Insights into Apolipoprotein E: understanding the major genetic risk factor for Alzheimer’s Disease

Sporadic, late-onset Alzheimer’s disease (LOAD) is the leading cause of dementia. Advanced age is the main culprit for LOAD, but the ε4 variant of APOE has been identified as a major genetic risk factor. APOE encodes Apolipoprotein E (ApoE), which exists as three isoforms. ApoE3, the most common one, confers a neutral chance for LOAD onset whereas ApoE4 presents a risk and ApoE2 is protective. This thesis explores different avenues taken to understand the differences between the three ApoE isoforms, with the ultimate goal being to answer why and how only ApoE4 increases an individual’s susceptibility for LOAD. In Chapter 3, the structure, stability, and propensity for aggregation for the isoforms were compared using a recombinant model of human ApoE. In Chapter 4, I conducted a thorough exploration of both the impact of the APOE genotype on vulnerability to oxidative stress and ApoE’s differential response to oxidative stress itself, using recombinant protein and cultures from human ApoE targeted- replacement mice. Finally, in Chapter 5, the interaction between ApoE and ageing was explored by quantifying longitudinal changes in ApoE expression in human ApoE3 and ApoE4- targeted replacement mice. Additionally, differences in markers of ageing between mice were examined. The results presented in this thesis highlight the fact that ApoE2 and ApoE3 outperform ApoE4. Out of the three isoforms, ApoE4 was the more prone to self-assembly, which is a pathogenic feature of LOAD. While no major differences could be spotted at the tissue level, significant dissimilarities appeared in how the structure of ApoE isoforms is affected by oxidative and reducing conditions, with results suggesting that ApoE2 and to a lesser extent ApoE3 could function as scavengers of oxidative agents. Finally, overall ApoE levels were found to be reduced in ApoE4 targeted-replacement mice compared to ApoE3-mice, especially at old age; and ApoE4 mice appeared to have increased markers of ageing compared to ApoE3 mice. Taken together, these result attest to the complexity of LOAD and show the great number of ways ApoE could be differentially implicated in its onset

The molecular basis for apolipoprotein E4 as the major risk factor for late onset Alzheimer's disease

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an -helical, 299-amino acid protein. Homozygosity for the ε4 allele is the major risk factor for developing late onset Alzheimer’s disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158 and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerisation state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small angle X-ray scattering, analytical ultracentrifugation, circular dichroism, X-ray fibre diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerise to form wavy filaments which do not share the characteristics of cross- amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis and these results open opportunities for uncovering new triggers for AD onset

Standardized immunoprecipitation protocol for efficient isolation of native apolipoprotein E particles utilizing HJ15.4 monoclonal antibody

The apolipoprotein E protein (apoE) confers differential risk for Alzheimer\u27s disease depending on which isoforms are expressed. Here, we present a 2-day immunoprecipitation protocol using the HJ15.4 monoclonal apoE antibody for the pull-down of native apoE particles. We describe major steps for apoE production via immortalized astrocyte culture and HJ15.4 antibody bead coupling for apoE particle pull-down, elution, and characterization. This protocol could be used to isolate native apoE particles from multiple model systems or human biospecimens

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer, studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory, a versatile observatory designed to address the Hot and Energetic Universe science theme, selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), it aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over an hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR, browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters. Finally we briefly discuss on the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, and touch on communication and outreach activities, the consortium organisation, and finally on the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. (abridged).Comment: 48 pages, 29 figures, Accepted for publication in Experimental Astronomy with minor editin

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory. Athena is a versatile observatory designed to address the Hot and Energetic Universe science theme, as selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), X-IFU aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over a hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR (i.e. in the course of its preliminary definition phase, so-called B1), browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters, such as the instrument efficiency, spectral resolution, energy scale knowledge, count rate capability, non X-ray background and target of opportunity efficiency. Finally, we briefly discuss the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, touch on communication and outreach activities, the consortium organisation and the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. The X-IFU will be provided by an international consortium led by France, The Netherlands and Italy, with ESA member state contributions from Belgium, Czech Republic, Finland, Germany, Poland, Spain, Switzerland, with additional contributions from the United States and Japan.The French contribution to X-IFU is funded by CNES, CNRS and CEA. This work has been also supported by ASI (Italian Space Agency) through the Contract 2019-27-HH.0, and by the ESA (European Space Agency) Core Technology Program (CTP) Contract No. 4000114932/15/NL/BW and the AREMBES - ESA CTP No.4000116655/16/NL/BW. This publication is part of grant RTI2018-096686-B-C21 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. This publication is part of grant RTI2018-096686-B-C21 and PID2020-115325GB-C31 funded by MCIN/AEI/10.13039/501100011033

An assay to evaluate the capacity of cholesterol acceptors using BODIPY-cholesterol in cells

Summary: Cholesterol is a structural component of cell membranes. Most cells are incapable of its catabolism, and intracellular cholesterol accumulation is linked to several disorders including cardiovascular and neurodegenerative diseases. Cholesterol efflux, essential to its metabolism, is dependent on acceptors such as apolipoproteins. Here, we describe an assay to evaluate the capacity of cholesterol acceptors. Cells are treated with an analog of cholesterol tagged with fluorescent BODIPY. Addition of an acceptor leads to BODIPY-cholesterol efflux, measured using a plate reader.For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

The Loss of α- and β-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing

Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann’s area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of α- and β-tubulins, and increased levels of acetylated α-tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased α-tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly

Impact of APOE on amyloid and tau accumulation in argyrophilic grain disease and Alzheimer’s disease

Abstract Alzheimer’s disease (AD), characterized by the deposition of amyloid-β (Aβ) in senile plaques and neurofibrillary tangles of phosphorylated tau (pTau), is increasingly recognized as a complex disease with multiple pathologies. AD sometimes pathologically overlaps with age-related tauopathies such as four repeat (4R)-tau predominant argyrophilic grain disease (AGD). While AGD is often detected with AD pathology, the contribution of APOE4 to AGD risk is not clear despite its robust effects on AD pathogenesis. Specifically, how APOE genotype influences Aβ and tau pathology in co-occurring AGD and AD has not been fully understood. Using postmortem brain samples (N = 353) from a neuropathologically defined cohort comprising of cases with AD and/or AGD pathology built to best represent different APOE genotypes, we measured the amounts of major AD-related molecules, including Aβ40, Aβ42, apolipoprotein E (apoE), total tau (tTau), and pTau181, in the temporal cortex. The presence of tau lesions characteristic of AD (AD-tau) was correlated with cognitive decline based on Mini-Mental State Examination (MMSE) scores, while the presence of AGD tau lesions (AGD-tau) was not. Interestingly, while APOE4 increased the risk of AD-tau pathology, it did not increase the risk of AGD-tau pathology. Although APOE4 was significantly associated with higher levels of insoluble Aβ40, Aβ42, apoE, and pTau181, the APOE4 effect was no longer detected in the presence of AGD-tau. We also found that co-occurrence of AGD with AD was associated with lower insoluble Aβ42 and pTau181 levels. Overall, our findings suggest that different patterns of Aβ, tau, and apoE accumulation mediate the development of AD-tau and AGD-tau pathology, which is affected by APOE genotype

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.Chan Zuckerberg Initiative Neurodegeneration Challenge Network, New York Stem Cell Foundatio