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Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells.

Cao Y, Staropoli JF, Biswas S, Espinola JA, MacDonald ME, Lee JM, Cotman SL - PLoS ONE (2011)

Bottom Line: To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells.However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution.Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells. CbCln6(nclf/nclf) cells and CbCln3(Δex7/8/Δex7/8) cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.

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Mitochondrial abnormalities in homozygous CbCln6nclf and CbCln3Δex7/8 cells.A. Representative micrographs of wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells immunostained with antibody recognizing the mitochondrial matrix protein, grp75, are shown. Note the elongated appearance of mitochondria in homozygous CbCln3Δex7/8 and CbCln6nclf cells, compared to wild-type cells in the zoomed insets. A lower magnification of a representative field of cells is also shown to demonstrate the subconfluent culture conditions. The altered mitochondrial morphology was also quantified by automated image analysis, showing a significantly reduced circularity index of the labeled mitochondria in the mutant cells, compared to wild-type cells (see Results). Wild-type panel is representative of both CbCln3+/+ and CbCln6+/+ cell lines. Heterozygous cell lines were indistinguishable from wild-type (not shown). All images were captured at 40× magnification and were taken on the same day with identical settings. B. The bar graph depicts relative total cellular ATP levels in wild-type, heterozygous, or homozygous CbCln3Δex7/8 and CbCln6nclf cells, determined using the CellTiter-GLO® Luminescent Cell assay. Relative luciferase units were normalized to the wild-type cell lines and were pooled from 2–3 independent assays per cell line, each tested in 3–10 wells per assay. For reference, absolute RLUs for CbCln3+/+ and CbCln6+/+ cell lines were 1995830±27506 and 626172±151671, respectively. *, p≤.01 in a Student's t-test; n.s. = not significant.
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pone-0017118-g001: Mitochondrial abnormalities in homozygous CbCln6nclf and CbCln3Δex7/8 cells.A. Representative micrographs of wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells immunostained with antibody recognizing the mitochondrial matrix protein, grp75, are shown. Note the elongated appearance of mitochondria in homozygous CbCln3Δex7/8 and CbCln6nclf cells, compared to wild-type cells in the zoomed insets. A lower magnification of a representative field of cells is also shown to demonstrate the subconfluent culture conditions. The altered mitochondrial morphology was also quantified by automated image analysis, showing a significantly reduced circularity index of the labeled mitochondria in the mutant cells, compared to wild-type cells (see Results). Wild-type panel is representative of both CbCln3+/+ and CbCln6+/+ cell lines. Heterozygous cell lines were indistinguishable from wild-type (not shown). All images were captured at 40× magnification and were taken on the same day with identical settings. B. The bar graph depicts relative total cellular ATP levels in wild-type, heterozygous, or homozygous CbCln3Δex7/8 and CbCln6nclf cells, determined using the CellTiter-GLO® Luminescent Cell assay. Relative luciferase units were normalized to the wild-type cell lines and were pooled from 2–3 independent assays per cell line, each tested in 3–10 wells per assay. For reference, absolute RLUs for CbCln3+/+ and CbCln6+/+ cell lines were 1995830±27506 and 626172±151671, respectively. *, p≤.01 in a Student's t-test; n.s. = not significant.

Mentions: The pathological hallmark of NCL is an abnormal lysosomal accumulation of the pore-forming subunit c of the mitochondrial F0 ATP synthase complex. At sub-confluent density, a mitochondrial marker, anti-grp75, which has revealed significant elongation of mitochondria in homozygous CbCln3Δex7/8 cells [20] (mean circularity index 0.81±.002), also revealed abnormally elongated mitochondria in homozygous CbCln6nclf cerebellar cells (mean circularity index 0.79±.003), compared to wild-type cells (CbCln3+/+ or CbCln6+/+; mean circularity index 0.85±.002) (Figure 1A). Similar to homozygous CbCln3Δex7/8 cells, the homozygous CbCln6nclf cerebellar cells also displayed a significant reduction (∼30–40%, p≤.01) in total cellular ATP levels, relative to wild-type or heterozygous cells (Figure 1B), strongly suggesting altered mitochondrial morphology and function.


Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells.

Cao Y, Staropoli JF, Biswas S, Espinola JA, MacDonald ME, Lee JM, Cotman SL - PLoS ONE (2011)

Mitochondrial abnormalities in homozygous CbCln6nclf and CbCln3Δex7/8 cells.A. Representative micrographs of wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells immunostained with antibody recognizing the mitochondrial matrix protein, grp75, are shown. Note the elongated appearance of mitochondria in homozygous CbCln3Δex7/8 and CbCln6nclf cells, compared to wild-type cells in the zoomed insets. A lower magnification of a representative field of cells is also shown to demonstrate the subconfluent culture conditions. The altered mitochondrial morphology was also quantified by automated image analysis, showing a significantly reduced circularity index of the labeled mitochondria in the mutant cells, compared to wild-type cells (see Results). Wild-type panel is representative of both CbCln3+/+ and CbCln6+/+ cell lines. Heterozygous cell lines were indistinguishable from wild-type (not shown). All images were captured at 40× magnification and were taken on the same day with identical settings. B. The bar graph depicts relative total cellular ATP levels in wild-type, heterozygous, or homozygous CbCln3Δex7/8 and CbCln6nclf cells, determined using the CellTiter-GLO® Luminescent Cell assay. Relative luciferase units were normalized to the wild-type cell lines and were pooled from 2–3 independent assays per cell line, each tested in 3–10 wells per assay. For reference, absolute RLUs for CbCln3+/+ and CbCln6+/+ cell lines were 1995830±27506 and 626172±151671, respectively. *, p≤.01 in a Student's t-test; n.s. = not significant.
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Related In: Results  -  Collection

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pone-0017118-g001: Mitochondrial abnormalities in homozygous CbCln6nclf and CbCln3Δex7/8 cells.A. Representative micrographs of wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells immunostained with antibody recognizing the mitochondrial matrix protein, grp75, are shown. Note the elongated appearance of mitochondria in homozygous CbCln3Δex7/8 and CbCln6nclf cells, compared to wild-type cells in the zoomed insets. A lower magnification of a representative field of cells is also shown to demonstrate the subconfluent culture conditions. The altered mitochondrial morphology was also quantified by automated image analysis, showing a significantly reduced circularity index of the labeled mitochondria in the mutant cells, compared to wild-type cells (see Results). Wild-type panel is representative of both CbCln3+/+ and CbCln6+/+ cell lines. Heterozygous cell lines were indistinguishable from wild-type (not shown). All images were captured at 40× magnification and were taken on the same day with identical settings. B. The bar graph depicts relative total cellular ATP levels in wild-type, heterozygous, or homozygous CbCln3Δex7/8 and CbCln6nclf cells, determined using the CellTiter-GLO® Luminescent Cell assay. Relative luciferase units were normalized to the wild-type cell lines and were pooled from 2–3 independent assays per cell line, each tested in 3–10 wells per assay. For reference, absolute RLUs for CbCln3+/+ and CbCln6+/+ cell lines were 1995830±27506 and 626172±151671, respectively. *, p≤.01 in a Student's t-test; n.s. = not significant.
Mentions: The pathological hallmark of NCL is an abnormal lysosomal accumulation of the pore-forming subunit c of the mitochondrial F0 ATP synthase complex. At sub-confluent density, a mitochondrial marker, anti-grp75, which has revealed significant elongation of mitochondria in homozygous CbCln3Δex7/8 cells [20] (mean circularity index 0.81±.002), also revealed abnormally elongated mitochondria in homozygous CbCln6nclf cerebellar cells (mean circularity index 0.79±.003), compared to wild-type cells (CbCln3+/+ or CbCln6+/+; mean circularity index 0.85±.002) (Figure 1A). Similar to homozygous CbCln3Δex7/8 cells, the homozygous CbCln6nclf cerebellar cells also displayed a significant reduction (∼30–40%, p≤.01) in total cellular ATP levels, relative to wild-type or heterozygous cells (Figure 1B), strongly suggesting altered mitochondrial morphology and function.

Bottom Line: To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells.However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution.Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf) cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8) cerebellar cells. CbCln6(nclf/nclf) cells and CbCln3(Δex7/8/Δex7/8) cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf) cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf) and CbCln3(Δex7/8/Δex7/8) cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8) and Cln6(nclf) mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.

Show MeSH
Related in: MedlinePlus