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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 fluid-phase endocytosis and LysoTracker® stain in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of CbCln3 cells (wild-type and homozygous) and CbCln6 (wild-type and homozygous) cells stained with the fluid-phase endocytic marker, dextran Alexa-488 (green), and the lysosomal marker, LysoTracker®-Red (red), are shown. Reduced vesicular staining is evident in the homozygous cells, compared to wild-type cells for both CbCln3 and CbCln6 cells. However, note the differential distribution pattern of the labeled vesicles in homozygous CbCln3Δex7/8 cells, compared to homozygous CbCln6nclf cells. Labeled vesicles in homozygous CbCln6nclf cells remain perinuclear localized, as they appear in wild-type cell lines. To the contrary, labeled vesicles in homozygous CbCln3Δex7/8 cells appear less perinuclear localized, compared to wild-type cells and homozygous CbCln6nclf cells. All images were captured at 40× magnification and were taken on the same day with identical settings. B. Bar graphs depicting quantification of labeled endosomes (left) and lysosomes (right) in CbCln3 cells (CbCln3+/+ and CbCln3Δex7/8/Δex7/8) and CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) cells are shown. Mean vesicle count/cell was significantly reduced (*p<.001) in both homozygous CbCln3Δex7/8and CbCln6nclf cells, compared to the respective wild-type or heterozygous cell lines. A student's t-test also revealed a significant difference (p<.001) between the homozygous cell lines of differing genotype (i.e. CbCln3Δex7/8/Δex7/8 versus CbCln6nclf/nclf), which was suggestive of a more dramatic reduction of stained vesicles in the CbCln3Δex7/8/Δex7/8 cells.
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pone-0017118-g004: Altered fluid-phase endocytosis and LysoTracker® stain in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of CbCln3 cells (wild-type and homozygous) and CbCln6 (wild-type and homozygous) cells stained with the fluid-phase endocytic marker, dextran Alexa-488 (green), and the lysosomal marker, LysoTracker®-Red (red), are shown. Reduced vesicular staining is evident in the homozygous cells, compared to wild-type cells for both CbCln3 and CbCln6 cells. However, note the differential distribution pattern of the labeled vesicles in homozygous CbCln3Δex7/8 cells, compared to homozygous CbCln6nclf cells. Labeled vesicles in homozygous CbCln6nclf cells remain perinuclear localized, as they appear in wild-type cell lines. To the contrary, labeled vesicles in homozygous CbCln3Δex7/8 cells appear less perinuclear localized, compared to wild-type cells and homozygous CbCln6nclf cells. All images were captured at 40× magnification and were taken on the same day with identical settings. B. Bar graphs depicting quantification of labeled endosomes (left) and lysosomes (right) in CbCln3 cells (CbCln3+/+ and CbCln3Δex7/8/Δex7/8) and CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) cells are shown. Mean vesicle count/cell was significantly reduced (*p<.001) in both homozygous CbCln3Δex7/8and CbCln6nclf cells, compared to the respective wild-type or heterozygous cell lines. A student's t-test also revealed a significant difference (p<.001) between the homozygous cell lines of differing genotype (i.e. CbCln3Δex7/8/Δex7/8 versus CbCln6nclf/nclf), which was suggestive of a more dramatic reduction of stained vesicles in the CbCln3Δex7/8/Δex7/8 cells.

Mentions: To determine whether subconfluent, non-stressed CbCln6nclf cerebellar neuronal precursor cells displayed abnormalities in the endosomal-lysosomal system, which is significantly disrupted in subconfluent homozygous CbCln3Δex7/8 cells [20], we assayed fluid phase endocytosis using a fluorescently labeled dextran uptake assay (dextran, Alexa Fluor® 488) and acidic organelles were probed using LysoTracker® Red. As expected, homozygous CbCln3Δex7/8 cells showed consistently reduced numbers of dextran-Alexa 488 labeled vesicles and LysoTracker® stained vesicles, along with a reduced perinuclear distribution of the labeled vesicles, compared to wild-type (CbCln3+/+) or heterozygous (CbCln3+/Δex7/8) cells (Figure 4). Homozygous CbCln6nclf cells also showed consistently reduced dextran-Alexa 488 labeled vesicles and LysoTracker® stained vesicles, compared to wild-type (CbCln6+/+) or heterozygous (CbCln6+/nclf) cells. However, in contrast to what was observed in homozygous CbCln3Δex7/8 cells, the distribution of the labeled vesicles in homozygous CbCln6nclf cells was not obviously altered (Figure 4A). Notably, the expanded and/or aggregated lysosomal compartment, which was evident by Lamp 1 immunostain in the confluent density aged homozygous CbCln3Δex7/8 and CbCln6nclf cells, as shown in Figure 2B, was not observed in the subconfluent, Lysotracker-stained homozygous CbCln3Δex7/8 and CbCln6nclf cells here. Automated image analysis demonstrated that the numbers of labeled vesicles were more dramatically decreased (p<.001) in the homozygous CbCln3Δex7/8 cells (∼65–75% reduced from wild-type levels), than in the homozygous CbCln6nclf cells (∼35–50% reduced from wild-type levels) (Figure 4B).


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 fluid-phase endocytosis and LysoTracker® stain in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of CbCln3 cells (wild-type and homozygous) and CbCln6 (wild-type and homozygous) cells stained with the fluid-phase endocytic marker, dextran Alexa-488 (green), and the lysosomal marker, LysoTracker®-Red (red), are shown. Reduced vesicular staining is evident in the homozygous cells, compared to wild-type cells for both CbCln3 and CbCln6 cells. However, note the differential distribution pattern of the labeled vesicles in homozygous CbCln3Δex7/8 cells, compared to homozygous CbCln6nclf cells. Labeled vesicles in homozygous CbCln6nclf cells remain perinuclear localized, as they appear in wild-type cell lines. To the contrary, labeled vesicles in homozygous CbCln3Δex7/8 cells appear less perinuclear localized, compared to wild-type cells and homozygous CbCln6nclf cells. All images were captured at 40× magnification and were taken on the same day with identical settings. B. Bar graphs depicting quantification of labeled endosomes (left) and lysosomes (right) in CbCln3 cells (CbCln3+/+ and CbCln3Δex7/8/Δex7/8) and CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) cells are shown. Mean vesicle count/cell was significantly reduced (*p<.001) in both homozygous CbCln3Δex7/8and CbCln6nclf cells, compared to the respective wild-type or heterozygous cell lines. A student's t-test also revealed a significant difference (p<.001) between the homozygous cell lines of differing genotype (i.e. CbCln3Δex7/8/Δex7/8 versus CbCln6nclf/nclf), which was suggestive of a more dramatic reduction of stained vesicles in the CbCln3Δex7/8/Δex7/8 cells.
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Related In: Results  -  Collection

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pone-0017118-g004: Altered fluid-phase endocytosis and LysoTracker® stain in homozygous CbCln3Δex7/8 and CbCln6nclf cells.A. Representative micrographs of CbCln3 cells (wild-type and homozygous) and CbCln6 (wild-type and homozygous) cells stained with the fluid-phase endocytic marker, dextran Alexa-488 (green), and the lysosomal marker, LysoTracker®-Red (red), are shown. Reduced vesicular staining is evident in the homozygous cells, compared to wild-type cells for both CbCln3 and CbCln6 cells. However, note the differential distribution pattern of the labeled vesicles in homozygous CbCln3Δex7/8 cells, compared to homozygous CbCln6nclf cells. Labeled vesicles in homozygous CbCln6nclf cells remain perinuclear localized, as they appear in wild-type cell lines. To the contrary, labeled vesicles in homozygous CbCln3Δex7/8 cells appear less perinuclear localized, compared to wild-type cells and homozygous CbCln6nclf cells. All images were captured at 40× magnification and were taken on the same day with identical settings. B. Bar graphs depicting quantification of labeled endosomes (left) and lysosomes (right) in CbCln3 cells (CbCln3+/+ and CbCln3Δex7/8/Δex7/8) and CbCln6 (CbCln6+/+ and CbCln6nclf/nclf) cells are shown. Mean vesicle count/cell was significantly reduced (*p<.001) in both homozygous CbCln3Δex7/8and CbCln6nclf cells, compared to the respective wild-type or heterozygous cell lines. A student's t-test also revealed a significant difference (p<.001) between the homozygous cell lines of differing genotype (i.e. CbCln3Δex7/8/Δex7/8 versus CbCln6nclf/nclf), which was suggestive of a more dramatic reduction of stained vesicles in the CbCln3Δex7/8/Δex7/8 cells.
Mentions: To determine whether subconfluent, non-stressed CbCln6nclf cerebellar neuronal precursor cells displayed abnormalities in the endosomal-lysosomal system, which is significantly disrupted in subconfluent homozygous CbCln3Δex7/8 cells [20], we assayed fluid phase endocytosis using a fluorescently labeled dextran uptake assay (dextran, Alexa Fluor® 488) and acidic organelles were probed using LysoTracker® Red. As expected, homozygous CbCln3Δex7/8 cells showed consistently reduced numbers of dextran-Alexa 488 labeled vesicles and LysoTracker® stained vesicles, along with a reduced perinuclear distribution of the labeled vesicles, compared to wild-type (CbCln3+/+) or heterozygous (CbCln3+/Δex7/8) cells (Figure 4). Homozygous CbCln6nclf cells also showed consistently reduced dextran-Alexa 488 labeled vesicles and LysoTracker® stained vesicles, compared to wild-type (CbCln6+/+) or heterozygous (CbCln6+/nclf) cells. However, in contrast to what was observed in homozygous CbCln3Δex7/8 cells, the distribution of the labeled vesicles in homozygous CbCln6nclf cells was not obviously altered (Figure 4A). Notably, the expanded and/or aggregated lysosomal compartment, which was evident by Lamp 1 immunostain in the confluent density aged homozygous CbCln3Δex7/8 and CbCln6nclf cells, as shown in Figure 2B, was not observed in the subconfluent, Lysotracker-stained homozygous CbCln3Δex7/8 and CbCln6nclf cells here. Automated image analysis demonstrated that the numbers of labeled vesicles were more dramatically decreased (p<.001) in the homozygous CbCln3Δex7/8 cells (∼65–75% reduced from wild-type levels), than in the homozygous CbCln6nclf cells (∼35–50% reduced from wild-type levels) (Figure 4B).

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