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Publications in Health Sciences by NOMIS researchers

Molecular foundations of prion strain diversity

NOMIS Researcher(s)

Published in

February 1, 2022

Despite being caused by a single protein, prion diseases are strikingly heterogenous. Individual prion variants, known as strains, possess distinct biochemical properties, form aggregates with characteristic morphologies and preferentially seed certain brain regions, causing markedly different disease phenotypes. Strain diversity is determined by protein structure, post-translational modifications and the presence of extracellular matrix components, with single amino acid substitutions or altered protein glycosylation exerting dramatic effects. Here, we review recent advances in the study of prion strains and discuss how a deeper knowledge of the molecular origins of strain heterogeneity is providing a foundation for the development of anti-prion therapeutics.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1016/j.conb.2021.07.010

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma

NOMIS Researcher(s)

Published in

February 1, 2022

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.26508/lsa.202101270

Genetic Mechanisms Underlying the Evolution of Connectivity in the Human Cortex

NOMIS Researcher(s)

Published in

January 7, 2022

One of the most salient features defining modern humans is our remarkable cognitive capacity, which is unrivaled by any other species. Although we still lack a complete understanding of how the human brain gives rise to these unique abilities, the past several decades have witnessed significant progress in uncovering some of the genetic, cellular, and molecular mechanisms shaping the development and function of the human brain. These features include an expansion of brain size and in particular cortical expansion, distinct physiological properties of human neurons, and modified synaptic development. Together they specify the human brain as a large primate brain with a unique underlying neuronal circuit architecture. Here, we review some of the known human-specific features of neuronal connectivity, and we outline how novel insights into the human genome led to the identification of human-specific genetic modifiers that played a role in the evolution of human brain development and function. Novel experimental paradigms are starting to provide a framework for understanding how the emergence of these human-specific genomic innovations shaped the structure and function of neuronal circuits in the human brain.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.3389/fncir.2021.787164

Disentangling cadherin-mediated cell-cell interactions in collective cancer cell migration

NOMIS Researcher(s)

Published in

January 4, 2022

Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single-cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling with in vitro observations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). The spreading of these colonies is driven by stochastic single-cell migration with frequent transient cell-cell contacts. We find that inhibition of E- and N-cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that in cancer cell migration, cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration.

Research field(s)
Health Sciences, Biomedical Research, Biophysics

DOI
10.1016/j.bpj.2021.12.006

FGF1 and insulin control lipolysis by convergent pathways

NOMIS Researcher(s)

Published in

January 4, 2022

Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.

Research field(s)
Health Sciences, Clinical Medicine, Endocrinology & Metabolism

DOI
10.1016/j.cmet.2021.12.004

Heat-shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

NOMIS Researcher(s)

Published in

January 1, 2022

While acetylated, RNA-binding-deficient TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) comprised of TDP-43-containing liquid outer shells and liquid centres of HSP70-family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we show that transient oxidative stress, proteasome inhibition or inhibition of the ATP-dependent chaperone activity of HSP70 provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independently of RNA binding or stress granules. Isotope labelling mass spectrometry was used to identify that phase-separated cytoplasmic TDP-43 is bound by the small heat-shock protein HSPB1. Binding is direct, mediated through TDP-43’s RNA binding and low-complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced TDP-43 droplets. A decrease in HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion was identified in spinal motor neurons of patients with ALS containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/s41556-022-00988-8

“Tackling the Substance Use Disorder Crisis: The Role of Access to Treatment Facilities”

NOMIS Researcher(s)

Published in

January 1, 2022

The continuing drug overdose crisis in the U.S. has highlighted the urgent need for greater access to treatment. This paper examines the impact of openings and closings of substance use disorder treatment facilities in New Jersey on emergency room visits for substance use disorder issues among nearby residents. We find that drug-related ER visits increase by 7.4% after a facility closure and decrease by 6.5% after an opening. The effects are smaller for the middle aged than for either younger or older people, and are also somewhat larger for Black residents, and for those on Medicaid. The results suggest that expanding access to treatment results in significant reductions in morbidity related to drugs.

Research field(s)
Health Sciences, Public Health & Health Services, Health Policy & Services

DOI
10.1016/j.jhealeco.2021.102579

Democratic Forecast: Small Groups Predict the Future Better Than Individuals and Crowds

NOMIS Researcher(s)

Published in

January 1, 2022

Predictions pose unique problems. Experts regularly get them wrong, and collective solutions (such as prediction markets and super-forecaster schemes) do better but remain selective and costly. Contrary to the idea that face-to-face discussion hinders collective intelligence, social deliberation improves the resolution of general knowledge problems, with four consensually agreed answers outperforming the aggregate knowledge of 5,000 nondeliberating individuals. Could discussion help predict the future in an efficient, cheap, and inclusive way? We show that smaller groups of lay individuals, when organized, come up with better predictions than those they provide alone. Deliberation and consensus made individual predictions significantly more accurate. Aggregating as few as two consensual predictions did better than classical “wisdom of crowds” aggregation of 100 individual ones. Against the view that discussion can impair decision-making, our results demonstrate that collective intelligence of small groups and consensus-seeking improves accuracy about yet unknown facts, opening the avenue for efficient, inclusive, and inexpensive group forecasting solutions.

Research field(s)
Health Sciences, Psychology & Cognitive Sciences, Experimental Psychology

DOI
10.1037/xap0000424

The effect of feedback valence and source on perception and metacognition: An fMRI investigation

NOMIS Researcher(s)

Published in

January 1, 2022

Receiving feedback from our environment that informs us about the outcomes of our actions helps us assess our abilities (e.g., metacognition) and to flexibly adapt our behavior, consequently increasing our chances of success. However, a detailed examination of the effect of feedback on the brain activation during perceptual and confidence judgments as well as the interrelations between perceptual accuracy, prospective and retrospective confidence remains unclear. Here we used functional magnetic resonance imaging (fMRI) to examine the neural response to feedback valence and source in visual contrast discrimination together with prospective confidence judgments at the beginning of each block and retrospective confidence judgments after every decision. Positive feedback was associated with higher activation (or lower deactivation depending on the area) in areas previously involved in attention, performance monitoring and visual regions during the perceptual judgment than during the confidence judgment. Changes in prospective confidence were positively related to changes in perceptual accuracy as well as to the corresponding retrospective confidence. Thus, feedback information impacted multiple, qualitatively different brain processing states, and we also revealed the dynamic interplay between prospective, perceptual accuracy and retrospective self-assessment.

Research field(s)
Health Sciences, Psychology & Cognitive Sciences, Experimental Psychology

DOI
10.1080/17588928.2020.1828323

Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity

NOMIS Researcher(s)

Published in

January 1, 2022

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples — so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample — a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.7554/eLife.68490

Long-Duration Progressive Supranuclear Palsy: Clinical Course and Pathological Underpinnings

NOMIS Researcher(s)

Published in

January 1, 2022

Objectives: To identify the clinical characteristics of the subgroup of benign progressive supranuclear palsy with particularly long disease duration; to define neuropathological determinants underlying variability in disease duration in progressive supranuclear palsy. Methods: Clinical and pathological features were compared among 186 autopsy-confirmed cases with progressive supranuclear palsy with ≥10 years and shorter survival times. Results: The 45 cases (24.2%) had a disease duration of ≥10 years. The absence of ocular motor abnormalities within the first 3 years from disease onset was the only significant independent clinical predictor of longer survival. Histopathologically, the neurodegeneration parameters in each survival group were paralleled anatomically by the distribution of neuronal cytoplasmic inclusions, whereas the tufted astrocytes displayed anatomically an opposite severity pattern. Most interestingly, we found significantly less coiled bodies in those who survive longer, in contrast to patients with less favorable course. Interpretation: A considerable proportion of patients had a more ”benign” disease course with ≥10 years survival. They had a distinct pattern and evolution of core symptoms compared to patients with short survival. The inverted anatomical patterns of astrocytic tau distribution suggest distinct implications of these cell types in trans-cellular propagation. The tempo of disease progression appeared to be determined mostly by oligodendroglial tau, where the high degree of oligodendroglial tau pathology might affect neuronal integrity and function on top of neuronal tau pathology. The relative contribution of glial tau should be further explored in cellular and animal models. ANN NEUROL 2022.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1002/ana.26455

Cultivation of a vampire: ‘Candidatus Absconditicoccus praedator’

NOMIS Researcher(s)

Published in

January 1, 2022

Halorhodospira halophila, one of the most-xerophilic halophiles, inhabits biophysically stressful and energetically expensive, salt-saturated alkaline brines. Here, we report an additional stress factor that is biotic: a diminutive Candidate-Phyla-Radiation bacterium, that we named ‘Ca. Absconditicoccus praedator’ M39-6, which predates H. halophila M39-5, an obligately photosynthetic, anaerobic purple-sulfur bacterium. We cultivated this association (isolated from the hypersaline alkaline Lake Hotontyn Nur, Mongolia) and characterized their biology. ‘Ca. Absconditicoccus praedator’ is the first stably cultivated species from the candidate class-level lineage Gracilibacteria (order-level lineage Absconditabacterales). Its closed-and-curated genome lacks genes for the glycolytic, pentose phosphate- and Entner–Doudoroff pathways which would generate energy/reducing equivalents and produce central carbon currencies. Therefore, ‘Ca. Absconditicoccus praedator’ is dependent on host-derived building blocks for nucleic acid-, protein-, and peptidoglycan synthesis. It shares traits with (the uncultured) ‘Ca. Vampirococcus lugosii’, which is also of the Gracilibacteria lineage. These are obligate parasitic lifestyle, feeding on photosynthetic anoxygenic Gammaproteobacteria, and absorption of host cytoplasm. Commonalities in their genomic composition and structure suggest that the entire Absconditabacterales lineage consists of predatory species which act to cull the populations of their respective host bacteria. Cultivation of vampire : host associations can shed light on unresolved aspects of their metabolism and ecosystem dynamics at life-limiting extremes.

Research field(s)
Health Sciences, Biomedical Research, Microbiology

DOI
10.1111/1462-2920.15823

A public resource of baseline data from the Alzheimer’s Prevention Initiative Autosomal-Dominant Alzheimer’s Disease Trial

NOMIS Researcher(s)

Published in

January 1, 2022

Introduction: The Alzheimer’s Prevention Initiative Autosomal-Dominant Alzheimer’s Disease (API ADAD) Trial evaluated the anti-oligomeric amyloid beta (Aβ) antibody therapy crenezumab in cognitively unimpaired members of the Colombian presenilin 1 (PSEN1) E280A kindred. We report availability, methods employed to protect confidentiality and anonymity of participants, and process for requesting and accessing baseline data. Methods: We developed mechanisms to share baseline data from the API ADAD Trial in consultation with experts and other groups sharing data from Alzheimer’s disease (AD) prevention trials, balancing the need to protect anonymity and trial integrity with making data broadly available to accelerate progress in the field. We pressure-tested deliberate and inadvertent potential threats under specific assumptions, employed a system to suppress or mask both direct and indirect identifying variables, limited and firewalled data managers, and put forth specific principles requisite to receive data. Results: Baseline demographic, PSEN1 E280A and apolipoprotein E genotypes, florbetapir and fluorodeoxyglucose positron emission tomography, magnetic resonance imaging, clinical, and cognitive data can now be requested by interested researchers. Discussion: Baseline data are publicly available; treatment data and biological samples, including baseline and treatment-related blood-based biomarker data will become available in accordance with our original trial agreement and subsequently developed Collaboration for Alzheimer’s Prevention principles. Sharing of these data will allow exploration of important questions including the differential effects of initiating an investigational AD prevention therapy both before as well as after measurable Aβ plaque deposition.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1002/alz.12843

Not on my team: Medial prefrontal cortex responses to ingroup fusion and unfair monetary divisions

NOMIS Researcher(s)

Published in

January 1, 2022

Objective: People are highly attuned to fairness, with people willingly suffering personal costs to prevent others benefitting from unfair acts. Are fairness judgments influenced by group alignments? A new theory posits that we favor ingroups and denigrate members of rival outgroups when our personal identity is fused to a group. Although the mPFC has been separately implicated in group membership and fairness processing, it is unclear whether group alignments affect medial prefrontal cortex (mPFC) activity in response to fairness. Here, we examine the contribution of different regions of the mPFC to processing from ingroup and outgroup members and test whether its response differs depending on how fused we are to an ingroup. Methods: Subjects performed rounds of the Ultimatum Game, being offered fair or unfair divisions of money from supporters of the same soccer team (ingroup), the fiercest rival (outgroup) or neutral individuals whilst undergoing functional Magnetic Resonance Imaging (fMRI). Results: Strikingly, people willingly suffered personal costs to prevent outgroup members benefitting from both unfair and fair offers. Activity across dorsal and ventral (VMPFC) portions of the mPFC reflected an interaction between fairness and group membership. VMPFC activity in particular was consistent with it coding one’s fusion to a group, with the fairness by group membership interaction correlating with the extent that the responder’s identity was fused to the ingroup. Conclusions: The influence of fusion on social behavior therefore seems to be linked to processing in the VMPFC.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1002/brb3.1030

Strategic disinformation outperforms honesty in competition for social influence

NOMIS Researcher(s)

Published in

December 17, 2021

Competition for social influence is a major force shaping societies, from baboons guiding their troop in different directions, to politicians competing for voters, to influencers competing for attention on social media. Social influence is invariably a competitive exercise with multiple influencers competing for it. We study which strategy maximizes social influence under competition. Applying game theory to a scenario where two advisers compete for the attention of a client, we find that the rational solution for advisers is to communicate truthfully when favored by the client, but to lie when ignored. Across seven pre-registered studies, testing 802 participants, such a strategic adviser consistently outcompeted an honest adviser. Strategic dishonesty outperformed truth-telling in swaying individual voters, the majority vote in anonymously voting groups, and the consensus vote in communicating groups. Our findings help explain the success of political movements that thrive on disinformation, and vocal underdog politicians with no credible program.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1016/j.isci.2021.103505

Exercise plasma boosts memory and dampens brain inflammation via clusterin

NOMIS Researcher(s)

Published in

December 16, 2021

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2–4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that ‘runner plasma’, collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer’s disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise.

Research field(s)
Health Sciences, Clinical Medicine, Neurology & Neurosurgery

DOI
10.1038/s41586-021-04183-x

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins

NOMIS Researcher(s)

Published in

December 1, 2021

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity in targeting phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or β2 glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by–among other factors–viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL with prothrombin (PT). The strength of the antibody response against SARS-CoV-2 was further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity, and aPL against PT in patients with SARS-CoV-2.

Research field(s)
Health Sciences, Biomedical Research, Virology

DOI
10.1371/journal.ppat.1010118

Wild cereal grain consumption among Early Holocene foragers of the Balkans predates the arrival of agriculture

NOMIS Researcher(s)

Published in

December 1, 2021

Forager focus on wild cereal plants has been documented in the core zone of domestication in southwestern Asia, while evidence for forager use of wild grass grains remains sporadic elsewhere. In this paper, we present starch grain and phytolith analyses of dental calculus from 60 Mesolithic and Early Neolithic individuals from five sites in the Danube Gorges of the central Balkans. This zone was inhabited by likely complex Holocene foragers for several millennia before the appearance of the first farmers ~6200 cal BC. We also analyzed forager ground stone tools for evidence of plant processing. Our results based on the study of dental calculus show that certain species of Poaceae (species of the genus Aegilops) were used since the Early Mesolithic, while ground stone tools exhibit traces of a developed grass grain processing technology. The adoption of domesticated plants in this region after ~6500 cal BC might have been eased by the existing familiarity with wild cereals.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.7554/eLife.72976

The role of anticipated regret in choosing for others

NOMIS Researcher(s)

Published in

December 1, 2021

In everyday life, people sometimes find themselves making decisions on behalf of others, taking risks on another’s behalf, accepting the responsibility for these choices and possibly suffering regret for what they could have done differently. Previous research has extensively studied how people deal with risk when making decisions for others or when being observed by others. Here, we asked whether making decisions for present others is affected by regret avoidance. We studied value-based decision making under uncertainty, manipulating both whether decisions benefited the participant or a partner (beneficiary effect) and whether the partner watched the participant’s choices (audience effect) and their factual and counterfactual outcomes. Computational behavioural analysis revealed that participants were less mindful of regret (and more strongly driven by bigger risks) when choosing for others vs for themselves. Conversely, they chose more conservatively (regarding both regret and risk) when being watched vs alone. The effects of beneficiary and audience on anticipated regret counteracted each other, suggesting that participants’ financial and reputational interests impacted the feeling of regret independently.

Research field(s)
Health Sciences, Psychology & Cognitive Sciences, Social Psychology

DOI
10.1038/s41598-021-91635-z

Alpha-Synuclein defects autophagy by impairing SNAP29-mediated autophagosome-lysosome fusion

NOMIS Researcher(s)

Published in

December 1, 2021

Dopaminergic (DA) cell death in Parkinson’s disease (PD) is associated with the gradual appearance of neuronal protein aggregates termed Lewy bodies (LBs) that are comprised of vesicular membrane structures and dysmorphic organelles in conjunction with the protein alpha-Synuclein (α-Syn). Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests α-Syn-mediated alterations in the lysosomal degradation of aggregated proteins and organelles – a process termed autophagy. Here, we used a combination of molecular biology and immunochemistry to investigate the effect of α-Syn on autophagy turnover in cultured human DA neurons and in human post-mortem brain tissue. We found α-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, α-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, α-Syn decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of α-Syn on autophagy whereas SNAP29 co-expression reversed the α-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In accord with our results from cultured neurons, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human post-mortem brain tissue of Lewy body pathology (LBP) cases. In summary, our results thus demonstrate a previously unknown effect of α-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autolysosome fusion. As such, our findings will therefore support the investigation of autophagy-associated pathological changes in PD

Research field(s)
Health Sciences, Biomedical Research, Biochemistry & Molecular Biology

DOI
10.1038/s41419-021-04138-0

A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

NOMIS Researcher(s)

Published in

December 1, 2021

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.1038/s41598-021-01689-2

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