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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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Altered PDI immunostain in homozygous CbCln6nclf cells.Representative micrographs of CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) and CbCln3 cells (CbCln3Δex7/8/Δex7/8; CbCln3+/+) immunostained for the ER marker protein PDI (red) are shown. Note the decreased PDI signal in CbCln6nclf/nclf cells, as compared to wild-type (CbCln6+/+ or CbCln3+/+) and CbCln3Δex7/8/Δex7/8 cells. PDI immunostain of normal and variant late-infantile (vLINCL) lymphoblast cells is also shown. BiP immunostain was comparable across all of the cerebellar cell lines, but is only shown for the CbCln6+/+ and CbCln6nclf/nclf cells. BiP was not assessed in lymphoblast cells. All images were taken at 40× magnification, and for like stains, on the same day, with identical instrument settings. DAPI nuclear counter stain is shown in blue.
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pone-0017118-g003: Altered PDI immunostain in homozygous CbCln6nclf cells.Representative micrographs of CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) and CbCln3 cells (CbCln3Δex7/8/Δex7/8; CbCln3+/+) immunostained for the ER marker protein PDI (red) are shown. Note the decreased PDI signal in CbCln6nclf/nclf cells, as compared to wild-type (CbCln6+/+ or CbCln3+/+) and CbCln3Δex7/8/Δex7/8 cells. PDI immunostain of normal and variant late-infantile (vLINCL) lymphoblast cells is also shown. BiP immunostain was comparable across all of the cerebellar cell lines, but is only shown for the CbCln6+/+ and CbCln6nclf/nclf cells. BiP was not assessed in lymphoblast cells. All images were taken at 40× magnification, and for like stains, on the same day, with identical instrument settings. DAPI nuclear counter stain is shown in blue.

Mentions: To determine whether similar accumulation of subunit c in Lamp 1-positive vesicles, might reflect similar or distinct membrane organelle perturbations, CbCln6nclf/nclf and CbCln3Δex7/8/Δex7/8 cerebellar cells were assessed using a panel of organelle markers [20]. We found no obvious morphological differences in the cis- and trans-Golgi in either homozygous CbCln3Δex7/8 or CbCln6nclf cerebellar cells (data not shown). However, the ER marker protein, PDI, consistently displayed reduced staining intensity in homozygous CbCln6nclf cerebellar cells, compared to wild-type (CbCln6+/+) cells (Figure 3, top panels). Homozygous CbCln3Δex7/8 cells did not display altered PDI immunostain (Figure 3). Interestingly, immunostain for another ER-associated protein, BiP [34], was not decreased in homozygous CbCln6nclf cerebellar cells and highlighted a morphologically intact ER network (Figure 3). Notably, PDI immunostain was also decreased in variant late-infantile NCL patient lymphoblast cells, compared to normal lymphoblasts (Figure 3, bottom panels), suggesting that the vLINCL mutation may specifically alter PDI epitope availability or distribution within the ER of diverse cell types. Total PDI levels were not obviously different in the homozygous CbCln6nclf cerebellar cells versus wild-type cells by immunoblot analysis (data not shown).


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)

Altered PDI immunostain in homozygous CbCln6nclf cells.Representative micrographs of CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) and CbCln3 cells (CbCln3Δex7/8/Δex7/8; CbCln3+/+) immunostained for the ER marker protein PDI (red) are shown. Note the decreased PDI signal in CbCln6nclf/nclf cells, as compared to wild-type (CbCln6+/+ or CbCln3+/+) and CbCln3Δex7/8/Δex7/8 cells. PDI immunostain of normal and variant late-infantile (vLINCL) lymphoblast cells is also shown. BiP immunostain was comparable across all of the cerebellar cell lines, but is only shown for the CbCln6+/+ and CbCln6nclf/nclf cells. BiP was not assessed in lymphoblast cells. All images were taken at 40× magnification, and for like stains, on the same day, with identical instrument settings. DAPI nuclear counter stain is shown in blue.
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pone-0017118-g003: Altered PDI immunostain in homozygous CbCln6nclf cells.Representative micrographs of CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) and CbCln3 cells (CbCln3Δex7/8/Δex7/8; CbCln3+/+) immunostained for the ER marker protein PDI (red) are shown. Note the decreased PDI signal in CbCln6nclf/nclf cells, as compared to wild-type (CbCln6+/+ or CbCln3+/+) and CbCln3Δex7/8/Δex7/8 cells. PDI immunostain of normal and variant late-infantile (vLINCL) lymphoblast cells is also shown. BiP immunostain was comparable across all of the cerebellar cell lines, but is only shown for the CbCln6+/+ and CbCln6nclf/nclf cells. BiP was not assessed in lymphoblast cells. All images were taken at 40× magnification, and for like stains, on the same day, with identical instrument settings. DAPI nuclear counter stain is shown in blue.
Mentions: To determine whether similar accumulation of subunit c in Lamp 1-positive vesicles, might reflect similar or distinct membrane organelle perturbations, CbCln6nclf/nclf and CbCln3Δex7/8/Δex7/8 cerebellar cells were assessed using a panel of organelle markers [20]. We found no obvious morphological differences in the cis- and trans-Golgi in either homozygous CbCln3Δex7/8 or CbCln6nclf cerebellar cells (data not shown). However, the ER marker protein, PDI, consistently displayed reduced staining intensity in homozygous CbCln6nclf cerebellar cells, compared to wild-type (CbCln6+/+) cells (Figure 3, top panels). Homozygous CbCln3Δex7/8 cells did not display altered PDI immunostain (Figure 3). Interestingly, immunostain for another ER-associated protein, BiP [34], was not decreased in homozygous CbCln6nclf cerebellar cells and highlighted a morphologically intact ER network (Figure 3). Notably, PDI immunostain was also decreased in variant late-infantile NCL patient lymphoblast cells, compared to normal lymphoblasts (Figure 3, bottom panels), suggesting that the vLINCL mutation may specifically alter PDI epitope availability or distribution within the ER of diverse cell types. Total PDI levels were not obviously different in the homozygous CbCln6nclf cerebellar cells versus wild-type cells by immunoblot analysis (data not shown).

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