrate. Nucleoplasts were fixed with 3.8% paraformaldehyde for 20 min, permeabilized, and then stained with a... more

Nuclear CD38 is involved in gluconeogenesis by glucagon.a After isolating nuclei with sucrose ultracentrifugation, the outer membrane of the nuclei was separated by treatment with sodium citrate. Nucleoplasts were fixed with 3.8% paraformaldehyde for 20 min, permeabilized, and then stained with anti-mouse CD38 antibody (green). Lamin B1 was used as a marker for the inner membrane of the nucleus. The nucleus was stained with DAPI (blue). Scale bar, 5 μm. b After nuclei were isolated from Cd38+/+ or Cd38−/− mouse primary hepatocytes, the outer membrane of the nucleus was removed by sodium citrate treatment. The nucleoplasts (outer membrane-deprived nuclei) were stained with an anti-mouse CD38 antibody (C90). Scale bar, 5 μm. c Adenoviral vector constructs of Control (Ad-NLS), catalytic active-nCD38 (wtCd38), and catalytic inactive-nCD38 (Cd38E230D). d Hepatocytes were transduced with adenoviral vectors expressing Ad-NLS, wtCd38-NLS, or Cd38E230D-NLS at an MOI of 50 for 24 h, and then cytoplasmic and nuclear extracts were isolated. Immunoblotting was performed to detect Myc, LAMP-1 (a marker of lysosomes), GAPDH (a cytoplasmic marker), and Lamin B1 (a nuclear marker). e NAD glycohydrolase activity. f ADPR levels in hepatocytes treated with glucagon (100 nM) for 30 sec. g, h qRT-PCR of G6pc (g) and Pck1 (h) after incubation with glucagon (100 nM) for 4 h. i Glucose production after incubation with glucagon (100 nM) for 5 h. e–in = 6 independent experiments, mean values ± SEMs are shown, and P values were calculated by one-way ANOVA followed by Tukey’s multiple comparison test. **P < 0.01, ***P < 0.001, and not significant (NS). For a, b, d, representative images of three independent experiments are shown. less

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CD38/ADP-ribose/TRPM2-mediated nuclear Ca2+ signaling is essential for hepatic gluconeogenesis in fasting and diabetes.

In Exp Mol Med on 1 July 2023 by Rah, S. Y., Joe, Y., et al.

Fig.2.E

Cx43 regulates NAD+ transport to CD38 in the perinuclear space via phosphorylation by PKCδ.a Glucagon-induced ADPR production after preincubation with oleamide (50 μM) for 30 min. n = 6 independent experiments. ***P < 0.001 and not significant (NS). b Effect of oleamide on glucagon-induced mRNA e... more

Cx43 regulates NAD+ transport to CD38 in the perinuclear space via phosphorylation by PKCδ.a Glucagon-induced ADPR production after preincubation with oleamide (50 μM) for 30 min. n = 6 independent experiments. ***P < 0.001 and not significant (NS). b Effect of oleamide on glucagon-induced mRNA expression of G6pc and Pck1. n = 6 independent experiments. **P < 0.01, ***P < 0.001. c After isolating nuclei, nucleoplasts were stained with anti-Cx43 antibody (green). Lamin B1 was used as a marker for the inner membrane of the nucleus. The nucleus was stained with DAPI (blue). Scale bar, 5 μm. d The bar graph represents the localization of Cx43. n = 6 independent experiments. ***P < 0.001. e Hepatocytes were transduced with adenoviral vectors expressing Ad-Cx43-NLS or Ad-Cx43S368A-NLS at an MOI of 50 for 24 h and then assayed for immunoblot analysis for Ad-NLS, Ad-Cx43-NLS, or Ad-Cx43S368A-NLS and qRT-PCR of G6pc and Pck1 in hepatocytes infected with Cx43 WT or S368A before and after treatment with glucagon. n = 8 independent experiments. **P < 0.01, ***P < 0.001. f, g Blood glucose levels during fasting (f) and after pyruvate challenge (g) in mice after treatment with Ad-NLS, Ad-Cx43-NLS, or Ad-Cx43S368A-NLS. n = 10 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05; Ad-NLS vs. Ad-Cx43-NLS, ##P < 0.001; Ad-Cx43-NLS vs. Ad-Cx43S368A-NLS. h Nucleoplasts were stained with anti-PKCδ antibody (green). Lamin B1 was used as a marker for the inner membrane of the nucleus. The nucleus was stained with DAPI (blue). Scale bar, 5 μm. i Glucagon-induced G6pc and Pck1 mRNA levels. GF109203X (1 μM), Go6976 (1 μM), or rottlerin (3 μM) was preincubated for 30 min. n = 6 independent experiments. **P < 0.01, ***P < 0.001. j Effects of PKCδ KD on glucagon-induced G6pc and Pck1 gene expression. Immunoblotting of PKCδ and β-actin (upper). n = 6 independent experiments. **P < 0.01, ***P < 0.001. k Nucleoplasts were incubated with OAG for the indicated times. Immunoblotting for pCx43 (S368), Cx43, and Lamin B1. l NAD and ADPR levels after nucleoplasts were incubated with OAG (100 μM) for 30 sec. Ara-2’-F-NAD (200 nM) was preincubated for 30 min. n = 6 independent experiments. ***P < 0.001. m NAD and ADPR levels in PKCδ-knockdown nucleoplasts. OAG (100 μM) was incubated for 30 sec. Immunoblotting of PKCδ and Lamin B1 (upper). n = 6 independent experiments. **P < 0.01, ***P < 0.001. Data are represented as the mean ± SEM. Statistics were determined by one-way ANOVA followed by Tukey’s multiple comparison test except d where unpaired t test was used. For confocal images and immunoblotting, representative images of three independent experiments are shown. less

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TMEM106B regulates microglial proliferation and survival in response to demyelination.

In Sci Adv on 5 May 2023 by Zhang, T., Pang, W., et al.

Fig.4.A

TMEM106B deficiency results in lysosomal abnormalities in microglia.(A) Western blot analysis of lysosomal proteins in the control and Tmem106b−/− BV2 cells using the indicated antibodies. The levels of each protein were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data from th... more

TMEM106B deficiency results in lysosomal abnormalities in microglia.(A) Western blot analysis of lysosomal proteins in the control and Tmem106b−/− BV2 cells using the indicated antibodies. The levels of each protein were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data from three independent experiments were plotted. (B) Reduced CathD/E activities in TMEM106B-deficient BV2 cells. CathD/E activities were measured in control and Tmem106b−/− BV2 cells using fluorogenic substrates. Data represent the means ± SEM. Statistical significance was analyzed by unpaired one-tailed Student’s t test (n = 3). *P < 0.05. (C) Lysosomal pH measurement in control and Tmem106b−/− BV2 cells. BV2 cells were incubated with dextran conjugated with pH-sensitive fluorescein (green) and pH-insensitive tetramethylrhodamine (red). Representative images were shown. Scale bar, 10 μm. FITC and TRITC intensity per cell were quantified and the ratio (FITC/TRITC) was calculated. Lysosomal pH was determined from the calibration curve generated from the FTIC/TRITC value of permeabilized cells in various calibration standard solutions of different pH values. Data represent the means ± SEM. Statistical significance was analyzed by unpaired one-tailed Student’s t test (n = 3). *P < 0.05. (D and E) WT and KO mice were fed with CPZ-containing chow (CPZ 5w) for 5 weeks or fed with CPZ for 5 weeks and a normal diet for additional 3 weeks after CPZ removal (Rec 3w). Brain sections were stained with LAMP1, CathD, and IBA1 antibodies. Representative confocal images in the corpus callosum region were shown (A). Scale bars, 10 μm. The LAMP1 or CathD levels in microglia were quantified (B). Data represent the means ± SEM. Statistical significance was analyzed by unpaired two-tailed Student’s t test (n = 3 mice per group). *P < 0.05 and **P < 0.01. less

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Mycobacterial protein PE_PGRS30 induces macrophage apoptosis through prohibitin 2 mitochondrial function interference.

In Front Microbiol on 14 February 2023 by Matsumura, K., Takaki, S., et al.

Fig.4.C

PE_PGRS30 induces apoptosis via PGRS domain. (A) Macrophages undergo apoptosis in response to recombinant PE_PGRS30 proteins. RAW264.7 cells were treated with 10 μg/ml of full-length (FL), PE, PGRS, CT domains and control GFP for 24 h. Cells were stained with Annexin V and analyzed by flow cytome... more

PE_PGRS30 induces apoptosis via PGRS domain. (A) Macrophages undergo apoptosis in response to recombinant PE_PGRS30 proteins. RAW264.7 cells were treated with 10 μg/ml of full-length (FL), PE, PGRS, CT domains and control GFP for 24 h. Cells were stained with Annexin V and analyzed by flow cytometry. Numbers of Annexin V+ cells are presented as percentages against total cells. More than 104 cells were analyzed in each experiment. (B) LDH release by cells treated with PE_PGRS30 proteins. LDH in culture supernatants at 24 h post treatment were detected. (C) Co-localization of PE_PGRS30 with PHB2, mitochondria and LAMP1 in macrophages. RAW264.7 cells were treated as in (A). Cells were stained with a combination of anti-Myc-tag antibody and anti-PHB2 antibody or MitoTracker Red CMXRos or anti-LAMP1 antibodies 16 h after treatment. Arrowheads denote co-localization with PE_PGRS30 and detected molecules. Scale bar, 10 μm. (D–F) Quantification of co-localization of PE_PGRS30 with PHB2 (D), mitochondria (E), and LAMP1 (F) in macrophages. More than 100 cells were analyzed in each experiment. (A–F) Data are representative of three independent experiments. (A,B,D–F) Means ± SD are shown. *p< 0.05 using one-way ANOVA with Tukey’s multiple comparisons. less

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TMEM106B deficiency impairs cerebellar myelination and synaptic integrity with Purkinje cell loss.

In Acta Neuropathol Commun on 14 March 2022 by Feng, T., Luan, L., et al.

Fig.4.A

Lysosome and lysosomal trafficking defects in the cerebellum of aged Tmem106b−/− mice. a, b Western blot analysis of lysosomal proteins and GAPDH in 16-month-old WT and Tmem106b−/− cerebellar lysates. Protein levels were quantified and normalized to GAPDH in b. HM: high molecular weight. n = 5, *... more

Lysosome and lysosomal trafficking defects in the cerebellum of aged Tmem106b−/− mice. a, b Western blot analysis of lysosomal proteins and GAPDH in 16-month-old WT and Tmem106b−/− cerebellar lysates. Protein levels were quantified and normalized to GAPDH in b. HM: high molecular weight. n = 5, **, p < 0.01, ***, p < 0.001, unpaired t-test. Asterisk indicates non‐specific bands. c, d Immunostaining of AnkG and Cath D in the cerebellar sections from 16‐month‐old WT and Tmem106b−/− mice. Representative images from the Purkinje cell layer are shown. The percentage of axon initial segments (AISs) with Cath D-positive lysosomal vesicles were quantified in d. n = 3. ****, p < 0.0001, unpaired t-test less

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Neuropathological and behavioral characterization of aged Grn R493X progranulin-deficient frontotemporal dementia knockin mice.

In Acta Neuropathol Commun on 1 April 2021 by Frew, J. & Nygaard, H. B.

Fig.3.A

Global dysregulation of lysosomal function in aged GrnR493X/R493X brain. Representative western blots of hemibrain RIPA-soluble lysates from 18 month old Grn+/+ and GrnR493X/R493X mice probed with the indicated antibodies (a). Expression of Lamp1, total/pro/mat-Ctsd, and LC3I/II in Grn+/+ and Grn... more

Global dysregulation of lysosomal function in aged GrnR493X/R493X brain. Representative western blots of hemibrain RIPA-soluble lysates from 18 month old Grn+/+ and GrnR493X/R493X mice probed with the indicated antibodies (a). Expression of Lamp1, total/pro/mat-Ctsd, and LC3I/II in Grn+/+ and GrnR493X/R493X hemibrain lysates was analyzed by western blotting, using actin as the loading control. Densitometric quantification of brain-wide Lamp1 (b) and Ctsd (c) expression was normalized to actin and Grn+/+ levels. The LC3 II:LC3 I densitometric expression ratio (d) was normalized to Grn+/+ levels. n = 6 mice were used per sex/genotype; values are shown as mean ± SEM; ns = not significant, **p < 0.01, ***p < 0.0001, Student’s t-test less

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The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors.

In Int J Mol Sci on 8 December 2020 by Tran, M. T., Okusha, Y., et al.

Fig.6.A

The effects of CLQ-mediated lysosomal function on endogenous levels of c-Fms and RANK in RAW-D cell-derived osteoclasts. (A) RAW-D cells expressing GFP or one of three different types of GFP-Rab11b (#1, #2, and #3) were treated with RANKL (300 ng/mL) for 3 days. The immunoblotting analysis of end... more

The effects of CLQ-mediated lysosomal function on endogenous levels of c-Fms and RANK in RAW-D cell-derived osteoclasts. (A) RAW-D cells expressing GFP or one of three different types of GFP-Rab11b (#1, #2, and #3) were treated with RANKL (300 ng/mL) for 3 days. The immunoblotting analysis of endogenous levels of LAMP1, a specific lysosomal receptor, and two lysosomal enzymes, Cathepsins B and D was done. GAPDH was used as a loading control. The shown data was representative of two independent repeats. (B) RAW-D cells expressing GFP or GFP-Rab11b (type #3) was pre-treated with RANKL (300 ng/mL) for 3 days before incubation with CLQ (0, 1, 5, 10, and 30 μM) for 10 h. The WB analysis of endogenous levels of c-Fms, RANK, and Rab11b was done. GAPDH was used as a loading control. The shown data was representative of two independent repeats. (C) RAW-D cells expressing GFP or GFP-Rab11b (type #3) were pre-treated with RANKL (300 ng/mL) for 3 days before incubation with CLQ (10 μM) over a time course (0, 1, 3, 5, and 8 h). The WB analysis of endogenous levels of c-Fms, RANK, and Rab11b was done. GAPDH was used as a loading control. (D,E) Total RNA was extracted, and cDNA was prepared from osteoclasts differentiated from RAW-D cells expressing GFP or GFP-Rab11b (type #3), following RANKL (300 ng/mL) stimulation for 3 days. Expression levels of c-Fms (D) and RANK (E) mRNAs were analyzed by qRT-PCR. Mean ± SD of two independent repeats. * p < 0.05, ** p < 0.01 (Student’s t-test). (F) RAW-D cells expressing GFP or one of three different types of GFP-Rab11b (#1, #2, and #3) were pre-treated with RANKL (300 ng/mL) for 3 days. The cells were subsequently treated with CHX (20 μg/mL) and simultaneously with or without CLQ (10 μM) for 5 h. The WB analysis of endogenous levels of c-Fms, RANK, and Rab11b was done. β-actin was used as a loading control. The shown data was the representative of two independent repeats. less

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Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice.

In EMBO Rep on 5 October 2020 by Feng, T., Mai, S., et al.

Fig.8.G

Increases in the levels of lipofuscin and lysosome proteins in Tmem106b−/−Grn−/− miceA, BLipofuscin accumulation in the retina: The number of autofluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform lay... more

Increases in the levels of lipofuscin and lysosome proteins in Tmem106b−/−Grn−/− miceA, BLipofuscin accumulation in the retina: The number of autofluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. n = 3–6. Scale bar = 50 μm.C–FTMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord (C, D) and thalamus (E, F) of 5‐month‐old mice. n = 3. Scale bar = 10 μm.G, HWestern blot analysis of lysosomal proteins in the spinal cord lysates from 5‐month‐old WT, Tmem106b−/−, Grn−/−, and Tmem106b−/−Grn−/− mice. Asterisk indicates non‐specific bands. n = 3.Data information: For all the analyses, data are presented as mean ± SEM. One‐way ANOVA tests with Bonferroni's multiple comparisons: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance. Source data are available online for this figure. less

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Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy.

In Elife on 4 May 2017 by Kleinecke, S., Richert, S., et al.

Fig.3.F

Hallmarks of lysosomal storage disorders in Pex5 mutant nerves.(A) Double-staining of sciatic teased fibers displayed giant GD1a+ (green) vesicles that colocalized with LAMP1 (red) in internodes only of mutant animals (rectangular inset magnified in right panels). Dotted lines indicate borders of... more

Hallmarks of lysosomal storage disorders in Pex5 mutant nerves.(A) Double-staining of sciatic teased fibers displayed giant GD1a+ (green) vesicles that colocalized with LAMP1 (red) in internodes only of mutant animals (rectangular inset magnified in right panels). Dotted lines indicate borders of myelinated fibers; scale bar in inset, 5 µm. (B) Electron microscopy of a teased and sectioned mutant sciatic nerve depicting paranodal vesicle accumulations. Scale bar, 500 nm. (C) Immuno-electron microscopy of sciatic nerves from mice aged 9 months identifies enlarged LAMP1+ vesicles (arrows) within paranodal loops (PNLs). M, myelin; Ax, axon. Scale bar, 500 nm. (D, E) Immunolabeling of teased sciatic nerves at the age of 2 months shows enlarged LAMP1+ (left, green) or LIMP-2+ (right, green) compartments close to CASPR+ paranodes (red) of mutant nerves. Scale bars, 10 µm. (F–H) Analysis of sciatic nerve lysates by Western blotting (n = 4; β-III Tubulin, served as loading control for normalization) and qRT-PCR (n = 6) for LAMP1 and LIMP-2 showing increased protein, but decreased mRNA levels in mutants. (I) Immunostaining of control sciatic fibers shows close association of peroxisomal PMP70 (green) and LAMP1 on lysosomal membranes (red) in the paranodal region, suggesting physical interactions. A wide-field flourescent image after deconvolution (250 nm z-stack width) is shown. Scale bar, 10 µm. Dotted lines indicate borders of the myelinated fiber. (J) Lysosomal enzyme activities of nerve lysates was enhanced in mutants compared to controls as assessed by assays using substrates of α-Mannosidase (left) and β-Hexosaminidase (right; n = 4). Statistics: means ± s.e.m. *p<0.05; **p<0.01; ***p<0.001; n.s., not significant, Student’s T-test (G, H, J). Blue arrows indicate nodes of Ranvier (D, E, I).DOI:http://dx.doi.org/10.7554/eLife.23332.01110.7554/eLife.23332.012Figure 3—source data 1.Lysosomal accumulation within paranodal loops.DOI:http://dx.doi.org/10.7554/eLife.23332.012Lysosomal accumulation within paranodal loops.DOI:http://dx.doi.org/10.7554/eLife.23332.012Characterization of paranodal vesicle inclusions in Pex5 mutant nerves.(A–C) Immunofluorescence showing normal abundance of early/late endosomal and autophagosomal vesicles (green) in the vicinity of nodes of Ranvier (indicated by paranodal CASPR or blue arrow) of teased fibers obtained from 9-month-old animals. RAB7, Ras-related protein 7 (late endosomes; top); EEA1, early endosome-associated protein 1 (middle); ATG5, autophagy-related protein 5 (autophagosomes; bottom). Scale bars, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.23332.013 less

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CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL.

In Nat Commun on 11 March 2016 by Bartuzi, P., Billadeau, D. D., et al.

Fig.5.A

The WASH complex is essential for endosomal sorting of LDLR.(a) Cellular localization of LDLR (green), COMMD1 (red) and LAMP1 (pink) in Wash1f/f and Wash1−/− MEFs was determined by immunofluorescence staining. Representative images are shown; scale bar, 5 μm. Relative colocalization of (b) LDLR a... more

The WASH complex is essential for endosomal sorting of LDLR.(a) Cellular localization of LDLR (green), COMMD1 (red) and LAMP1 (pink) in Wash1f/f and Wash1−/− MEFs was determined by immunofluorescence staining. Representative images are shown; scale bar, 5 μm. Relative colocalization of (b) LDLR and (c) COMMD1 with VPS35, EEA1 and LAMP1 was quantified. (d) LDLR, WASH1, VPS35 and COMMD1 levels in Wash1f/f and Wash1−/− MEFs analysed by western blot. (e) Representative images (n=3) of immunoblot analysis of total LDLR levels in Wash1f/f and Wash1−/− MEFs treated with bafilomycin A (100 nM) for 0, 4 and 6 h. (f) Densitometry revealed the relative levels of LDLR in bafilomycin A-treated cells (n=3). (g) Representative images (n=3) of LDLR on the surface of Wash1f/f and Wash1−/− MEFs determined by surface biotinylation assay. (h) Densitometry revealed relative LDLR surface levels (n=3). (i) Wash1f/f and Wash1−/− MEFs were incubated with DiI-LDL for 30 min and imaged by fluorescence microscope. (j) Fluorescence intensity was quantified using ImageJ software and was normalized to the number of DAPI nuclei per image; >30 cells per condition were recorded. The results are presented as mean±s.e.m.; significance was calculated relative to the control group by unpaired Student's t-test; ***P<0.001. less

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