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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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Subunit c deposits co-localize with Lamp 1 in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and the mitochondrial marker, grp75 (red). Note the large accumulations of subunit c immunostain (white arrows) in both CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, but which are not common in the wild-type cells. Moderate overlap of the subunit c and grp75 immunostains is observed in wild-type cells (yellow in overlay), but little to no overlap is seen in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Also, again note the elongated mitochondrial morphology revealed by the grp75 immunostain in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, compared to wild-type cells. B. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8, and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and Lamp 1 (red). Limited overlap of subunit c and Lamp 1 immunostain is observed in wild-type cells (yellow in overlay), but Lamp 1 strongly, though not perfectly, overlaps with the accumulated subunit c in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Note that in confluency aged cultures, the Lamp 1-labeled compartment appears expanded and/or aggregated in the mutant cells, which was not observed under sub-confluent culture conditions (not shown). A,B. Insets provide a zoomed view of the degree of immunostain overlap (yellow). Blue = DAPI stain. Images were captured with a 40X objective and, for like stains, were taken on the same day with identical settings.
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pone-0017118-g002: Subunit c deposits co-localize with Lamp 1 in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and the mitochondrial marker, grp75 (red). Note the large accumulations of subunit c immunostain (white arrows) in both CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, but which are not common in the wild-type cells. Moderate overlap of the subunit c and grp75 immunostains is observed in wild-type cells (yellow in overlay), but little to no overlap is seen in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Also, again note the elongated mitochondrial morphology revealed by the grp75 immunostain in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, compared to wild-type cells. B. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8, and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and Lamp 1 (red). Limited overlap of subunit c and Lamp 1 immunostain is observed in wild-type cells (yellow in overlay), but Lamp 1 strongly, though not perfectly, overlaps with the accumulated subunit c in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Note that in confluency aged cultures, the Lamp 1-labeled compartment appears expanded and/or aggregated in the mutant cells, which was not observed under sub-confluent culture conditions (not shown). A,B. Insets provide a zoomed view of the degree of immunostain overlap (yellow). Blue = DAPI stain. Images were captured with a 40X objective and, for like stains, were taken on the same day with identical settings.

Mentions: Homozygous CbCln3Δex7/8 cells, when aged at confluent cell density, accumulated subunit c-positive puncta [20]. Assessment of the wild-type and CbCln6nclf cerebellar cells revealed subunit c-positive puncta in aged homozygous mutant CbCln6nclf cerebellar cells, similar to that observed in the aged homozygous CbCln3Δex7/8 cells (Figure 2). Consistent with the abnormal accumulation occurring in a lysosomal compartment, little to no overlap of subunit c signal was observed with mitochondrial grp75 in confluent aged homozygous CbCln3Δex7/8 cells and only limited overlap of the subunit c and grp75 immunostain was observed in the confluent aged homozygous CbCln6nclf cerebellar cells (Figure 2A), while the subunit c immunostain in confluent aged wild-type cells exhibited moderate overlap with grp75 immunostain (Figure 2A). Moreover, in the aged wild-type cerebellar cells, the subunit c immunostain did not overlap with Lamp 1 immunostain, whereas the strongly immunopositive subunit c puncta in both the homozygous CbCln3Δex7/8 and CbCln6nclf cerebellar cells aged at confluent density were Lamp 1-positive (Figure 2B), though the overlap was imperfect, particularly in the homozygous CbCln6nclf cerebellar cells. In both homozygous CbCln3Δex7/8 and CbCln6nclf cells aged at confluent density, the Lamp 1 immunostain also revealed enlarged or aggregated lysosomes, which were not often observed in the aged wild-type cells, consistent with lysosomal defects.


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)

Subunit c deposits co-localize with Lamp 1 in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and the mitochondrial marker, grp75 (red). Note the large accumulations of subunit c immunostain (white arrows) in both CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, but which are not common in the wild-type cells. Moderate overlap of the subunit c and grp75 immunostains is observed in wild-type cells (yellow in overlay), but little to no overlap is seen in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Also, again note the elongated mitochondrial morphology revealed by the grp75 immunostain in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, compared to wild-type cells. B. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8, and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and Lamp 1 (red). Limited overlap of subunit c and Lamp 1 immunostain is observed in wild-type cells (yellow in overlay), but Lamp 1 strongly, though not perfectly, overlaps with the accumulated subunit c in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Note that in confluency aged cultures, the Lamp 1-labeled compartment appears expanded and/or aggregated in the mutant cells, which was not observed under sub-confluent culture conditions (not shown). A,B. Insets provide a zoomed view of the degree of immunostain overlap (yellow). Blue = DAPI stain. Images were captured with a 40X objective and, for like stains, were taken on the same day with identical settings.
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Related In: Results  -  Collection

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pone-0017118-g002: Subunit c deposits co-localize with Lamp 1 in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and the mitochondrial marker, grp75 (red). Note the large accumulations of subunit c immunostain (white arrows) in both CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, but which are not common in the wild-type cells. Moderate overlap of the subunit c and grp75 immunostains is observed in wild-type cells (yellow in overlay), but little to no overlap is seen in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Also, again note the elongated mitochondrial morphology revealed by the grp75 immunostain in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, compared to wild-type cells. B. Representative micrographs of confluency aged wild-type, CbCln3Δex7/8/Δex7/8, and CbCln6nclf/nclf cells, co-immunostained with antibodies recognizing subunit c (green) and Lamp 1 (red). Limited overlap of subunit c and Lamp 1 immunostain is observed in wild-type cells (yellow in overlay), but Lamp 1 strongly, though not perfectly, overlaps with the accumulated subunit c in CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells. Note that in confluency aged cultures, the Lamp 1-labeled compartment appears expanded and/or aggregated in the mutant cells, which was not observed under sub-confluent culture conditions (not shown). A,B. Insets provide a zoomed view of the degree of immunostain overlap (yellow). Blue = DAPI stain. Images were captured with a 40X objective and, for like stains, were taken on the same day with identical settings.
Mentions: Homozygous CbCln3Δex7/8 cells, when aged at confluent cell density, accumulated subunit c-positive puncta [20]. Assessment of the wild-type and CbCln6nclf cerebellar cells revealed subunit c-positive puncta in aged homozygous mutant CbCln6nclf cerebellar cells, similar to that observed in the aged homozygous CbCln3Δex7/8 cells (Figure 2). Consistent with the abnormal accumulation occurring in a lysosomal compartment, little to no overlap of subunit c signal was observed with mitochondrial grp75 in confluent aged homozygous CbCln3Δex7/8 cells and only limited overlap of the subunit c and grp75 immunostain was observed in the confluent aged homozygous CbCln6nclf cerebellar cells (Figure 2A), while the subunit c immunostain in confluent aged wild-type cells exhibited moderate overlap with grp75 immunostain (Figure 2A). Moreover, in the aged wild-type cerebellar cells, the subunit c immunostain did not overlap with Lamp 1 immunostain, whereas the strongly immunopositive subunit c puncta in both the homozygous CbCln3Δex7/8 and CbCln6nclf cerebellar cells aged at confluent density were Lamp 1-positive (Figure 2B), though the overlap was imperfect, particularly in the homozygous CbCln6nclf cerebellar cells. In both homozygous CbCln3Δex7/8 and CbCln6nclf cells aged at confluent density, the Lamp 1 immunostain also revealed enlarged or aggregated lysosomes, which were not often observed in the aged wild-type cells, consistent with lysosomal defects.

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