scholarly journals Quantitative Study of Alpha-Synuclein Prion-Like Spreading in Fully Oriented Reconstructed Neural Networks Reveals Non-synaptic Dissemination of Seeding Aggregates

2021 ◽  
Author(s):  
Josquin COURTE ◽  
Ngoc Anh LE ◽  
Luc BOUSSET ◽  
Ronald MELKI ◽  
Catherine VILLARD ◽  
...  
Keyword(s):  
Neural Networks ◽  
Culture System ◽  
Transfer Rate ◽  
Alpha Synuclein ◽  
Transfer Efficiency ◽  
Synaptic Connectivity ◽  
In Vivo Models ◽  
Low Efficiency ◽  
The Impact

Abstract Background: The trans-neuronal spread of protein aggregation in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson’s disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. Indeed, in vivo models of aSyn prion-like propagation yield results whose interpretation is hindered by the complexity of brain networks, and in vitro models have for now failed to properly model trans-neuronal propagation in synaptically connected neural networks.Results: To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic neuronal culture system. This system is the first to guide in vitro the development of fully oriented, synaptically connected and fluidically isolated neural networks. It thus uniquely permitted us to model the trans-neuronal propagation of aggregation: we selectively exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size (Fibrils and Oligomers), and quantitatively followed their dissemination to postsynaptic neurons. Both aggregates were anterogradely transferred to through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates, and found that while endogenous aSyn expression level controlled the seeding of aSyn aggregation, it did not affect the transfer rate of exogenous aggregates. Conclusion: Overall, we describe here the first culture system for reconstructing in vitro fully oriented and synaptically connected neural networks, and demonstrate it is uniquely suitable to quantitatively interrogate original aspects of the trans-neuronal propagation of prion-like aggregates.

scholarly journals Quantitative study of alpha-synuclein prion-like spreading in fully oriented reconstructed neural networks reveals non-synaptic dissemination of seeding aggregates

2021 ◽  
Author(s):  
Josquin Courte ◽  
Ngoc Anh Le ◽  
Luc Bousset ◽  
Ronald Melki ◽  
Catherine Villard ◽  
...  
Keyword(s):  
Neural Networks ◽  
Alpha Synuclein ◽  
Transfer Efficiency ◽  
Synaptic Connectivity ◽  
Low Efficiency ◽  
Synaptic Structures ◽  
The Impact ◽  
First Time ◽  
The Brain

The trans-neuronal spread of protein aggregates in a prion-like manner underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's disease. Despite being studied actively, the mechanisms of alpha-synuclein (aSyn) aggregates propagation remain poorly understood. This hinders the development of therapeutic approaches aiming at preventing the spatial progression of intracellular inclusions in neural networks. To assess the role of synaptic structures and neuron characteristics in the transfer efficiency of aggregates with seeding propensity, we developed a novel microfluidic culture system which allows for the first time to reconstruct in vitro fully oriented and synaptically connected neural networks. This is achieved by filtering axonal growth with unidirectional "axon valves" microchannels. We exposed the presynaptic compartment of reconstructed networks to well characterized human aSyn aggregates differing in size: Fibrils and Oligomers. Both aggregates were transferred to postsynaptic neurons through active axonal transport, albeit with poor efficiency. By manipulating network maturity, we compared the transfer rate of aggregates in networks with distinct levels of synaptic connectivity. Surprisingly, we found that transfer efficiency was lower in mature networks with higher synaptic connectivity. We then investigated the seeding efficiency of endogenous aSyn in the postsynaptic population. We found that exposure to Fibrils, and not Oligomers, resulted in low efficiency trans-neuronal seeding which was restricted to postsynaptic axons. Finally, we assessed the impact of neuron characteristics and aSyn expression on the propagation of aSyn aggregates. By reconstructing chimeric networks, we found that neuron characteristics, such as the brain region from which they originate or aSyn expression levels, did not significantly impact aggregates transfer, and observed no trans-neuronal seeding where the presynaptic population did not express aSyn. Overall, we demonstrate that this novel platform uniquely allows the quantitative interrogation of original aspects of the trans-neuronal propagation of seeding pathogenic entities.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

scholarly journals Knockdown of Leptin Receptor Affects Macrophage Phenotype in the Tumor Microenvironment Inhibiting Breast Cancer Growth and Progression

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2078
Author(s):  
Luca Gelsomino ◽  
Giuseppina Daniela Naimo ◽  
Rocco Malivindi ◽  
Giuseppina Augimeri ◽  
Salvatore Panza ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Microenvironment ◽  
Leptin Receptor ◽  
Macrophage Phenotype ◽  
In Vivo Models ◽  
Monocyte Chemoattractant Protein 1 ◽  
Arginase 1 ◽  
The Impact

Aberrant leptin (Ob) signaling, a hallmark of obesity, has been recognized to influence breast cancer (BC) biology within the tumor microenvironment (TME). Here, we evaluated the impact of leptin receptor (ObR) knockdown in affecting BC phenotype and in mediating the interaction between tumor cells and macrophages, the most abundant immune cells within the TME. The stable knockdown of ObR (ObR sh) in ERα-positive and ERα-negative BC cells turned the tumor phenotype into a less aggressive one, as evidenced by in vitro and in vivo models. In xenograft tumors and in co-culture experiments between circulating monocytes and BC cells, the absence of ObR reduced the recruitment of macrophages, and also affected their cytokine mRNA expression profile. This was associated with a decreased expression and secretion of monocyte chemoattractant protein-1 in ObR sh clones. The loss of Ob/ObR signaling modulated the immunosuppressive TME, as shown by a reduced expression of programmed death ligand 1/programmed cell death protein 1/arginase 1. In addition, we observed increased phagocytic activity of macrophages compared to control Sh clones in the presence of ObR sh-derived conditioned medium. Our findings, addressing an innovative role of ObR in modulating immune TME, may open new avenues to improve BC patient health care.

Commentary on: L-Methylfolate, Methylcobolamine, and N-Acetylcysteine in the Treatment of Alzheimer's Disease-Related Cognitive Decline

CNS Spectrums ◽  
2010 ◽  
Vol 15 (S1) ◽  
pp. 7-7 ◽  
Author(s):  
Jeffrey L Cummings
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Methylation Status ◽  
Neurofibrillary Tangle ◽  
Amyloid Β ◽  
Vitamin B ◽  
Amyloid Β Protein ◽  
In Vivo Models ◽  
The Impact

Drs. McCaddon and Hudson provide a thorough review of the multiple ways in which vitamin B12, vitamin B6, folate, and homocysteine (Hey) are implicated in the pathogenesis of Alzheimer's disease (AD). They noted that Hey is more often elevated in AD and in mild cognitive impairment (MCI) than in cognitively healthy elderly; phosphatases needed to limit tau hyperphosphoryalation and neurofibrillary tangle formation require methylation and are dependent on folate and methylation status; cerebrospinal fluid (CSF) tau levels correlated with markers of methylation status; reduced folate and B12 levels lead to increase β-secretase and pesenilin 1 (PS1) actions leading to greater amyloid-β production in in vitro models; elevated Hey levels in rates are associated with increased PS1 activity and spatial memory deficits that are reversed following treatment with B12 and folate; raised Hey levels in vitro increase amyloid-β protein neurotoxicity; methylation impacts transmitters and transmitter function relevant to AD; in cultured neurons, Hey induces injury in DNA and stimulates cell death pathways. B12 deficiency leads to accumulation of methyl malonic acid, which inhibits mitochondrial function and may compromise energy generation and impair maintenance of synaptic plasticity. Methylation abnormalities result in excessive generation of reactive oxygen species that contribute importantly to cell injury. Biomarkers of oxidative injury, such as isoprostanes, are elevated in AD and suggest excess oxidation. Thus, there are multiple pathways through which deficient methylation may contribute to AD. In some cases, the observations are derived from models with B12 or folate deficiency and some in vitro observations have not been tested in in vivo models. There are no biomarkers specific to some of the pathways implicated and the magnitude of the impact of the deficiency or its treatment has not been established for all the relationships. Two open-label experiments in early- and late-stage AD patients have suggested benefit.

scholarly journals Alpha-Synuclein Aggregation Induced by Brief Ischemia Negatively Impacts Neuronal Survival in vivo: A Study in [A30P]alpha-Synuclein Transgenic Mouse

2010 ◽  
Vol 31 (3) ◽  
pp. 913-923 ◽  
Author(s):  
Isin Unal-Cevik ◽  
Yasemin Gursoy-Ozdemir ◽  
Muge Yemisci ◽  
Sevda Lule ◽  
Gunfer Gurer ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neuronal Survival ◽  
Alpha Synuclein ◽  
In Vivo Model ◽  
Neuronal Necrosis ◽  
Mca Occlusion ◽  
Nitrative Stress ◽  
The Impact

Alpha-synuclein oligomerization and aggregation are considered to have a role in the pathogenesis of neurodegenerative diseases. However, despite numerous in vitro studies, the impact of aggregates in the intact brain is unclear. In vitro, oxidative/nitrative stress and acidity induce α-synuclein oligomerization. These conditions favoring α-synuclein fibrillization are present in the ischemic brain, which may serve as an in vivo model to study α-synuclein aggregation. In this study, we show that 30-minute proximal middle cerebral artery (MCA) occlusion and 72 hours reperfusion induce oligomerization of wild-type α-synuclein in the ischemic mouse brain. The nonamyloidogenic isoform β-synuclein did not form oligomers. Alpha-synuclein aggregates were confined to neurons and colocalized with ubiquitin immunoreactivity. We also found that 30 minutes proximal MCA occlusion and 24 hours reperfusion induced larger infarcts in C57BL/6(Thy1)-h[A30P]alphaSYN transgenic mice, which have an increased tendency to form synuclein fibrils. Trangenics also developed more selective neuronal necrosis when subjected to 20 minutes distal MCA occlusion and 72 hours reperfusion. Enhanced 3-nitrotyrosine immunoreactivity in transgenic mice suggests that oxidative/nitrative stress may be one of the mechanisms mediating aggregate toxicity. Thus, the increased vulnerability of transgenic mice to ischemia suggests that α-synuclein aggregates not only form during ischemia but also negatively impact neuronal survival, supporting the idea that α-synuclein misfolding may be neurotoxic.

scholarly journals The Puzzling Potential of Carbon Nanomaterials: General Properties, Application, and Toxicity

Nanomaterials ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1508
Author(s):  
Danica Jović ◽  
Vesna Jaćević ◽  
Kamil Kuča ◽  
Ivana Borišev ◽  
Jasminka Mrdjanovic ◽  
...  
Keyword(s):  
Carbon Nanomaterials ◽  
Wide Spectrum ◽  
Chemical Properties ◽  
Carbon Nanomaterial ◽  
In Vivo Models ◽  
Physico Chemical ◽  
The Impact ◽  
Cell Signaling Pathways

Being a member of the nanofamily, carbon nanomaterials exhibit specific properties that mostly arise from their small size. They have proved to be very promising for application in the technical and biomedical field. A wide spectrum of use implies the inevitable presence of carbon nanomaterials in the environment, thus potentially endangering their whole nature. Although scientists worldwide have conducted research investigating the impact of these materials, it is evident that there are still significant gaps concerning the knowledge of their mechanisms, as well as the prolonged and chronic exposure and effects. This manuscript summarizes the most prominent representatives of carbon nanomaterial groups, giving a brief review of their general physico-chemical properties, the most common use, and toxicity profiles. Toxicity was presented through genotoxicity and the activation of the cell signaling pathways, both including in vitro and in vivo models, mechanisms, and the consequential outcomes. Moreover, the acute toxicity of fullerenol, as one of the most commonly investigated members, was briefly presented in the final part of this review. Thinking small can greatly help us improve our lives, but also obliges us to deeply and comprehensively investigate all the possible consequences that could arise from our pure-hearted scientific ambitions and work.

scholarly journals Chagas Disease Drug Discovery

2014 ◽  
Vol 20 (1) ◽  
pp. 22-35 ◽  
Author(s):  
Eric Chatelain
Keyword(s):  
Chagas Disease ◽  
New Technologies ◽  
Low Cost ◽  
Screening Strategy ◽  
New Drugs ◽  
In Vivo Models ◽  
The Right ◽  
The Impact

American trypanosomiasis, or Chagas disease, is the result of infection by the Trypanosoma cruzi parasite. Endemic in Latin America where it is the major cause of death from cardiomyopathy, the impact of the disease is reaching global proportions through migrating populations. New drugs that are safe, efficacious, low cost, and adapted to the field are critically needed. Over the past five years, there has been increased interest in the disease and a surge in activities within various organizations. However, recent clinical trials with azoles, specifically posaconazole and the ravuconazole prodrug E1224, were disappointing, with treatment failure in Chagas patients reaching 70% to 90%, as opposed to 6% to 30% failure for benznidazole-treated patients. The lack of translation from in vitro and in vivo models to the clinic observed for the azoles raises several questions. There is a scientific requirement to review and challenge whether we are indeed using the right tools and decision-making processes to progress compounds forward for the treatment of this disease. New developments in the Chagas field, including new technologies and tools now available, will be discussed, and a redesign of the current screening strategy during the discovery process is proposed.

scholarly journals AB0158 IMPACT OF JAK INHIBITORS ON MACROPHAGE POLARISATION: PERSPECTIVES FOR SYSTEMIC SCLEROSIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1380.3-1380
Author(s):  
A. Lescoat ◽  
A. Ballerie ◽  
M. Lelong ◽  
C. Morzadec ◽  
S. Jouneau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Systemic Sclerosis ◽  
Blood Monocytes ◽  
Jak Inhibitors ◽  
In Vivo Models ◽  
Macrophage Polarisation ◽  
Healthy Donors ◽  
The Impact

Background:Macrophage can adopt various phenotypes and activation states according to their surrounding microenvironment. M1 or inflammatory macrophages are generated under IFNɣ/LPS signaling and express the membrane marker CD86. Different subtypes of M2 macrophages are also described: M2a macrophages (generated under IL4/IL13 signaling) and characterized by a high expression of CD206 and pro-fibrotic properties and, M2c macrophages (generated under IL10 and/or glucorticoid signaling), considered as anti-inflammatory resolving macrophages. There is growing interest in the role of macrophages in the pathogenesis of Systemic Sclerosis (SSc). Recent studies highlight that macrophages from fibrotic tissues such as lung or skin from SSc patients have a M2 phenotype whereas, in blood-monocytes derived macrophages (MDM), SSc MDM have a mixed signature associating M1 and M2 characteristics. Jak inhibitors are treatments used in rheumatoid arthritis and that can variously target signals that could be involved both in M1 and in M2 polarisation.Objectives:This study evaluates the impact of three Jak inhibitors on the polarisation state of human MDM in vitro.Methods:Blood monocytes form healthy donors (HD) were differentiated with M-CSF (for 7 days) in MDM and pre-treated by ruxolitinib (Jak2-Jak1 inhibitor), tofacitinib (Jak3 inhibitor) or itacitinib (Jak1 inhibitor) (1µM for all). They were then polarised into M1i (IFNɣ, 20µg/mL), M1Li (IFNɣ+LPS, 20µg/mL), M2a (IL4+IL13; 20µg/ML), M2c (IL10, 20µg/mL) and M2c(dex) (IL10+dexamethasone, 10 nM). The impact of each Jak inhibitor on phenotype (flow cytometry), gene expression (qPCR) and cytokine secretion (ELISA) was evaluated in each polarisation state.Results:Concerning phenotypes, all Jak inhibitors reduced the expression of the M1i and M1Li marker CD86, but ruxolitinib had a higher effect. Only ruxolitinib reduced the expression of the M1i marker MHCII. All Jak inhibitors reduced the expression of CD206 in M2a. They had no impact on the expression of CD163, CD204 in any M2 conditions. Key M1 genes were repressed by all Jak inhibitors, such as CXCL10, IL6 or TNFα with a more significant effect of ruxolitinib. All Jak inhibitors reduced the gene expression of CXCL13 and SOCS3 in M2c. Secretion levels of IL6 and CCL18 were also repressed, with a more significant effect of ruxolitinib.Conclusion:Jak inhibitors can limit M1 and M2 polarisation state in vitro, with a more significant effect of the Jak2-Jak1 inhibitor ruxolitinib. The relevance of these results in MDM from SSc patients and in vivo models of SSc is still to be determined.Disclosure of Interests:Alain LESCOAT: None declared, Alice Ballerie: None declared, Marie Lelong: None declared, Claudie Morzadec: None declared, Stéphane Jouneau Grant/research support from: AIRB, Boehringer Ingelheim, LVL Medical, Novartis, Roche, Bellorophon Therapeutics, Biogen, Fibrogen, Galecto Biotech, Gilead Sciences, Pharm-Olam, Pliant Therapeutics, Savara Pharmaceuticals/Serendex Pharmaceuticals, Consultant of: Actelion, AIRB, AstraZeneca, Bristol-Myers Squibb, Boehringer Ingelheim, Chiesi, Genzyme, GlazoSmithKline, LVL Medical, Mundipharma, Novartis, Pfizer, Roche, Sanofi, Patrick Jégo: None declared, Laurent Vernhet: None declared, Olivier Fardel: None declared, Valérie Lecureur: None declared

scholarly journals Rab27 GTPases regulate alpha-synuclein uptake, cell-to-cell transmission, and toxicity

2020 ◽  
Author(s):  
Rachel Underwood ◽  
Bing Wang ◽  
Aneesh Pathak ◽  
Laura Volpicelli-Daley ◽  
Talene A. Yacoubian
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Lewy Bodies ◽  
Neuronal Loss ◽  
Alpha Synuclein ◽  
Rab Gtpases ◽  
Double Knockout ◽  
The Impact

SUMMARYParkinson’s disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein α-synuclein. α-Synuclein is hypothesized to have prion-like properties, by which misfolded α-synuclein induces the pathological aggregation of endogenous α-synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate α-synuclein secretion, clearance, and toxicity in vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic α-synuclein. Double knockout of both Rab27 isoforms eliminated α-synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary α-synuclein. In vivo, Rab27 double knockout mice lacked fibril-induced α-synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled α-synuclein fibrils revealed that Rab27a/b knockout prevented α-synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of α-synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson’s disease and identifies Rab27s as key players in the progression of synucleinopathies.

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