The impact of ESCRT on Aβ1-42 induced membrane lesions in a yeast model for Alzheimer’s disease

Aβ metabolism plays a pivotal role in Alzheimer’s disease. Here, we used a yeast model to monitor Aβ42 toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full Aβ42 toxic potential. Consistent with previously reported data, our data suggests that Aβ42 interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing Aβ42 toxicity, based on screening of the yeast knockout collection. This revealed a reduced Aβ42 toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased Aβ42 toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that Aβ42 heavily disturbed the plasma membrane integrity in a strain lacking the ESCRT-III accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of Aβ42. Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders

Recent insights on Alzheimer’s disease originating from yeast models

In this review article, yeast model-based research advances regarding the role of Amyloid-? (A?), Tau and frameshift Ubiquitin UBB+1 in Alzheimer’s disease (AD) are discussed. Despite having limitations with regard to intercellular and cognitive AD aspects, these models have clearly shown their added value as a complementary model for the study of the molecular aspects of these proteins, including their interplay with AD related cellular processes such as mitochondrial dysfunction and altered proteostasis. Moreover, these yeast models have also shown their importance in translational research, e.g. in compound screenings and for AD diagnostics development. In addition to well-established Saccharomyces cerevisiae models, new upcoming Schizosaccharomyces pombe, Candida glabrata and Kluyveromyces lactis yeast models for A? and Tau are briefly described. Finally, traditional and more innovative research methodologies, e.g. for studying protein oligomerization/aggregation, are highlighted

Neuroserpin Inclusion Bodies in a FENIB Yeast Model.

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is a human monogenic disease caused by point mutations in the SERPINI1 gene, characterized by the intracellular deposition of polymers of neuroserpin (NS), which leads to proteotoxicity and cell death. Despite the different cell and animal models developed thus far, the exact mechanism of cell toxicity elicited by NS polymers remains unclear. Here, we report that human wild-type NS and the polymerogenic variant G392E NS form protein aggregates mainly localized within the endoplasmic reticulum (ER) when expressed in the yeast S. cerevisiae. The expression of NS in yeast delayed the exit from the lag phase, suggesting that NS inclusions cause cellular stress. The cells also showed a higher resistance following mild oxidative stress treatments when compared to control cells. Furthermore, the expression of NS in a pro-apoptotic mutant strain-induced cell death during aging. Overall, these data recapitulate phenotypes observed in mammalian cells, thereby validating S. cerevisiae as a model for FENIB

Front. Mol. Neurosci.,

A beta metabolism plays a pivotal role in Alzheimer's disease. Here, we used a yeast model to monitor A beta(42) toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full A beta(42) toxic potential. Consistent with previously reported data, our data suggests that A beta(42) interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing A beta(42) toxicity, based on screening of the yeast knockout collection. This revealed a reduced A beta(42) toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased A beta(42) toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that A beta(42) heavily disturbed the plasma membrane integrity in a strain lacking the ESCRT-III accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of A beta(42). Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders

The Impact of ESCRT on Aβ1-42 Induced Membrane Lesions in a Yeast Model for Alzheimer’s Disease

Ab metabolism plays a pivotal role in Alzheimer’s disease. Here, we used a yeast model to monitor Aß42 toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full Aß42 toxic potential. Consistent with previously reported data, our data suggests that Aß42 interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing Aß42 toxicity, based on screening of the yeast knockout collection. This revealed a reduced Aß42 toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased Aß42 toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that Aß42 heavily disturbed the plasma membrane integrity in a strain lacking the ESCRTIII accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of Aß42. Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders.status: Published onlin

Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis

Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.

<p>Cell cycle analysis of transiently transfected HeLa cells after double thymidine block.</p

Localization of Nup98 fusion proteins.

<p>HeLa cells were transiently transfected with GFP constructs and visualized after 24 hours by direct fluorescence microscopy. All fusion proteins localize to the nucleus. (<b>A</b>) GFP-Nup98 is found at the nuclear rim and in the nucleoplasm, whereas Nup98 homeodomain fusions exhibit a punctate pattern: (<b>B</b>) GFP-Nup98-HOXA9, (<b>C</b>) GFP-Nup98-HOXA10, (<b>D</b>) GFP-Nup98-HHEX, and (<b>E</b>) GFP-Nup98-PMX1. Nup98 fusions with other chromatin-binding motifs show a different punctate distribution: (<b>F</b>) GFP-Nup98-JARID1A, and (<b>G</b>) GFP-Nup98-PHF23 (<b>H</b>) GFP-Nup98-NSD1, (<b>I</b>) GFP-Nup98-NSD3 and (<b>K</b>) GFP-Nup98-RARG. Nup98 fused to partners that lack chromatin-binding domains localize more dispersed to the nucleoplasm: (<b>L</b>) GFP-Nup98-LEDGF. Disruption of the FG, the HD or the PHD domain disrupts the localization of the Nup98 chimeras: (<b>M</b>) GFP-Nup98-PMX1 N51S, (<b>N</b>) GFP-Nup98-HOXA9 ΔFG, (<b>O</b>) GFP-Nup98-HOXA9 N51S, (<b>P</b>) GFP-Nup98-HHEX ΔFG, (<b>Q</b>) GFP-Nup98-HHEX ΔHD, and (<b>R</b>) GFP-Nup98-JARID1A W1625A. (<b>S</b>) GFP-HOXA9 and (<b>T</b>) GFP-HHEX localize to the nucleoplasm. Shown are representative confocal images. Scale bars, 5 μm.</p

Expression of Nup98 fusions perturbs the nuclear distribution of lamina-associated polypeptide 2α (LAP2α).

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) Lamin A/C (LA/C, red) concentrates at the nuclear envelope in HeLa control cells and in Nup98 expressing cells (green), while LAP2α (magenta) is found throughout the nucleoplasm. In HeLa cells expressing Nup98-HOXA9 (<b>A</b>) and Nup98-HHEX (<b>B</b>), LAP2α diminished from the nucleoplasm and aggregates at the nuclear periphery. Disruption of the homeodomain in HOXA9 (<b>A</b>) and HHEX (<b>B</b>) and the FG domain of Nup98 (<b>A</b> and <b>B</b>) prevent the relocation of the lamina proteins. DAPI was used to visualize DNA (merge). (<b>B</b>) Fluorescence intensity of LAP2α staining was determined along the axis shown as line in the fluorescence images and plotted as a graph. (<b>D</b>) Quantification of cells with altered LA/C and LAP2α distribution. About 400 cells were analyzed for each sample. (<b>E</b>) Western blot analysis of the expression levels of LA/C, LAP2α, and LB1.</p

Mitotic localization of Nup98 chimeras.

<p>HeLa cells were transiently transfected with GFP constructs and fixed and stained after 24 hours for immunofluorescence microscopy. (<b>A</b>) CREST serum, which in particular recognizes CENP-B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152321#pone.0152321.ref036" target="_blank">36</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152321#pone.0152321.ref055" target="_blank">55</a>], was used to detect the inner kinetochore and DAPI to visualize DNA. Nup98-HD fusion proteins (Nup98-HOXA9, Nup98-HHEX, Nup98-PMX1; green) associate with chromatin (blue), but not with the inner kinetochore (red) during prometaphase. No association with chromatin was found for Nup98 or Nup98 fused to non-HD partners (i.e. Nup98-JARID1A and Nup98-RARG). Disruption of the HD domain of Nup98-HOXA9 (Nup98-HOXA9 N51S), but not of the FG domain (Nup98-HOXA9 ΔFG) affects chromatin association of the fusion protein. Shown are single confocal sections. Scale bars, 5 μm. (<b>B</b>) Anti-Hec1 antibodies were used to detect the outer kinetochore (red), but no co-localization of Nup98-HOXA9, Nup98-HHEX, and Nup98-PMX1, respectively (green) was observed in prometaphase cells. The fusion proteins exclusively associated with chromatin (blue). Shown are single confocal sections. Scale bars, 5 μm.</p