Biology subjects

Aguzzi, A.

Publications and source records attributed to Aguzzi, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies

While the initial pathology of Parkinsons disease and other -synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of -synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analyzed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with -synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of -synuclein pathology ex vivo. The overall survival of A53T -synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of -synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of -synucleinopathies is not universally valid.

neuroscience↗

bioRxiv2021

Ligands binding to the cellular prion protein induce its protective proteolytic release with therapeutic potential in neurodegenerative proteinopathies

The cellular prion protein (PrPC) is a central player in neurodegenerative diseases caused by protein misfolding, such as prion diseases or Alzheimers disease (AD). Expression levels of this GPI-anchored glycoprotein, especially at the neuronal cell surface, critically correlate with various pathomechanistic aspects underlying these diseases, such as templated misfolding (in prion diseases) and neurotoxicity and, hence, with disease progression and severity. In stark contrast to cell-associated PrPC, soluble extracellular forms or fragments of PrP are linked with neuroprotective effects, which is likely due to their ability to interfere with neurotoxic disease-associated protein conformers in the interstitial fluid. Fittingly, the endogenous proteolytic release of PrPC by the metalloprotease ADAM10 ( shedding) was characterized as a protective mechanism. Here, using a recently generated cleavage-site specific antibody, we shed new light on earlier studies by demonstrating that shed PrP (sPrP) negatively correlates with conformational conversion (in prion disease) and is markedly redistributed in murine brain in the presence of prion deposits or AD-associated amyloid plaques indicating a blocking and sequestrating activity. Importantly, we reveal that administration of certain PrP-directed antibodies and other ligands results in increased PrP shedding in cells and organotypic brain slice cultures. We also provide mechanistic and structural insight into this shedding-stimulating effect. In addition, we identified a striking exception to this, as one particular neuroprotective antibody, due to its special binding characteristics, did not cause increased shedding but rather strong surface clustering followed by fast endocytosis and degradation of PrPC. Both mechanisms may contribute to the beneficial action described for some PrP-directed antibodies/ligands and pave the way for new therapeutic strategies against devastating and currently incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Behavioral deficits, learning impairment, and enhanced hippocampal excitability in co-isogenic PrnpZH3/ZH3 mice

BackgroundThe cellular prion protein (PrPC) has been associated with numerous cellular processes, such as cell differentiation and neurotransmission. Moreover, it was recently demonstrated that some functions were misattributed to PrPC since results were obtained from mouse models with genetic artifacts. Here we elucidate the role of PrPC in the hippocampal circuitry and its related functions, like learning and memory, using the new strictly co-isogenic Prnp0/0 mouse (PrnpZH3/ZH3). Behavioral and operant conditioning tests were performed to evaluate memory and learning capabilities. In vivo electrophysiological recordings were carried out at CA3-CA1 synapses in living behaving mice, and spontaneous neuronal firing and network formation were monitored in primary neuronal cultures of PrnpZH3/ZH3 vs. wild-type mice. ResultsResults showed decreased motility, impaired operant conditioning learning, and anxiety-related behavior in PrnpZH3/ZH3 animals. PrPC absence enhanced susceptibility to high-intensity stimulations and kainate-induced seizures. However, long-term potentiation (LTP) was not enhanced in the PrnpZH3/ZH3 hippocampus. In addition, we observed a delay in neuronal maturation and network formation in PrnpZH3/ZH3 cultures. ConclusionIn conclusion, PrPC mediates synaptic function and protects the synapse from excitotoxic insults. Its deletion might evoke a susceptible epileptogenic brain that would fail to perform highly cognitive-demanding tasks such as associative learning and anxiety-like behaviors.

neuroscience↗

bioRxiv2021

The landscape of PrPC biosynthesis revealed by an arrayed genome-wide interference screen

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

neuroscience↗

bioRxiv2021

Quantitative 3D microscopy reveals a genetic network predicting the local activity of anti-Aβ compounds

Many efforts targeting amyloid-{beta} (A{beta}) plaques for the treatment of Alzheimers Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-A{beta} treatments through high-resolution light-sheet imaging of electrophoretically-cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to {beta}-secretase inhibitors, polythiophenes, or anti-A{beta} antibodies. Each treatment showed unique spatiotemporal A{beta} clearance, with polythiophenes emerging as a potent anti-A{beta} compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each A{beta} therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of A{beta} plaques. This may also contribute to the clinical trial failures of A{beta}-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.

neuroscience↗

bioRxiv2021

Loss of PIKfyve drives the spongiform degeneration in prion diseases

Brain-matter vacuolation is a defining trait of all prion diseases, yet its cause is unknown. Here we report that prion infection and prion-mimetic antibodies deplete the phosphatidylinositol kinase PIKfyve in mouse brains, cultured cells, organotypic brain slices, and in brains of Creutzfeldt-Jakob Disease victims. We found that PIKfyve, an inositol kinase involved endolysosomal maturation, is acylated by zDHHC9 and zDHHC21, whose juxtavesicular topology is disturbed by prion infection, resulting in PIKfyve deacylation and destabilization. A protracted unfolded protein response (UPR), typical of prion diseases, also induced PIKfyve deacylation and degradation. Conversely, UPR antagonists restored PIKfyve levels in prion-infected cells. Overexpression of zDHHC9 and zDHHC21, administration of the antiprion polythiophene LIN5044, or supplementation with the PIKfyve reaction product PI(3,5)P2, suppressed prion-induced vacuolation. Thus, PIKfyve emerges as a central mediator of vacuolation and neurotoxicity in prion diseases.

neuroscience↗

bioRxiv2021

Glial activation in prion diseases is strictly nonautonomous and requires neuronal PrPSc

Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons, astrocytes or oligodendrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC-deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.

neuroscience↗

bioRxiv2020

In vitro measurements of protein-protein interactions show that antibody affinity governs the inhibition of SARS-CoV-2 spike/ACE2 binding in convalescent serum

The humoral immune response plays a key role in suppressing the pathogenesis of SARS-CoV-2. The molecular determinants underlying the neutralization of the virus remain, however, incompletely understood. Here, we show that the ability of antibodies to disrupt the binding of the viral spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell, the key molecular event initiating SARS-CoV-2 entry into host cells, is controlled by the affinity of these antibodies to the viral antigen. By using microfluidic antibody-affinity profiling, we were able to quantify the serum-antibody mediated inhibition of ACE2-spike binding in two SARS-CoV-2 seropositive individuals. Measurements to determine the affinity, concentration, and neutralization potential of antibodies were performed directly in human serum. Using this approach, we demonstrate that the level of inhibition in both samples can be quantitatively described using the binding energies of the binary interactions between the ACE2 receptor and the spike protein, and the spike protein and the neutralizing antibody. These experiments represent a new type of in-solution receptor binding competition assay, which has further potential areas of application ranging from decisions on donor selection for convalescent plasma therapy, to identification of lead candidates in therapeutic antibody development, and vaccine development.

biochemistry↗

bioRxiv2020

The role of macrophage scavenger receptor 1 (Msr1) in prion pathogenesis

The progression of prion diseases is accompanied by the accumulation of prions in the brain. Ablation of microglia enhances prion accumulation and accelerates disease progression, suggesting that microglia play a neuroprotective role by clearing prions. However, the mechanisms underlying the phagocytosis and clearance of prion are largely unknown. The macrophage scavenger receptor 1 (Msr1) is an important phagocytic receptor expressed by microglia in the brain, and is involved in the uptake and clearance of soluble amyloid-{beta}. We therefore asked whether Msr1 might play a role in prion clearance, and assessed the scavenger function of Msr1 in prion pathogenesis. We found that Msr1 expression was upregulated in prion-infected mouse brains. However, Msr1 deficiency did not change prion disease progression or lesion patterns. Prion deposition in Msr1 deficient mice was similar to their wild type littermates. In addition, prion-induced neuroinflammation was not affected by Msr1 ablation. We conclude that Msr1 does not play a major role in prion pathogenesis.

neuroscience↗

bioRxiv2020

The prion protein is not required for peripheral nerve repair after crush injury

The cellular prion protein (PrP) is essential to the long-term maintenance of myelin sheaths in peripheral nerves. PrP activates the adhesion G-protein coupled receptor Adgrg6 on Schwann cells and initiates a pro-myelination cascade of molecular signals. Because Adgrg6 is crucial for peripheral myelin development and regeneration after nerve injury, we investigated the role of PrP in peripheral nerve repair. We performed experimental sciatic nerve crush injuries in co-isogenic wild-type and PrP-deficient mice, and examined peripheral nerve repair processes. Generation of repair Schwann cells, macrophage recruitment and remyelination were similar in PrP-deficient and wild-type mice. We conclude that PrP is dispensable for sciatic nerve regeneration after crush injury. Adgrg6 may sustain its function in peripheral nerve repair independently of its activation by PrP.

neuroscience↗

bioRxiv2020

Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the peripheral demyelinating neuropathy was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands. Summary blurbA dimeric prion protein ligand activates Adgrg6 but fails to induce pro-myelination signaling upon chronic treatment in a mouse model of peripheral demyelination.

neuroscience↗

bioRxiv2020

Prions induce minor genome-wide translational changes in neurons compared to glia

Prion diseases are caused by PrPSc, a self-replicating pathologically misfolded protein that exerts toxicity predominantly in the brain. The administration of PrPSc causes a robust, reproducible and specific disease manifestation. Here we have applied a combination of translating ribosome affinity purification and ribosome profiling to identify biologically relevant prion-induced changes during disease progression in a cell-type specific and genome-wide manner. Terminally diseased mice with severe neurological symptoms showed extensive alterations in astrocytes and microglia. Surprisingly, we detected only minor changes in the translational profiles of neurons. Prion-induced alterations in glia overlapped with those identified in other neurodegenerative diseases, suggesting that similar events occur in a broad spectrum of pathologies. Our results suggest that aberrant translation within glia may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease.

neuroscience↗

bioRxiv2020

Systemic and mucosal antibody secretion specific to SARS-CoV-2 during mild versus severe COVID-19

BackgroundInfection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an acute illness termed coronavirus disease 2019 (COVID-19). Humoral immune responses likely play an important role in containing SARS-CoV-2, however, the determinants of SARS-CoV-2-specific antibody responses are unclear. MethodsUsing immunoassays specific for the SARS-CoV-2 spike protein, we determined SARS-CoV-2-specific immunoglobulin A (IgA) and immunoglobulin G (IgG) in sera and mucosal fluids of two cohorts, including patients with quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR)-confirmed SARS-CoV-2 infection (n = 56; median age 61 years) with mild versus severe COVID-19, and SARS-CoV-2-exposed healthcare workers (n = 109; median age 36 years) with or without symptoms and tested negative or positive by RT-qPCR. FindingsOn average, SARS-CoV-2-specific serum IgA titers in mild COVID-19 cases became positive eight days after symptom onset and were often transient, whereas serum IgG levels remained negative or reached positive values 9-10 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers as a function of duration since symptom onset, independent of patient age and comorbidities. Very high levels of SARS-CoV-2-specific serum IgA correlated with severe acute respiratory distress syndrome (ARDS). Interestingly, some of the SARS-CoV-2-exposed healthcare workers with negative SARS-CoV-2-specific IgA and IgG serum titers had detectable SARS-CoV-2-specific IgA antibodies in their nasal fluids and tears. Moreover, SARS-CoV-2-specific IgA levels in nasal fluids of these healthcare workers were inversely correlated with patient age. InterpretationThese data show that systemic IgA and IgG production against SARS-CoV-2 develops mainly in severe COVID-19, with very high IgA levels seen in patients with severe ARDS, whereas mild disease may be associated with transient serum titers of SARS-CoV-2-specific antibodies but stimulate mucosal SARS-CoV-2-specific IgA secretion. The findings suggest four grades of antibody responses dependent on COVID-19 severity.

immunology↗

bioRxiv2020

Developmental Divergence of Sensory Stimulus Representation in Cortical Interneurons

Two inhibitory cell types involved in modulating barrel cortex activity and perception during active whisking in adult mice, are the VIP+ and SST+ interneurons. Here we identify a developmental transition point of structural and functional rearrangements onto these interneuron types around the start of active sensation at P14. Using in vivo two-photon Ca2+ imaging, we find that before P14, both interneuron types respond stronger to a multi-whisker stimulus, whereas after P14 their responses diverge, with VIP+ cells losing their multi-whisker preference and SST+ neurons enhancing theirs. Rabies virus tracings followed by tissue clearing, as well as photostimulation-coupled electrophysiology reveal that SST+ cells receive higher cross-barrel inputs compared to VIP+ at both time points. In addition, we also uncover that whereas prior to P14 both cell types receive direct input from the sensory thalamus, after P14 VIP+ cells show reduced inputs and SST+ cells largely shift to motor-related thalamic nuclei.

neuroscience↗

bioRxiv2020

Prion infection, transmission and cytopathology modelled in a low-biohazard human cell line

Transmission of prion infectivity to susceptible murine cell lines has simplified prion titration assays and has greatly reduced the need for animal experimentation. However, murine cell models suffer from technical and biological constraints. Human cell lines might be more useful, but they are much more biohazardous and are often poorly infectible. Here we describe the human clonal cell line hovS, which lacks the human PRNP gene and expresses instead the ovine PRNP VRQ allele. HovS cells were highly susceptible to the PG127 strain of sheep-derived murine prions, reaching up to 90% infected cells in any given culture, and were maintained in a continuous infected state for at least 14 passages. Infected hovS cells produced proteinase K-resistant prion protein (PrPSc), pelletable PrP aggregates and bona fide infectious prions capable of infecting further generations of naive hovS cells and mice expressing the VRQ allelic variant of ovine PrPC. Infection in hovS led to prominent cytopathic vacuolation akin to the spongiform changes observed in individuals suffering from prion diseases. In addition to expanding the toolbox for prion research to human experimental genetics, the hovS cell line provides a human-derived system that does not require human prions. Hence, the manipulation of scrapie-infected hovS cells may present fewer biosafety hazards than that of genuine human prions.

neuroscience↗

bioRxiv2020

Whole brain optoacoustic tomography reveals strain-specific regional beta-amyloid densities in Alzheimer`s disease amyloidosis models

Deposition of beta-amyloid (A{beta}) deposits is one major histopathological hallmark of Alzheimers disease (AD). Here, we introduce volumetric multi-spectral optoacoustic tomography (vMSOT), which covers 10x10x10 mm3 field-of-view, capable of 3D whole mouse brain imaging. We show for the first time the optoacoustic properties of oxazine-derivative AOI987 probe, which binds to A{beta}, and the application of vMSOT for the quantification of brain-wide A{beta} deposition. Administration of AOI987 to two common transgenic mouse strains of AD amyloidosis led to a retention of the probe in A{beta}-laden brain regions. Co-registered of vMSOT data to a brain atlas revealed strain-specific pattern of AOI987 uptake. A comparison with ex vivo light-sheet microscopy in cleared mouse brains showed a good correspondence in A{beta} distribution. Lastly, we demonstrate the specificity of the AOI987 probe by immunohistochemistry. vMSOT with AOI987 facilitates preclinical brain region-specific studies of A{beta} spread and accumulation, and the monitoring of putative treatments targeting A{beta}.

neuroscience↗

bioRxiv2020

Protective anti-prion antibodies in human immunoglobulin repertoires

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here we identified >6000 PrP-binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage-derived antibodies. When expressed recombinantly, these antibodies exhibited anti-PrP reactivity. Furthermore, we surveyed 48718 samples from 37894 hospital patients for the presence of anti-PrP IgGs, and found 21 high-titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti-PrP autoimmunity is innocuous. The existence of protective anti-prion antibodies in unbiased human immunological repertoires, combined with the reported lack of such antibodies in carriers of disease-associated PRNP mutations, suggests a link to the low incidence of spontaneous prion diseases in human populations.

neuroscience↗

bioRxiv2020

The uptake of tau amyloid fibrils is facilitated by the cellular prion protein and hampers prion propagation in cultured cells

Tauopathies are prevalent, invariably fatal brain diseases for which no cure is available. Tauopathies progressively affect the brain through cell-to-cell transfer of tau protein amyloids, yet the spreading mechanisms are unknown. Here we show that the cellular prion protein (PrPC) facilitates the uptake of tau aggregates by cultured cells, possibly by acting as an endocytic receptor. In mouse neuroblastoma cells, we found that tau amyloids bind to PrPC; internalization of tau fibrils was reduced in isogenic cells devoid of the gene encoding PrPC. Antibodies against N-proximal epitopes of PrPC impaired the binding of tau amyloids and decreased their uptake. Surprisingly, exposure of chronically prion-infected cells to tau amyloids reduced the accumulation of aggregated prion protein; this effect lasted for more than 72 hours after amyloid removal. These results point to bidirectional interactions between the two proteins: whilst PrPC mediates the entrance of tau fibrils in cells, PrPSc buildup is greatly reduced in their presence, possibly because of an impairment in the prion conversion process.

neuroscience↗