Journal: Human molecular genetics

Article Title: Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage.

doi: 10.1093/hmg/ddn043

Figure Lengend Snippet: Figure 1. Effect of Neu4 transfection on cultured neuroglia cells from a Tay- Sachs patient. (A) TLC analysis of the gangliosides extracted from the culture medium (Medium) or from the cell homogenates (Cells) of the control neuro- glia cells (C) or the cells from a Tay-Sachs patient transfected with pCMV-Neu4 (Neu4) and empty pCMV (Mock) plasmids. The cells were incu- bated in the presence of full medium containing 10% fetal calf serum and 3.3 nmol/ml of lissamine–rhodamine-labeled GM1 ganglioside for 72 h. The culture medium almost exclusively contains labeled GM1 ganglioside. Control cells, contain the labeled GM1 and GM2 gangliosides as well as lacto- sylceramide (LacCer), whereas in the Tay-Sachs cells the conversion stops at the level of GM2 ganglioside. Neu4-transfected Tay-Sachs cells show a ganglioside pattern similar to that of the normal control cells. (B) Ganglioside metabolism in neuroglia cells loaded with [3H]-labeled dihydroganglioside GM2. The cells were labeled in the presence of full medium containing 10% fetal calf serum and 10 mCi/ml of [3H]-labeled GM2 72 h. Histograms show the total amount of radioactivity found in glycolipids on the TLC plates and the amount of radioactivity associated with glycolipid GA2, GM3 ganglioside and its metabolites (LacCer, GlcCer) in the extracts from the control neuroglia cells (Control) or the cells from a Tay-Sachs patient transfected with

Article Snippet: The macrophages from the knockout and wild-type mice were loaded for 72 h with total porcine brain gangliosides (Avanti Polar Lipids).

Techniques: Transfection, Cell Culture, Control, Labeling, Radioactivity

Journal: Human molecular genetics

Article Title: Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage.

doi: 10.1093/hmg/ddn043

Figure Lengend Snippet: Figure 2. Effect of Neu4 and Neu1 silencing in HeLa cells. (A) Sialidase activity in lysates of non-transfected HeLa cells (Control) and the cells stably transfected with pSilencer-Neu1 (siNeu1), pSilencer-Neu4 (siNeu4) or pSilencer (Mock) vectors against the total porcine brain gangliosides or against the artificial sialidase substrate, 4MU-NeuAc. Values represent means+SD of triplicate experiments. P , 0.05 and P , 0.01 when com- pared with the mock-transfected cells. Effect of the Neu4 silencing on the cell morphology as seen on light (B) and electron (C) micrographs. HeLa cells transfected with pSilencer-Neu4 and loaded for 72 h with 0.2 mg of total gangliosides from porcine brain per milliliter of medium present the extensive vacuolation (B, arrows) and are filled with large vacuoles containing vesicular profiles (C, inset). Mock-transfected HeLa cells loaded with gangliosides do not present vacuolation (B) and contain normal lysosomes (C). Magnification 1200 (B) and 35 000 (C). All microphotographs represent typical images obtained in triplicate experiments; from 70 to 150 cells were studied for each condition in each experiment.

Article Snippet: The macrophages from the knockout and wild-type mice were loaded for 72 h with total porcine brain gangliosides (Avanti Polar Lipids).

Techniques: Activity Assay, Transfection, Control, Stable Transfection

Journal: Human molecular genetics

Article Title: Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage.

doi: 10.1093/hmg/ddn043

Figure Lengend Snippet: Figure 4. Pathological changes in Neu4-deficient mice. (A) Microscopic investigation of tissue sections from lungs and spleen shows a marked vacuolization in the Neu42/2 mice consistent with the lysosomal storage phenotype. While the cells from the wild-type mouse show dense lysosomes stained with osmium the cells from the Neu42/2 mice contain light vacuoles (arrows) different from the normal lipid granules stained with toluidin blue. Magnification 1200; bar equals 12 mm and applies to all images. (B) Electron micrographs showing a large accumulation of vacuoles in the cytoplasm of lymphocytes (L) in the red pulp of spleen from the Neu42/2 mouse (arrows). The cells from wild-type show homogeneous lysosomal content. Magnification 8000; bar equals 1 mm. (C) Electron micrographs of cultured splenocyte-derived macrophages supplemented with gangliosides. The cells from the knockout mice show a heterogeneous population of lysosomes (L) and a moderate accumulation of lysosomes containing lamellated or whorls of membrane-like structures (arrows). The wild-type splenocytes show a homogenous population of lysosomes (L). N, nucleus. Magnification 16 000; bar equals 1 mm. (D) Electron micrographs of septal macrophages from lungs of wild-type and knockout mice. Wild-type macrophages show a homogenous population of lysosomes (arrows). Conversely, knockout macrophages present a moderate accumulation of lysosomes containing whorls of membrane (L). N, nuclei of septal macrophages. Magnification 10 000; bar equals 1 mm. (E) Elec- tron micrograms of Type II pneumocytes from the lungs of the wild-type and knockout mice. The lamellar bodies in the cells of Neu42/2 mice are larger in diameter (mean diameter 0.8 mm+0.1 mm) than those in the lungs of the wild-type mice (mean diameter 0.4 mm+0.05 mm). Magnification 20 000; bar equals 1 mm.

Article Snippet: The macrophages from the knockout and wild-type mice were loaded for 72 h with total porcine brain gangliosides (Avanti Polar Lipids).

Techniques: Staining, Cell Culture, Derivative Assay, Knock-Out, Membrane

Journal: Human molecular genetics

Article Title: Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage.

doi: 10.1093/hmg/ddn043

Figure Lengend Snippet: Figure 5. Alteration of ganglioside metabolism in the tissues of Neu4-deficient mice. (A) Representative TLC images of orcinol-stained gangliosides from brain and resorcinol-stained gangliosides from lungs and spleen of the Neu4þ/þ, and Neu42/2 animals at 1 and 9 months of age. The positions of the ganglioside standards (S) are indicated on the left. (B) Histograms show the ratio of GM1 and GD1a gangliosides as measured by TLC in the extracts of brain tissues from Neu4þ/þ; Neu4þ/2 and Neu42/2 mice. Values represent means+SD of duplicate measurements.P,0.01 when compared with the Neu4þ/þ or Neu4þ/2 animals by the two-tailed t-test.

Article Snippet: The macrophages from the knockout and wild-type mice were loaded for 72 h with total porcine brain gangliosides (Avanti Polar Lipids).

Techniques: Staining, Two Tailed Test