Les amyloïdoses : détection à l'aide de nanoparticules et propriétés optiques originales

Amyloidosis are diseases characterised by self-agregation of misfolded proteins in fibrillary forms, called amyloid fibrils. They are associated with many diseases, and their early diagnosis remains a clinical challenge. In this work, we show the targeting and the detection of amyloid fibrils, from in vitro to in vivo experiments in mice models, useful for early diagnostic of amyloidosis. For that purpose, we use multimodal nanoparticles, further functionalized with specific molecules against amyloid fibrils. This multimodality for imaging (fluorescence, MRI, PET) represent a breakthrough in modern medicine, to add structural and functional informations. After negative toxicity tests on various cell lines, these nanoparticles have been tested on three different amyloid fibrils, formed with amyloid β(1-42) peptide (Alzheimer’s disease), amylin (type II diabetes mellitus), or mutated Val30Met transthyretin (familial polyneuropathy). As it is shown by spectroscopy and surface plasmon resonance experiments, nanoparticles grafted with generic targeters (Pittsburgh compound B or a nanobody) target the three amyloid fibrils, with good affinity, whereas nanoparticles vectorised with peptides show specific targeting for amyloid β(1-42) or Val30Met mutated transthyretin, but with lower affinity. The targeting by nanoparticles have been confirmed ex vivo on pathological tissues with each amyloid burden, thanks to fluorescence microscopy. Generic nanoparticles have been injected in Alzheimer’s mice model, and the monitoring in vivo by IRM and post-mortem by optical microscopy supposed a targeting of amyloid β(1-42) aggregates in brain. Furthermore, we demonstrate the possibility to detect amyloid fibrils without labelling. Spectroscopic measurements show original, and specific optical properties of amyloid fibrils, around the UV-visible and the near infra-red regions. Interestingly, these properties are also observed on pathological tissues by ex vivo fluorescence microscopy, and in vivo ongoing analysis by photoacoustic imagery and real-time imaging seems to be very promising. By multimodality of non-toxic grafted nanoparticles or by intrinsic properties of amyloid fibrils suggesting completely non-invasive tests, these two strategies can be useful for early diagnostic of amyloidoses in humans.Les amyloïdoses sont des maladies caractérisées par l’agrégation structurée de protéines, sous forme de fibres amyloïdes. Le diagnostic précoce de ces maladies représente un enjeu important, pour la prise en charge de nombreuses pathologies associées. Dans ce travail, nous avons montré le ciblage et la détection de plusieurs fibres amyloïdes, de l’in vitro à l’in vivo. Pour cela, nous avons utilisé des nanoparticules multimodales pour l’imagerie médicale (TEP, IRM), greffées avec diverses molécules ciblant les fibres. Trois types de fibres amyloïdes ont été testées, formées à partir du peptide amyloïde β (maladie d’Alzheimer), d’amyline (diabète de type 2), et de la transthyrétine (polyneuropathie familiale). Comme montré par des techniques de spectroscopie et de résonance plasmonique de surface (fluorescence et Biacore), des nanoparticules génériques (dû au greffage du Pittsburgh compound B ou d’un nanocorps) ciblent avec une bonne affinité les trois types de fibres in vitro, tandis que des nanoparticules spécifiques (dû au greffage de peptides) ciblent avec une affinité moindre les fibres d’amyloïde β ou de transthyrétine. Le ciblage et la détection des dépôts amyloïdes par les nanoparticules ont été confirmés par microscopie à fluorescence, sur des tissus de souris présentant chacune des trois maladies. Le suivi par imageries in vivo (par IRM) et post-mortem (par microscopie optique), après injection de nanoparticules génériques chez la souris Alzheimer, supposent un ciblage des dépôts amyloïdes intracérébraux. Par ailleurs, nous avons détecté les fibres amyloïdes sans aucun marquage. Des études spectroscopiques in vitro ont permis de montrer des propriétés luminescentes intrinsèques des fibres amyloïdes, dans l’UV-visible et le proche infrarouge. Ces caractéristiques ont été observées sur des coupes de tissus de cerveau de souris Alzheimer par microscopie à fluorescence, et les études in vivo en cours semblent prometteuses (par imagerie photo-acoustique, et en temps résolu). Que cela soit par l’utilisation de nanoparticules fonctionnalisées multimodales, ou de propriétés intrinsèques des fibres amyloïdes suggérant une détection complètement non-invasive, ces deux stratégies innovantes semblent adaptées pour le diagnostic précoce des amyloïdoses chez l’Homme

Amyloidosis : detection with functionalised nanoparticles and original optical properties

Les amyloïdoses sont des maladies caractérisées par l’agrégation structurée de protéines, sous forme de fibres amyloïdes. Le diagnostic précoce de ces maladies représente un enjeu important, pour la prise en charge de nombreuses pathologies associées. Dans ce travail, nous avons montré le ciblage et la détection de plusieurs fibres amyloïdes, de l’in vitro à l’in vivo. Pour cela, nous avons utilisé des nanoparticules multimodales pour l’imagerie médicale (TEP, IRM), greffées avec diverses molécules ciblant les fibres. Trois types de fibres amyloïdes ont été testées, formées à partir du peptide amyloïde β (maladie d’Alzheimer), d’amyline (diabète de type 2), et de la transthyrétine (polyneuropathie familiale). Comme montré par des techniques de spectroscopie et de résonance plasmonique de surface (fluorescence et Biacore), des nanoparticules génériques (dû au greffage du Pittsburgh compound B ou d’un nanocorps) ciblent avec une bonne affinité les trois types de fibres in vitro, tandis que des nanoparticules spécifiques (dû au greffage de peptides) ciblent avec une affinité moindre les fibres d’amyloïde β ou de transthyrétine. Le ciblage et la détection des dépôts amyloïdes par les nanoparticules ont été confirmés par microscopie à fluorescence, sur des tissus de souris présentant chacune des trois maladies. Le suivi par imageries in vivo (par IRM) et post-mortem (par microscopie optique), après injection de nanoparticules génériques chez la souris Alzheimer, supposent un ciblage des dépôts amyloïdes intracérébraux. Par ailleurs, nous avons détecté les fibres amyloïdes sans aucun marquage. Des études spectroscopiques in vitro ont permis de montrer des propriétés luminescentes intrinsèques des fibres amyloïdes, dans l’UV-visible et le proche infrarouge. Ces caractéristiques ont été observées sur des coupes de tissus de cerveau de souris Alzheimer par microscopie à fluorescence, et les études in vivo en cours semblent prometteuses (par imagerie photo-acoustique, et en temps résolu). Que cela soit par l’utilisation de nanoparticules fonctionnalisées multimodales, ou de propriétés intrinsèques des fibres amyloïdes suggérant une détection complètement non-invasive, ces deux stratégies innovantes semblent adaptées pour le diagnostic précoce des amyloïdoses chez l’Homme.Amyloidosis are diseases characterised by self-agregation of misfolded proteins in fibrillary forms, called amyloid fibrils. They are associated with many diseases, and their early diagnosis remains a clinical challenge. In this work, we show the targeting and the detection of amyloid fibrils, from in vitro to in vivo experiments in mice models, useful for early diagnostic of amyloidosis. For that purpose, we use multimodal nanoparticles, further functionalized with specific molecules against amyloid fibrils. This multimodality for imaging (fluorescence, MRI, PET) represent a breakthrough in modern medicine, to add structural and functional informations. After negative toxicity tests on various cell lines, these nanoparticles have been tested on three different amyloid fibrils, formed with amyloid β(1-42) peptide (Alzheimer’s disease), amylin (type II diabetes mellitus), or mutated Val30Met transthyretin (familial polyneuropathy). As it is shown by spectroscopy and surface plasmon resonance experiments, nanoparticles grafted with generic targeters (Pittsburgh compound B or a nanobody) target the three amyloid fibrils, with good affinity, whereas nanoparticles vectorised with peptides show specific targeting for amyloid β(1-42) or Val30Met mutated transthyretin, but with lower affinity. The targeting by nanoparticles have been confirmed ex vivo on pathological tissues with each amyloid burden, thanks to fluorescence microscopy. Generic nanoparticles have been injected in Alzheimer’s mice model, and the monitoring in vivo by IRM and post-mortem by optical microscopy supposed a targeting of amyloid β(1-42) aggregates in brain. Furthermore, we demonstrate the possibility to detect amyloid fibrils without labelling. Spectroscopic measurements show original, and specific optical properties of amyloid fibrils, around the UV-visible and the near infra-red regions. Interestingly, these properties are also observed on pathological tissues by ex vivo fluorescence microscopy, and in vivo ongoing analysis by photoacoustic imagery and real-time imaging seems to be very promising. By multimodality of non-toxic grafted nanoparticles or by intrinsic properties of amyloid fibrils suggesting completely non-invasive tests, these two strategies can be useful for early diagnostic of amyloidoses in humans

Charge detection mass spectrometry on human-amplified fibrils from different synucleinopathies

International audienceAmyloid fibrils are self-assembled mesoscopic protein aggregates, which can accumulate to form deposits or plaques in the brain. In vitro amplification of fibrils can be achieved with real-time quaking-induced conversion (RT-QuIC). However, this emerging technique would benefit from a complementary method to assess structural properties of the amplification products. This work demonstrates the feasibility of nanospray-charge-detection-mass-spectrometry (CDMS) performed on α-synuclein (αSyn) fibrils amplified from human brains with Parkinson's disease (PD) or Dementia with Lewy bodies (DLB) and its synergistic combination with RT-QuIC

Magnetic Nanoparticles Applications for Amyloidosis Study and Detection: A Review

Magnetic nanoparticles (MNPs) have great potential in biomedical and clinical applications because of their many unique properties. This contribution provides an overview of the MNPs mainly used in the field of amyloid diseases. The first part discusses their use in understanding the amyloid mechanisms of fibrillation, with emphasis on their ability to control aggregation of amyloidogenic proteins. The second part deals with the functionalization by various moieties of numerous MNPs’ surfaces (molecules, peptides, antibody fragments, or whole antibodies of MNPs) for the detection and the quantification of amyloid aggregates. The last part of this review focuses on the use of MNPs for magnetic-resonance-based amyloid imaging in biomedical fields, with particular attention to the application of gadolinium-based paramagnetic nanoparticles (AGuIX), which have been recently developed. Biocompatible AGuIX nanoparticles show favorable characteristics for in vivo use, such as nanometric and straightforward functionalization. Their properties have enabled their application in MRI. Here, we report that AGuIX nanoparticles grafted with the Pittsburgh compound B can actively target amyloid aggregates in the brain, beyond the blood–brain barrier, and remain the first step in observing amyloid plaques in a mouse model of Alzheimer’s disease

Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting

International audienceGadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses.MATERIALS & METHODS:The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy.RESULTS:Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues.CONCLUSION:Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy

Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting.

AmylDi

The influence of HLA-DRB1*15 on the relationship between microglia and neurons in multiple sclerosis normal appearing cortical grey matter

Cortical tissue injury is common in multiple sclerosis (MS) and associates with disability progression. We have previously shown that HLA-DRB1*15 genotype status associates with the extent of cortical inflammatory pathology. In the current study, we sought to examine the influence of HLA-DRB1*15 on relationships between inflammation and neurodegeneration in MS. Human post-mortem MS cases (n = 47) and controls (n = 10) were used. Adjacent sections of motor cortex were stained for microglia (Iba1+, CD68+, TMEM119+), lymphocytes (CD3+, CD8+), GFAP+ astrocytes, and neurons (NeuN+). A subset of MS cases (n = 20) and controls (n = 7) were double-labeled for neurofilament and glutamic acid decarboxylase 65/67 (GAD+) to assess the extent of the inhibitory synaptic loss. In MS cases, microglial protein expression positively correlated with neuron density (Iba1+: r = 0.548, p < 0.001, CD68+: r = 0.498, p = 0.001, TMEM119+ r = 0.437, p = 0.003). This finding was restricted to MS cases not carrying HLA-DRB1*15. Evidence of a 14% reduction in inhibitory synapses in MS was detected (MS: 0.299 ± 0.006 synapses/μm2 neuronal membrane versus control: 0.348 ± 0.009 synapses/μm2 neuronal membrane, p = 0.005). Neurons expressing inhibitory synapses were 24% smaller in MS cases compared to the control (MS: 403 ± 15 μm2 versus control: 531 ± 29 μm2, p = 0.001), a finding driven by HLA-DRB1*15+ cases (15+: 376 ± 21 μm2 vs. 15−: 432 ± 22 μm2, p = 0.018). Taken together, our results demonstrate that HLA-DRB1*15 modulates the relationship between microglial inflammation, inhibitory synapses, and neuronal density in the MS cortex

Mass and charge distributions of amyloid fibers involved in neurodegenerative diseases: Mapping heterogeneity and polymorphism

International audienceHeterogeneity and polymorphism are generic features of amyloid fibers with some important effects on the related disease development. We report here the characterization, by charge detection mass spectrometry, of amyloid fibers made of three polypeptides involved in neurodegenerative diseases: A beta(1-42) peptide, tau and alpha-synuclein. Beside the mass of individual fibers, this technique enables to characterize the heterogeneity and the polymorphism of the population. In the case of A beta(1-42) peptide and tau protein, several coexisting species could be distinguished and characterized. In the case of alpha-synuclein, we show how the polymorphism affects the mass and charge distributions

Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation is inhibited by chaperones targeting pathological misfolding and if so by what mechanism. Here we analyze how four entirely different human chaperones or protein modulators (transthyretin, S100A9, Bri2 BRICHOS and DNAJB6) and bacterial CsgC affect CsgA and FapC fibrillation. CsgA is more susceptible to inhibition than FapC and the chaperones vary considerably in the efficiency of their inhibition. However, mechanistic analysis reveals that all predominantly target primary nucleation rather than elongation or secondary nucleation, while stoichiometric considerations suggest that DNAJB6 and CsgC target nuclei rather than monomers. Inhibition efficiency broadly scales with the chaperones' affinity for monomeric CsgA and FapC. The chaperones tend to target the most aggregation-prone regions of CsgA, but do not display such tendencies towards the more complex FapC sequence. Importantly, the most efficient inhibitors (Bri2 BRICHOS and DNAJB6) significantly reduce bacterial biofilm formation. This commonality of chaperone action may reflect the simplicity of functional amyloid formation, driven largely by primary nucleation, as well as the ability of non-bacterial chaperones to deploy their proteostatic capacities across biological kingdoms