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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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Venn diagram depicting degree of overlap in gene expression changes in homozygous CbCln3Δex7/8 and CbCln6nclf cells.981 probes were significantly changed in CbCln3Δex7/8/Δex7/8 cells, compared to CbCln3+/+ cells (red). 718 probes were significantly changed in CbCln6nclf/nclf cells, compared to CbCln6+/+ cells (blue). A ±1.4-fold change and p≤0.01 cut-off was applied to generate these datasets. As represented by the pie chart, among the 36 shared probes, 8 (22%) were concordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, while 28 (78%) were discordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells.
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pone-0017118-g005: Venn diagram depicting degree of overlap in gene expression changes in homozygous CbCln3Δex7/8 and CbCln6nclf cells.981 probes were significantly changed in CbCln3Δex7/8/Δex7/8 cells, compared to CbCln3+/+ cells (red). 718 probes were significantly changed in CbCln6nclf/nclf cells, compared to CbCln6+/+ cells (blue). A ±1.4-fold change and p≤0.01 cut-off was applied to generate these datasets. As represented by the pie chart, among the 36 shared probes, 8 (22%) were concordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, while 28 (78%) were discordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells.

Mentions: In a probe-level analysis, we identified 981 significantly changed probes in the homozygous CbCln3Δex7/8 cerebellar cells (p<.01, absolute fold-change>1.5; Figure 5, Table S1) and 718 significantly changed probes in the homozygous CbCln6nclf cerebellar cells (p<.01, absolute fold-change>1.5; Figure 5, Table S2). Among these, only a small number, 36 probes, were significantly changed in both homozygous CbCln3Δex7/8 and CbCln6nclf cells, with the majority (28) discordant in the direction of change (Figure 5, Table 1). The reliability of the datasets was assessed by principal components analysis (PCA) (Figure S2) and real-time qRT-PCR of selected genes (Table S3).


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)

Venn diagram depicting degree of overlap in gene expression changes in homozygous CbCln3Δex7/8 and CbCln6nclf cells.981 probes were significantly changed in CbCln3Δex7/8/Δex7/8 cells, compared to CbCln3+/+ cells (red). 718 probes were significantly changed in CbCln6nclf/nclf cells, compared to CbCln6+/+ cells (blue). A ±1.4-fold change and p≤0.01 cut-off was applied to generate these datasets. As represented by the pie chart, among the 36 shared probes, 8 (22%) were concordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, while 28 (78%) were discordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040763&req=5

pone-0017118-g005: Venn diagram depicting degree of overlap in gene expression changes in homozygous CbCln3Δex7/8 and CbCln6nclf cells.981 probes were significantly changed in CbCln3Δex7/8/Δex7/8 cells, compared to CbCln3+/+ cells (red). 718 probes were significantly changed in CbCln6nclf/nclf cells, compared to CbCln6+/+ cells (blue). A ±1.4-fold change and p≤0.01 cut-off was applied to generate these datasets. As represented by the pie chart, among the 36 shared probes, 8 (22%) were concordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells, while 28 (78%) were discordant in their direction of change in the CbCln3Δex7/8/Δex7/8 and CbCln6nclf/nclf cells.
Mentions: In a probe-level analysis, we identified 981 significantly changed probes in the homozygous CbCln3Δex7/8 cerebellar cells (p<.01, absolute fold-change>1.5; Figure 5, Table S1) and 718 significantly changed probes in the homozygous CbCln6nclf cerebellar cells (p<.01, absolute fold-change>1.5; Figure 5, Table S2). Among these, only a small number, 36 probes, were significantly changed in both homozygous CbCln3Δex7/8 and CbCln6nclf cells, with the majority (28) discordant in the direction of change (Figure 5, Table 1). The reliability of the datasets was assessed by principal components analysis (PCA) (Figure S2) and real-time qRT-PCR of selected genes (Table S3).

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