Polarised Asymmetric Inheritance of Accumulated Protein Damage in Higher Eukaryotes

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny

A dnajb chaperone subfamily with hdac-dependent activities suppresses toxic protein aggregation

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Polyglutamine Aggregates Are Present in Committed Crypt Cells but Absent in Stem Cells in the Small Intestine of SCA3 Patients

<p>(A) Schematic representation of an intestinal crypt for visualisation of the different cell types present in this tissue. Light micrographs of a 4-μm (B) and 1-μm (C) section of a crypt from an SCA3 patient showing positive staining for anti-Musashi antibody. Note that stem cells are localised in between and on both sides of the morphologically recognizable Paneth cells residing at the base of the crypt. (D) A light micrograph of a crypt of a SCA3 patient showing positive staining for the anti-polyglutamine antibody IC2 in some epithelial cells (arrowheads). The asterisks (marked E–J) show representative positions of cells analyzed by subsequent electron microscopy. (E–J) show digitally modified, pseudo-coloured images of electron micrographs indicating the polyglutamine staining in blue. Differentiated epithelial cells (E), transit epithelial cells (F and I), and Paneth cells (J) contain polyglutamine aggregates (arrowheads). Stem cells (G) are negative for polyglutamine aggregates but occasionally contain micro-aggregates (H). Note that also some electron dense material is stained blue by this digital processing. In (G and H), contours are provided in black dashed lines to indicate the stem cells. Bars: D, 20 μm; E and H, 2 μm; F, 1 μm; G, I and J, 5 μm. (K) Quantification of cells with aggregates in the crypts of two SCA3 patients. As double labelling for aggregates and stem cells failed, only the stem cells that were adjacent to the Paneth cells were counted, because these could be easily identified on this basis.</p

Polyglutamine-Expanded Proteins Form Aggresomes in Hamster O23 Cells and Human HEK293 Cells

<div><p>(A) Percentage of cells containing inclusions 24 h after transfection with a fluorescently tagged huntingtin fragment containing a stretch of either 74 (O23: EGFP-HDQ74) or 119 (HEK293: HDQ119-EYFP) glutamines.</p> <p>(B) Fraction of cells showing either aggresome-like inclusions or non–aggresome-like inclusions (nuclear and/or multiple scattered inclusions). Bars represent standard errors of the mean.</p> <p>(C) Aggresome-like inclusions are either close to (upper panel) or co-localise (lower panel) with the centrosomes (decorated with γ-tubulin antibodies) in interphase O23 cells (likewise in HEK293 cells, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040417#pbio-0040417-sg001" target="_blank">Figure S1</a>).</p> <p>(D) Vimentin microfilaments are redistributed in a cage-like manner around the inclusion, consistent with aggresome morphology. Note that also microtubules (decorated with α-tubulin antibodies) showed partial redistribution to the aggresome.</p> <p>(E) Sequential confocal planes of an aggresome showing both co-localisation with the centrosome and the cage of vimentin. For a full image of this cell see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040417#pbio-0040417-sg001" target="_blank">Figure S1</a>B. DNA is stained with DAPI (blue) and only shown in the overlay images. Bars in (C) and (D) represent 10 μm. Bar in (E) represents 2 μm.</p></div

Polyglutamine Aggregates Are Inherited by De Novo Generated Neuroblast Cells after Mitosis in <i>D. Melanogaster</i>

<div><p>(A) Expression of Htt-Q128 (red) and Pon-GFP (green) was assessed by confocal laser scanning microscopy in whole embryos (Stage 11, in which anterior is at the top). Occasionally, Htt-Q128 aggregates were observed (inset).</p> <p>(B) During mitosis, the aggregated protein Htt-Q128 is associated with only one of the poles in metaphase, anaphase, and telophase, opposing the Pon-GFP crescent, indicative of asymmetric inheritance to de novo generated neuroblast.</p> <p>(C) Spindle pole–associated aggregates were more clearly visualised after α-tubulin (red) staining in Htt-Q128 (cyan), Pon-GFP (green) neuroblasts. DNA is stained with DAPI (blue).</p></div

Aggresomes Do Not Impair Mitotic Cell Division

<div><p>(A) Quantitative analysis of total mitoses in wild-type HEK293 cells and polyglutamine-expressing HEK293-HDQ119 cells. Bars represent standard error of the mean.</p> <p>(B) Relative fraction of mitoses in each population (diffuse, aggresome-containing, and non–aggresome-containing) of HEK293-HDQ119 cells.</p> <p>(C–G) Representative pictures of fixed O23 (C and D) and HEK293 (E–G) aggresome-containing cells in different mitotic phases. The aggresome is associated with only one of the poles during metaphase, anaphase, and telophase. (C) shows that alignment of chromosomes in metaphase appears normal, and (D and F) show that segregation during anaphase-telophase appears to be normal. Similarly, (C and D) show that positioning of the centrosomes is normal, and (E–F) show that distribution of microtubules and (G) cytokinesis are normal. DNA is stained with DAPI (blue). Bars, 5 μm.</p></div

Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects

The biogenesis of the multi-subunit vacuolar-type H(+)-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy.status: publishe

Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects

Rujano MA, Serio MC, Panasyuk G, et al. Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects. JOURNAL OF EXPERIMENTAL MEDICINE. 2017;214(12):3707-3729.The biogenesis of the multi-subunit vacuolar-type H+-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy

WDR81 mutations cause extreme microcephaly and impair mitotic progression in human fibroblasts and Drosophila neural stem cells.

International audienceMicrolissencephaly is a rare brain malformation characterized by congenital microcephaly and lissencephaly. Microlissencephaly is suspected to result from abnormalities in the proliferation or survival of neural progenitors. Despite the recent identification of six genes involved in microlissencephaly, the pathophysiological basis of this condition remains poorly understood. We performed trio-based whole exome sequencing in seven subjects from five non-consanguineous families who presented with either microcephaly or microlissencephaly. This led to the identification of compound heterozygous mutations in WDR81, a gene previously associated with cerebellar ataxia, intellectual disability and quadrupedal locomotion. Patient phenotypes ranged from severe microcephaly with extremely reduced gyration with pontocerebellar hypoplasia to moderate microcephaly with cerebellar atrophy. In patient fibroblast cells, WDR81 mutations were associated with increased mitotic index and delayed prometaphase/metaphase transition. Similarly, in vivo, we showed that knockdown of the WDR81 orthologue in Drosophila led to increased mitotic index of neural stem cells with delayed mitotic progression. In summary, we highlight the broad phenotypic spectrum of WDR81-related brain malformations, which include microcephaly with moderate to extremely reduced gyration and cerebellar anomalies. Our results suggest that WDR81 might have a role in mitosis that is conserved between Drosophila and humans