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Aguzzi, A.

Publications and source records attributed to Aguzzi, A..

At least 37 records · Page 2Linked to original sources
bioRxiv2023

A screen of alkaline and oxidative formulations for their inactivation efficacy of metal surface adsorbed prions using a steel-bead seed amplification assay

Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the development of TESSA (sTainlESs steel-bead Seed Amplification assay), a prion seed amplification assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for the bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

neuroscience↗
bioRxiv2023

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 {micro}m laterally, 3.3 {micro}m axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

neuroscience↗
bioRxiv2023

NG2 glia protect against prion neurotoxicity by inhibiting prostaglandin E2 signaling

Oligodendrocyte-lineage cells, including NG2 glia, undergo prominent changes in various neurodegenerative disorders. This raises the question of how myelinating cells interact with neurodegenerative processes. Here, we found that NG2 glia were activated after prion infection in cerebellar organotypic cultured slices (COCS) and in brains of prion-inoculated mice. In both model systems, depletion of NG2 glia exacerbated prion-induced neurodegeneration and accelerated prion pathology. Loss of NG2 glia unleashed a microglial reaction promoting the biosynthesis of prostaglandin E2 (PGE2), which augmented prion neurotoxicity in the HovS cell line, primary neurons and COCS through binding to the EP4 receptor. Single-cell RNA sequencing revealed molecular signatures of inflammatory, disease-associated and MHC+ microglia but not of interferon-responsiveness in PGE2-producing microglia of prion-inoculated mice. Pharmacological or genetic inhibition of PGE2 biosynthesis attenuated prion-induced neurodegeneration in COCS and mice, reduced the enhanced neurodegeneration in NG2-glia-depleted COCS after prion infection, and dampened the acceleration of prion disease in NG2-glia-depleted mice. These data unveil a non-cell-autonomous interaction between NG2 glia and microglia in prion disease and suggest that PGE2 signaling may represent an actionable target against prion diseases.

neuroscience↗
bioRxiv2023

Leptomeningeal collaterals regulate reperfusion in ischemic stroke

Recanalization is the mainstay of ischemic stroke treatment. However, even with timely clot removal, many stroke patients recover poorly. Leptomeningeal collaterals (LMCs) are pial anastomotic vessels with yet unknown functions. Utilizing a thrombin-based mouse model of stroke and the gold standard fibrinolytic treatment rt-PA, we here show that LMCs play a critical role in preserving vascular function in ischemic territories. We applied laser speckle contrast imaging, ultrafast ultrasound, and two-photon microscopy, to show that after thrombolysis, LMCs allow for gradual reperfusion resulting in small infarcts. On the contrary, in mice with poor LMCs, distal segments of recanalized arteries collapse and deleterious hyperemia causes hemorrhage and mortality. Accordingly, in stroke patients with poor collaterals undergoing thrombectomy, rapid reperfusion resulted in hemorrhagic transformation and unfavorable recovery. Thus, we identify LMCs as key components regulating reperfusion after stroke. Future therapeutic interventions should aim to enhance collateral function, allowing for gradual reperfusion of ischemic tissues after stroke.

neuroscience↗
bioRxiv2023

Distinct hypothalamus-habenula circuits govern risk preference

Appropriate risk evaluation is essential for survival in complex, uncertain environments. Confronted with choosing between certain (safe) and uncertain (risky) options, animals show strong preference for either option consistently across extended time periods. How such risk preference is encoded in the brain remains elusive. A candidate region is the lateral habenula (LHb), which is prominently involved in value-guided behavior. Here, using a balanced two-alternative choice task and longitudinal two-photon calcium imaging, we identify LHb neurons with risk-preference-selective activity reflecting individual risk preference prior to action selection. By employing whole-brain anatomical tracing, multi-fiber photometry, and projection- and cell-type-specific optogenetics, we find that glutamatergic LHb projections from the medial (MH) but not lateral (LH) hypothalamus provide behavior-relevant synaptic input before action selection. Optogenetic stimulation of MH{lozenge}LHb axons evoked excitatory and inhibitory postsynaptic responses, whereas LH{lozenge}LHb projections were excitatory. We thus reveal functionally distinct hypothalamus-habenula circuits for risk preference in habitual economic decision-making.

neuroscience↗
bioRxiv2022

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope

Rapid advances in tissue clearing protocols have begun to outpace the capabilities of existing microscope objectives: High-resolution imaging inside cm-sized cleared samples is often not possible as it requires multi-immersion objectives with high numerical aperture (NA > 0.7), long working distance (WD > 10 mm) and a large field-of-view (FOV > 1 mm). Here, we introduce a novel mirror-based optical design, the "Schmidt objective", which meets all these criteria despite containing only two optical elements. It consists of a spherical mirror in contact with the immersion medium and an aspherical correction plate. We showcase a multi-photon variant of a Schmidt objective that reaches NA 1.08 at an refractive index of 1.56 and demonstrate its versatility by imaging fixed samples in a wide range of immersion media ranging from air and water to BABB, DBE, and ECI. In addition, we demonstrate in vivo imaging by recording neuronal activity in larval zebrafish.

neuroscience↗
bioRxiv2022

Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208-H140 hydrogen bond ("H-latch") between the 2-3 and {beta}2-2 loops of PrPC. Importantly, GDL that suppress the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced nineteen individual PrPC variants to PrPC-deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27) within PrPC. Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-type PrPC. K27 may represent an actionable target for compounds aimed at preventing prion-related neurodegeneration.

neuroscience↗
bioRxiv2022

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrPC. Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.

genomics↗
bioRxiv2022

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35364 ternary pools of 52746 siRNAs targeting 17582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrPSc, and its cellular counterpart PrPC, 40 genes affected selectively PrPSc. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

neuroscience↗
bioRxiv2022

Brain aging is faithfully modelled in organotypic brain slices and accelerated by prions

Mammalian models are essential for brain aging research. However, the long lifespan and limited amenability to genetic and pharmacological perturbations have hindered the use of mammals for dissecting aging-regulatory molecular networks and discovering new anti-aging interventions. To circumvent these limitations, we developed an ex vivo model system that faithfully mimics the aging process of the mammalian brain using cultured mouse brain slices. Genome-wide gene expression analyses showed that brain slices spontaneously upregulated senescence-associated genes over time and reproduced many of the transcriptional characteristics of aged brains. Treatment with rapamycin, a classical anti-aging compound, largely abolished the time-dependent transcriptional changes in brain slices. Using this model system, we discovered that prions drastically accelerated the development of age-related molecular signatures and the pace of brain aging. We confirmed this finding in mouse models and human victims of Creutzfeldt-Jakob disease. These data establish a novel, eminently tractable mammalian model of brain aging, and uncover a surprising acceleration of brain aging in prion diseases.

neuroscience↗
bioRxiv2021

Dendritic integration of sensory and reward information facilitates learning

Learning goal-directed behavior requires association of pertinent sensory stimuli with behaviorally relevant outcomes. In the mammalian neocortex, dendrites of pyramidal neurons are suitable association sites1-3 but how their activities adapt during learning remains elusive. Here, we track calcium signals in apical dendrites of layer 5 (L5) pyramidal neurons in mouse barrel cortex during texture discrimination learning4. We observe diverse task-related responses, either localized to branches or widespread throughout the apical tuft. However, even in expert mice, the tufts capability to discriminate go/no-go stimuli remains poor. Yet, we identify two prevailing response types in dendritic branches: 1) responses to unexpected outcome (reward) in naive mice that decrease with growing task proficiency, and 2) responses associated with salient sensory stimuli, especially the outcome-predicting texture touch, that strengthen upon learning. We demonstrate that these response types match distinct unsigned components of the temporal difference error5 by replicating our results with a reinforcement learning model. Moreover, optogenetic apical inhibition of L5 neurons during the outcome time window prevents naive animals from learning the task, consistent with the effect of equivalent model perturbation. Our findings suggest that salience signals in L5 apical dendrites facilitate the recruitment of task-relevant neurons via dendritic gain modulation.

neuroscience↗
bioRxiv2021

Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1,2, which involve human-specific mechanisms3-6 that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors7. Single-cell transcriptomics (scRNA-seq) and comparison to independent NSCs8, showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks. Neuronal and glial maturation in iCoMoNSC-derived cultures was similar to that of cortical organoids9. Overexpression of wild-type TDP-43 in a minority of iCoMoNSC-derived neurons led to progressive fragmentation and aggregation, resulting in loss of function and neurotoxicity. scRNA-seq revealed a novel set of misregulated RNA targets coinciding in both TDP-43 overexpressing neurons and patient brains exhibiting loss of nuclear TDP-43. The strongest misregulated target encoded for the synaptic protein NPTX2, which was consistently misaccumulated in ALS and FTLD patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby highlighting a new pathway of neurotoxicity.

neuroscience↗
bioRxiv2021

A conformational switch controlling the toxicity of the prion protein

Prion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ("H-latch") altering the flexibility of the 2-3 and {beta}2-2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

molecular biology↗
bioRxiv2021

Mutations in two SARS-CoV-2 variants of concern reflect two distinct strategies of antibody escape

Understanding the factors that contribute to antibody escape of SARS-CoV-2 and its variants is key for the development of drugs and vaccines that provide broad protection against a variety of virus variants. Using microfluidic diffusional sizing, we determined the dissociation constant (KD) for the interaction between receptor binding domains (RBDs) of SARS-CoV-2 in its original version (WT) as well as alpha and beta variants with the host-cell receptor angiotensin converting enzyme 2 (ACE2). For RBD-alpha, the ACE2-binding affinity was increased by a factor of ten when compared with RBD-WT, while ACE2-binding of RBD-beta was largely unaffected. However, when challenged with a neutralizing antibody that binds to both RBD-WT and RBD-alpha with low nanomolar KD values, RBD-beta displayed no binding, suggesting a substantial epitope change. In SARS-CoV-2 convalescent sera, RBD-binding antibodies showed low nanomolar affinities to both wild-type and variant RBD proteins--strikingly, the concentration of antibodies binding to RBD-beta was half that of RBD-WT and RBD-alpha, again indicating considerable epitope changes in the beta variant. Our data therefore suggests that one factor contributing to the higher transmissibility and antibody evasion of SARS-CoV-2 alpha and beta is a larger fraction of viruses that can form a complex with ACE2. However, the two variants employ different mechanisms to achieve this goal. While SARS-CoV-2 alpha RBD binds with greater affinity to ACE2 and is thus more difficult to displace from the receptor by neutralizing antibodies, RBD-beta is less accessible to antibodies due to epitope changes which increases the chances of ACE2-binding and infection.

biochemistry↗
bioRxiv2021

Understanding the role of memory re-activation and cross-reactivity in the defense against SARS-CoV-2

Recent efforts in understanding the course and severity of SARS-CoV-2 infections have highlighted both potential beneficial as well as detrimental effects of cross-reactive antibodies derived from memory immunity. Specifically, due to a significant degree of sequence similarity between SARS-CoV-2 and other members of the coronavirus family, memory B-cells that emerged from previous infections with endemic human coronaviruses (HCoVs) could be re-activated upon encountering the newly emerged SARS-CoV-2, thus prompting the production of cross-reactive antibodies. Understanding the affinity and concentration of these potentially cross-reactive antibodies to the new SARS-CoV-2 antigens is therefore particularly important when assessing both existing immunity against common HCoVs and adverse effects like antibody-dependent enhancement (ADE) in COVID-19. However, these two fundamental parameters cannot easily be deconvoluted by surface-based assays like enzyme-linked immunosorbent assays (ELISAs) which are routinely used to assess cross-reactivity. Here, we have used microfluidic antibody-affinity profiling (MAAP) to quantitatively evaluate the humoral immune response in COVID-19 convalescent patients by determining both antibody affinity and concentration against spike antigens of SARS-CoV-2 directly in nine convalescent COVID-19 patient and three pre-pandemic sera that were seropositive for common HCoVs. All 12 sera contained low concentrations of high affinity antibodies against spike antigens of HCoV-NL63 and HCoV-HKU1, indicative of past exposure to these pathogens, while the affinity against the SARS-CoV-2 spike protein was lower. These results suggest that cross-reactivity as a consequence of memory re-activation upon an acute SARS-CoV-2 infection may not be a significant factor in generating immunity against SARS-CoV-2.

biophysics↗
bioRxiv2021

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

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity and detect phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or {beta}2 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 to prothrombin (PT) with the strength of the antibody response against SARS-CoV-2 and that it is 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.

immunology↗
bioRxiv2021

The ASC Protein Controls the Severity of Systemic AA Amyloidosis in Mice

ASC-containing inflammasomes form specks, extracellular aggregates which enhance the aggregation of A{beta} amyloid in Alzheimers disease. This raises the question whether ASC participates to additional aggregation proteinopathies. Here we show that ASC controls the extent of inflammation-associated AA amyloidosis, a systemic disease caused by the aggregation of the acute-phase reactant serum amyloid A (SAA). Using superresolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Purified recombinant ASC specks accelerated SAA fibril formation in vitro. Mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain. In a murine model of inflammation-associated AA amyloidosis, splenic AA amyloid load was conspicuously decreased in Pycardtm1Vmd/tm1Vmd mice which lack ASC. This reduction was not a consequence of enhanced amyloid phagocytosis, as SAA stimulation increased phagocytic activity in Pycard+/+, but not in Pycard-/- macrophages. Treatment with anti-ASC antibodies decreased the amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (-logEC50 [&ge;] 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. Higher anti-ASC titers did not correlate with any specific disease, suggesting that anti-ASC immunotherapy may be well-tolerated. These findings expand the role played by ASC to extraneural proteinopathies of humans and experimental animals and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.

molecular biology↗