Molecular dissection of A-type lamin-regulated pathways

Datum: 2 oktober 2017

Locatie: Universiteit Gent, Campus Coupure, lokaal A0.030 - Coupure 653 - 9000 Gent

Tijdstip: 18 uur

Organisatie / co-organisatie: Departement Bio-ingenieurswetenschappen

Promovendus: Tobias Corne

Promotor: Winnok De Vos (UA) & Els Van Damme (UGent)

Korte beschrijving: Doctoraatsverdediging Tobias Corne - Faculteit Wetenschappen - Departement Bio-ingenieurswetenschappen

Abstract (in English)

The nuclear lamina, a thin filamentous protein layer beneath the nuclear envelope, physically supports the cell nucleus and has a central role in nuclear organization and gene regulation. The major constituents of this meshwork are type V intermediate filament proteins, known as lamins. Mutations in the LMNA gene, which encodes A-type lamins, or in the ZMPSTE24 gene, which encodes a zinc metalloprotease involved in the maturation of A-type lamins, are linked to a wide spectrum of tissue-specific and systemic diseases collectively called laminopathies. Disease manifestations include muscular dystrophies, lipodystrophies, dilated cardiomyopathies and the premature aging syndromes Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). To unveil A-type regulated pathways, we have optimized and used cellular models in which we blocked the expression of LMNA or ZMPSTE24. This was achieved through sustained siRNA- mediated knockdown in human dermal fibroblasts or by CRISPR/Cas9-mediated genome editing in HeLa cells.

First, we investigated the effect on redox biology. Sustained knockdown revealed that both persistent prelamin A accumulation and lamin A/C depletion elevated intracellular ROS levels, but to a different extent, and with different effects on cell fate. LMNA knockdown eventually induced apoptosis, while ZMPSTE24 knockdown triggered a senescence pathway. Next, to further our knowledge on the molecular effects of LMNA deficiency, we compared the proteome of LMNA knockdown fibroblasts with mock-treated controls using quantitative stable isotope labelling-based shotgun proteomics. This revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization. Interestingly, functional validation showed that loss of A- type lamins perturbed the coordination between focal adhesion formation and cytoskeletal tension. Finally, to identify molecular changes associated with ZMPSTE24 deficiency at the cellular level we analysed genome-edited ZMPSTE24 knockout cells with label-free confocal Raman microscopy. We identified a decreased lipid content in ZMPSTE24-deficient HeLa cells attributed to a significant reduction in lipid droplet number and size.

In summary, we show that redox balance, focal adhesion and cytoskeletal tension are affected by loss of A-type lamins. We hypothesize that these pathways are interlinked and that ROS can be partly responsible for the uncoupling between cell adhesion and cytoskeletal tension. Furthermore, reduced focal adhesion and high ROS levels trigger apoptosis. Persistent prelamin A accumulation on the other hand, triggers a senescence pathway and interferes with lipid storage, in line with prelamin A-linked lipodystrophies. These findings open up new treatment strategies for laminopathies, in particular for muscular dystrophies, dilated cardiomyopathies and mandibuloacral dysplasia type B. These treatments strategies include reducing ROS levels, restoring mitochondrial function, increasing proteasome activity and increasing autophagy.