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

Emmenegger, Marc; Cecco, Elena De; Hruška-Plocháň, Marián; Eninger, Timo; Schneider, Matthias M.; Barth, Michael; Tantardini, Elena; Rossi, Pierre De; Bacioglu, Mehtap; Langston, Rebekah G.; Kaganovich, Alice; Bengoa-Vergniory, Nora; González-Guerra, Andrés; Avar, Merve; Heinzer, Daniel; Reimann, Regina; Häsler, Lisa M.; Herling, Therese W.; Matharu, Naunehal S.; Landeck, Natalie; Luk, Kelvin C.; Melki, Ronald; Kahle, Philipp J.; Hornemann, Simone; Knowles, Tuomas P. J.; Cookson, Mark; Polymenidou, Magdalini; Jucker, Mathias; Aguzzi, Adriano

doi:10.15252/emmm.202114745

Cited by 48 publications

(36 citation statements)

References 79 publications

(92 reference statements)

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“…The results we report here using a novel microfluidic culture system to generate synaptically mature and fully unidirectional neural networks, in which the trans-neuronal transfer of aSyn aggregates can unequivocally be attributed to active anterograde axonal transport are in sharp contrast with reports of efficient trans-neuronal spreading in networks reconstructed with conventional non-filtering microfluidic devices [29,52]. Some of those results have since then been contradicted [9]. This difference in propagation efficiency might arise from direct exposure of axons originating from neurons in the distal chamber of the microfluidic device that invade the proximal chamber, where aSyn aggregates are added.…”

Section: Efficiency Of Trans-neuronal Seedingmentioning

confidence: 70%

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

Courte

Bousset

et al. 2021

Preprint

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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.

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Section: Efficiency Of Trans-neuronal Seedingmentioning

confidence: 70%

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

Courte

Bousset

et al. 2021

Preprint

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“…Several mechanisms have been implicated in the internalisation of α-synuclein fibrils, principally via endocytosis [ 66 ], non-conventional pathways such as tunnelling nanotubes [ 67 ] or heparan sulfate proteoglycan mediated micropinocytosis [ 68 ]. It is likely that some mechanisms are cell-type specific or peculiar to a subcellular domain [ 68 , 69 ] but the existence of a “receptor” for α-synuclein fibrils is controversial [ 70 ]. The strength of connections has emerged as a primary determinant of the spread of fibrillar forms of α-synuclein pathology when injected in different brain regions [ 71 – 73 ].…”

Section: α-Synuclein Pathology Progression Involves Cell Non-autonomo...mentioning

confidence: 99%

Initiation and progression of α-synuclein pathology in Parkinson’s disease

Tofaris

2022

Cell. Mol. Life Sci.

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Abstractα-Synuclein aggregation is a critical molecular process that underpins the pathogenesis of Parkinson’s disease. Aggregates may originate at synaptic terminals as a consequence of aberrant interactions between α-synuclein and lipids or evasion of proteostatic defences. The nature of these interactions is likely to influence the emergence of conformers or strains that in turn could explain the clinical heterogeneity of Parkinson’s disease and related α-synucleinopathies. For neurodegeneration to occur, α-synuclein assemblies need to exhibit seeding competency, i.e. ability to template further aggregation, and toxicity which is at least partly mediated by interference with synaptic vesicle or organelle homeostasis. Given the dynamic and reversible conformational plasticity of α-synuclein, it is possible that seeding competency and cellular toxicity are mediated by assemblies of different structure or size along this continuum. It is currently unknown which α-synuclein assemblies are the most relevant to the human condition but recent advances in the cryo-electron microscopic characterisation of brain-derived fibrils and their assessment in stem cell derived and animal models are likely to facilitate the development of precision therapies or biomarkers. This review summarises the main principles of α-synuclein aggregate initiation and propagation in model systems, and their relevance to clinical translation.

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“…While the trypan blue exclusion assay only results in a very rough, unprecise estimation of the number of living and dead cells, there are better options, for example, using an automated cell counting instrument such as the CASY ® Cell Counter & Analyzer (OLS OMNI Life Science, Bremen, Germany) [ 97 , 98 ]. This is a highly accurate and precise analyzer for cell lines, primary cells, and all stem cell types, including iPSC and ESC [ 98 , 99 , 100 ]. The CASY ® counter can measure the total cell number.…”

Section: Thawing Of Ipscmentioning

confidence: 99%

Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells

Uhrig

Ezquer

2022

Cells

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Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4–7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2–3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.

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

Cited by 48 publications

References 79 publications

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

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

Initiation and progression of α-synuclein pathology in Parkinson’s disease

Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells

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