Abstract
Karyoptosis is a newly discovered form of non-apoptotic cell death triggered by proteotoxic stress and defined by nuclear breakdown and the release of large extracellular vesicles (EVs) containing nuclear LaminB1 and the autophagy protein, p62. Its characteristic nuclear lamina morphology and the presence of LaminB1 puncta accumulation in the cytoplasm have been used thus far as key markers for karyoptotic cell death, leading to its implication in multiple diseases, including dentatorubral-pallidoluysian atrophy (DRPLA), Vici Syndrome, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD) and cancer. As research continues to enlighten the intrinsic and extrinsic factors and molecular mechanisms involved in karyoptosis, the EVs that are released during this process remain to be fully understood. Given the recent surge in interest for EVs as important intercellular messengers and their involvement in disease pathophysiology, studying the karyoptosis-derived EVs represents an important research priority.First characterised in a DRPLA mouse model, karyoptosis was seen to arise as a result of impaired autophagic processing, leading to nucleophagy, cell atrophy and neurodegeneration. This formed the basis for the development of an in vitro model of karyoptosis to further our knowledge of this mechanism’s involvement in neuronal death.
This thesis uses brain sections from the DRPLA mouse model to profile microglia phenotypes in vivo in the disease-relevant anatomical brain areas exhibiting high levels of neuronal karyoptosis and the in vitro model of karyoptosis to study the karyoptosis-derived EVs. By gaining new knowledge on the EVs’ form and content and using them as an applied treatment, this thesis aims to explore the possible effects of karyoptosis-derived EVs on other non-neuronal cells.
In the DRPLA mouse model, immunohistochemical analysis of the microglia in the dentate nucleus (DN) of the cerebellum revealed Iba1-positive cells with large cell somas and fewer, shorter processes, as well as an increased presence in the DN at end-stage. This morphometric observation was age-dependent, progressing in line with symptoms, and matched the sexual dimorphism seen at the behavioural level in these mice. Previous analysis confirming the presence of neurons undergoing karyoptosis in the region and cell work showing the ability of neighbouring cells to internalise karyoptosis-derived EVs led to the hypothesis that the observed changes in microglia morphology may be an effect of these EVs.
In vitro, the pharmacological block of autophagy with Bafilomycin-A1 (BafA1) in human neuroblastoma cells (SH-SY5Y) models karyoptosis and enables the generation of vast quantities of karyoptosis-derived EVs by sequential ultracentrifugation of conditioned culture medium. Using a combination of nanoparticle tracking analysis (NTA), flow cytometry, UV-spectrophotometry, immunoblotting and enzyme-linked immunosorbent assay (ELISA), it was shown that the “20K” fraction (after 20,000 xg centrifugation) contains membrane-bound EVs and is comparatively enriched in large EVs that, although fewer in number, are more protein- and double-strand DNA (dsDNA)-rich than the small EVs in the “100K” fraction (after 100,000 xg centrifugation). These large EVs (in “BafA1 20K”) show an increased level of p62 and LaminB1, confirming the presence of the EVs of interest—karyoptosis-derived EVs. Upon proteomics and gene ontology analysis, the BafA1 20K also contains fewer classical exosomal markers like CD9 and CD81 but an increased abundance of apolipoproteins (B-100 and C-III). These are typically involved in VLDL but could be novel markers for the larger EVs of interest. Furthermore, the 20K fractions are enriched in nuclear proteins and endoplasmic reticulum (ER) stress markers, suggesting the involvement of the ER in karyoptosis-derived EV biogenesis, which may possibly be VLDL-like particles.
Finally, this thesis explores karyoptosis in the wider context of the complex cell-to-cell interactions known to play a role in neurodegenerative diseases by collecting and applying EVs as a treatment to BV2 microglia-like cells. Using flow cytometry, the uptake of EVs into BV2 cells was detected, which was increased in the BafA1 20K-treated condition. Because the morphometric analysis in DRPLA mouse brain sections revealed changes compatible with an “activated” phenotype, the expectation was that the EVs might induce a pro-inflammatory response in the BV2 cells. However, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to demonstrate that the BafA1 20K fraction containing karyoptosis-derived EVs did not induce a pro-inflammatory effect in BV2 cells. In contrast, the 20K vehicle control (“DMSO 20K”) elicited an increase in fold-change for pro-inflammatory cytokines interleukin-6 (IL-6), interleukin 1-beta (IL1-β), tumor necrosis factor-alpha (TNFα), and transforming growth factor-beta (TGFβ), that was absent in the BafA1 20K counterpart. This, along with the observation of cell loss in the BafA1 20K treatment condition, led to toxicity studies utilising the lactate dehydrogenase (LDH) release and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and immunoblotting for apoptosis marker, cleaved Caspase-3. The proteomics data was reviewed by performing gene ontology searches for cell death. Increased LDH release was measured, indicative of a loss of membrane integrity, with BafA1 20K treatment, but BV2 cells showed no signs of DNA damage or apoptosis. No obvious clustering to explain the specific mechanism for cell death was identified, although functional annotation of gene ontology did show a cluster for the “negative regulation of apoptotic processes”.
Taken together, this thesis presents evidence that the microglia in DRPLA mouse DN exhibit a de-ramified morphology indicative of activation. However, the findings in vitro suggest that the effect of the EVs is not pro-inflammatory but toxic. The data describes a distinct population of large, membrane-bound EVs that are protein- and dsDNA-rich, containing the before-described karyoptosis markers p62 and LaminB1. These karyoptosis derived-EVs are abundant in apolipoproteins, nuclear proteins, and ER stress proteins. When incubated with BV2 microglia-like cells, they induce some form of necrotic cell death that may be DNA-stimulated or mediated through some other unknown pathway. This requires further investigation to understand the cell non-autonomous aspects of neuronal karyoptosis and its consequences in disease.
Overall, this thesis is the first in-depth analysis of karyoptosis-derived EVs and sets the stage for further research, which may improve our understanding of karyoptosis and, more widely, potentially provide an explanation that links together autophagy defects, neuron-glia signalling, and EVs in neurodegeneration.
| Date of Award | Oct 2024 |
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| Original language | English |
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| Supervisor | Manolis Fanto (Supervisor), Marzia Malcangio (Supervisor) & Maria Jimenez Sanchez (Supervisor) |