Limits...
Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane.

Brandstaetter H, Kruppa AJ, Buss F - Dis Model Mech (2014)

Bottom Line: Although the progression of Huntington's disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood.Interestingly, heterozygous fibroblasts from a Huntington's disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway.Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK.

Show MeSH

Related in: MedlinePlus

ER-to-Golgi transport of a fluorescent reporter is reduced in homozygous Htt140Q/140Q KI MEFs but not in heterozygous patient fibroblasts with a 180Q expansion. (A) Human fibroblasts from a healthy control and a patient with juvenile-onset HD (HTT+/180Q) were transiently transfected with the GFP-hGH reporter construct. Cells were untreated or treated with the ligand AP21998 for 0, 15 and 30 minutes, fixed and stained with antibodies to GFP and the Golgi protein GM130. The amount of fluorescent reporter present in the Golgi region was quantified in confocal images using Volocity image-analysis software in >350 cells from four independent experiments and is illustrated as a scatter plot with the line representing the mean. There was no significant difference in reporter movement from the ER to Golgi in HD patient fibroblasts compared with control. (B) Mouse embryonic fibroblasts (MEFs) were isolated from wild-type and Htt140Q/140Q knock-in mice at E17/18 and transiently transfected with the GFP-hGH reporter construct. At 0, 15 and 30 minutes after AP21998 ligand addition, GFP and GM130 colocalisation images were analysed by calculating the Pearson’s coefficient using Volocity image-analysis software. Compared with wild-type MEFs, significantly less cargo accumulated in the Golgi of homozygous Htt140Q/140Q knock-in MEFs in >120 cells in four independent experiments and is illustrated as a box-and-whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum. A one-way ANOVA followed by a post-hoc Bonferroni multiple comparison test was conducted as a statistical test. **P≤0.01. ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257002&req=5

f4-0071335: ER-to-Golgi transport of a fluorescent reporter is reduced in homozygous Htt140Q/140Q KI MEFs but not in heterozygous patient fibroblasts with a 180Q expansion. (A) Human fibroblasts from a healthy control and a patient with juvenile-onset HD (HTT+/180Q) were transiently transfected with the GFP-hGH reporter construct. Cells were untreated or treated with the ligand AP21998 for 0, 15 and 30 minutes, fixed and stained with antibodies to GFP and the Golgi protein GM130. The amount of fluorescent reporter present in the Golgi region was quantified in confocal images using Volocity image-analysis software in >350 cells from four independent experiments and is illustrated as a scatter plot with the line representing the mean. There was no significant difference in reporter movement from the ER to Golgi in HD patient fibroblasts compared with control. (B) Mouse embryonic fibroblasts (MEFs) were isolated from wild-type and Htt140Q/140Q knock-in mice at E17/18 and transiently transfected with the GFP-hGH reporter construct. At 0, 15 and 30 minutes after AP21998 ligand addition, GFP and GM130 colocalisation images were analysed by calculating the Pearson’s coefficient using Volocity image-analysis software. Compared with wild-type MEFs, significantly less cargo accumulated in the Golgi of homozygous Htt140Q/140Q knock-in MEFs in >120 cells in four independent experiments and is illustrated as a box-and-whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum. A one-way ANOVA followed by a post-hoc Bonferroni multiple comparison test was conducted as a statistical test. **P≤0.01. ns, not significant.

Mentions: Finally, we analysed the secretory pathway in human fibroblasts from a healthy control (GM23976A) and an individual with juvenile-onset HD [heterozygous for an expanded polyQ tract of 180Q (HTT+/180Q); GM09197B], and in primary MEFs isolated from wild-type mice (typically around E17/18) and homozygous Htt140Q/140Q KI mice. The human and mouse fibroblasts were transiently transfected with the GFP-hGH reporter construct and the amount of this reporter molecule in the Golgi area was analysed at 0, 15 and 30 minutes after AP21998 ligand addition. As shown in Fig. 4A, there was no significant difference in reporter movement from the ER to the Golgi in control compared with heterozygous HTT+/180Q patient fibroblasts. However, significantly less cargo accumulated in the Golgi 15 minutes after ligand addition in homozygote Htt140Q/140Q KI MEFs when compared with wild-type MEFs (Fig. 4B). Thus, efficient cargo transport in the early secretory pathway requires at least one copy of wild-type huntingtin, which cannot be replaced by the overexpression of mutant 140Q huntingtin in primary MEFs. At least in fibroblasts, the presence of one copy of mutant huntingtin with a 180Q tract does not seem to disrupt cargo transport in the secretory pathway.


Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane.

Brandstaetter H, Kruppa AJ, Buss F - Dis Model Mech (2014)

ER-to-Golgi transport of a fluorescent reporter is reduced in homozygous Htt140Q/140Q KI MEFs but not in heterozygous patient fibroblasts with a 180Q expansion. (A) Human fibroblasts from a healthy control and a patient with juvenile-onset HD (HTT+/180Q) were transiently transfected with the GFP-hGH reporter construct. Cells were untreated or treated with the ligand AP21998 for 0, 15 and 30 minutes, fixed and stained with antibodies to GFP and the Golgi protein GM130. The amount of fluorescent reporter present in the Golgi region was quantified in confocal images using Volocity image-analysis software in >350 cells from four independent experiments and is illustrated as a scatter plot with the line representing the mean. There was no significant difference in reporter movement from the ER to Golgi in HD patient fibroblasts compared with control. (B) Mouse embryonic fibroblasts (MEFs) were isolated from wild-type and Htt140Q/140Q knock-in mice at E17/18 and transiently transfected with the GFP-hGH reporter construct. At 0, 15 and 30 minutes after AP21998 ligand addition, GFP and GM130 colocalisation images were analysed by calculating the Pearson’s coefficient using Volocity image-analysis software. Compared with wild-type MEFs, significantly less cargo accumulated in the Golgi of homozygous Htt140Q/140Q knock-in MEFs in >120 cells in four independent experiments and is illustrated as a box-and-whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum. A one-way ANOVA followed by a post-hoc Bonferroni multiple comparison test was conducted as a statistical test. **P≤0.01. ns, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257002&req=5

f4-0071335: ER-to-Golgi transport of a fluorescent reporter is reduced in homozygous Htt140Q/140Q KI MEFs but not in heterozygous patient fibroblasts with a 180Q expansion. (A) Human fibroblasts from a healthy control and a patient with juvenile-onset HD (HTT+/180Q) were transiently transfected with the GFP-hGH reporter construct. Cells were untreated or treated with the ligand AP21998 for 0, 15 and 30 minutes, fixed and stained with antibodies to GFP and the Golgi protein GM130. The amount of fluorescent reporter present in the Golgi region was quantified in confocal images using Volocity image-analysis software in >350 cells from four independent experiments and is illustrated as a scatter plot with the line representing the mean. There was no significant difference in reporter movement from the ER to Golgi in HD patient fibroblasts compared with control. (B) Mouse embryonic fibroblasts (MEFs) were isolated from wild-type and Htt140Q/140Q knock-in mice at E17/18 and transiently transfected with the GFP-hGH reporter construct. At 0, 15 and 30 minutes after AP21998 ligand addition, GFP and GM130 colocalisation images were analysed by calculating the Pearson’s coefficient using Volocity image-analysis software. Compared with wild-type MEFs, significantly less cargo accumulated in the Golgi of homozygous Htt140Q/140Q knock-in MEFs in >120 cells in four independent experiments and is illustrated as a box-and-whisker plot. The box represents the median, 25th and 75th percentiles and whiskers represent the maximum and minimum. A one-way ANOVA followed by a post-hoc Bonferroni multiple comparison test was conducted as a statistical test. **P≤0.01. ns, not significant.
Mentions: Finally, we analysed the secretory pathway in human fibroblasts from a healthy control (GM23976A) and an individual with juvenile-onset HD [heterozygous for an expanded polyQ tract of 180Q (HTT+/180Q); GM09197B], and in primary MEFs isolated from wild-type mice (typically around E17/18) and homozygous Htt140Q/140Q KI mice. The human and mouse fibroblasts were transiently transfected with the GFP-hGH reporter construct and the amount of this reporter molecule in the Golgi area was analysed at 0, 15 and 30 minutes after AP21998 ligand addition. As shown in Fig. 4A, there was no significant difference in reporter movement from the ER to the Golgi in control compared with heterozygous HTT+/180Q patient fibroblasts. However, significantly less cargo accumulated in the Golgi 15 minutes after ligand addition in homozygote Htt140Q/140Q KI MEFs when compared with wild-type MEFs (Fig. 4B). Thus, efficient cargo transport in the early secretory pathway requires at least one copy of wild-type huntingtin, which cannot be replaced by the overexpression of mutant 140Q huntingtin in primary MEFs. At least in fibroblasts, the presence of one copy of mutant huntingtin with a 180Q tract does not seem to disrupt cargo transport in the secretory pathway.

Bottom Line: Although the progression of Huntington's disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood.Interestingly, heterozygous fibroblasts from a Huntington's disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway.Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK.

Show MeSH
Related in: MedlinePlus