Synapses do not facilitate prion-like transfer of alpha-synuclein: a quantitative study in reconstructed unidirectional neural networks.

Alpha-synuclein (aSyn) aggregation spreads between cells and underlies the progression of neuronal lesions in the brain of patients with synucleinopathies such as Parkinson's diseases. The mechanisms of cell-to-cell propagation of aggregates, which dictate how aggregation progresses at the network level, remain poorly understood. Notably, while prion and prion-like spreading is often simplistically envisioned as a "domino-like" spreading scenario where connected neurons sequentially propagate protein aggregation to each other, the reality is likely to be more nuanced. Here, we demonstrate that the spreading of preformed aSyn aggregates is a limited process that occurs through molecular sieving of large aSyn seeds. We further show that this process is not facilitated by synaptic connections. This was achieved through the development and characterization of a new microfluidic platform that allows reconstruction of binary fully oriented neuronal networks in vitro with no unwanted backward connections, and through the careful quantification of fluorescent aSyn aggregates spreading between neurons. While this allowed us for the first time to extract quantitative data of protein seeds dissemination along neural pathways, our data suggest that prion-like dissemination of proteinopathic seeding aggregates occurs very progressively and leads to highly compartmentalized pattern of protein seeding in neural networks.

Controlling the force and the position of acoustic traps with a tunable acoustofluidic chip: Application to spheroid manipulations.

A multi-node acoustofluidic chip working on a broadband spectrum and beyond the resonance is designed for cell manipulations. A simple one-dimensional (1D) multi-layer model is used to describe the stationary standing waves generated inside a cavity. The transmissions and reflections of the acoustic wave through the different layers and interfaces lead to the creation of pressure nodes away from the resonance condition. A transparent cavity and a broadband ultrasonic transducer allow the measurement of the acoustic energy over a wide frequency range using particle image velocimetry measurements and the relation between acoustic energy and the particles velocity. The automation of the setup allows the acquisition over a large spectrum with a high frequency definition. The results show a wide continuous operating range for the acoustofluidic chip, which compares well with the 1D model. The variation of the acoustic radiation force when varying the frequency can be compensated to ensure a constant amplitude for the ARF. This approach is finally applied to mesenchymal stem cell (MCS) spheroids cultured in acoustic levitation. The MSC spheroids can be moved and merged just by varying the acoustic frequency. This approach opens the path to various acoustic manipulations and to complex 3D tissue engineering in acoustic levitation.

Cellular mechanisms responsible for cell-to-cell spreading of prions.

Prions are infectious agents that cause fatal neurodegenerative diseases. Current evidence indicates that they are essentially composed of an abnormally folded protein (PrPSc). These abnormal aggregated PrPSc species multiply in infected cells by recruiting and converting the host PrPC protein into new PrPSc. How prions move from cell to cell and progressively spread across the infected tissue is of crucial importance and may provide experimental opportunity to delay the progression of the disease. In infected cells, different mechanisms have been identified, including release of infectious extracellular vesicles and intercellular transfer of PrPSc-containing organelles through tunneling nanotubes. These findings should allow manipulation of the intracellular trafficking events targeting PrPSc in these particular subcellular compartments to experimentally address the relative contribution of these mechanisms to in vivo prion pathogenesis. In addition, such information may prompt further experimental strategies to decipher the causal roles of protein misfolding and aggregation in other human neurodegenerative diseases.

Correction to: Cellular mechanisms responsible for cell-to-cell spreading of prions.

In the original publication, part of acknowledgement text was missing. The complete acknowledgement section should read as follows.

Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation.

In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.

Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential.

To favor their replication, viruses express proteins that target diverse mammalian cellular pathways. Due to the limited size of many viral genomes, such proteins are endowed with multiple functions, which require targeting to different subcellular compartments. One salient example is the X protein of Borna disease virus, which is expressed both at the mitochondria and in the nucleus. Moreover, we recently demonstrated that mitochondrial X protein is neuroprotective. In this study, we sought to examine the mechanisms whereby the X protein transits between subcellular compartments and to define its localization signals, to enhance its mitochondrial accumulation and thus, potentially, its neuroprotective activity. We transfected plasmids expressing fusion proteins bearing different domains of X fused to enhanced green fluorescent protein (eGFP) and compared their subcellular localization to that of eGFP. We observed that the 5-16 domain of X was responsible for both nuclear export and mitochondrial targeting and identified critical residues for mitochondrial localization. We next took advantage of these findings and constructed mutant X proteins that were targeted only to the mitochondria. Such mutants exhibited enhanced neuroprotective properties in compartmented cultures of neurons grown in microfluidic chambers, thereby confirming the parallel between mitochondrial accumulation of the X protein and its neuroprotective potential.-Ferré C. A., Davezac, N., Thouard, A., Peyrin, J. M., Belenguer, P., Miquel, M.-C., Gonzalez-Dunia, D., Szelechowski, M. Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential.

NAD+ acts on mitochondrial SirT3 to prevent axonal caspase activation and axonal degeneration.

In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.

Influence of microgravity on spontaneous calcium activity of primary hippocampal neurons grown in microfluidic chips.

The influence of variations of gravity, either hypergravity or microgravity, on the brain of astronauts is a major concern for long journeys in space, to the Moon or to Mars, or simply long-duration missions on the ISS (International Space Station). Monitoring brain activity, before and after ISS missions already demonstrated important and long term effects on the brains of astronauts. In this study, we focus on the influence of gravity variations at the cellular level on primary hippocampal neurons. A dedicated setup has been designed and built to perform live calcium imaging during parabolic flights. During a CNES (Centre National d'Etudes Spatiales) parabolic flight campaign, we were able to observe and monitor the calcium activity of 2D networks of neurons inside microfluidic devices during gravity changes over different parabolas. Our preliminary results clearly indicate a modification of the calcium activity associated to variations of gravity.

Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death.

Prions cause fatal neurodegenerative conditions and result from the conversion of host-encoded cellular prion protein (PrP(C)) into abnormally folded scrapie PrP (PrP(Sc)). Prions can propagate both in neurons and astrocytes, yet neurotoxicity mechanisms remain unclear. Recently, PrP(C) was proposed to mediate neurotoxic signaling of ß-sheet-rich PrP and non-PrP conformers independently of conversion. To investigate the role of astrocytes and neuronal PrP(C) in prion-induced neurodegeneration, we set up neuron and astrocyte primary cocultures derived from PrP transgenic mice. In this system, prion-infected astrocytes delivered ovine PrP(Sc) to neurons lacking PrP(C) (prion-resistant), or expressing a PrP(C) convertible (sheep) or not (mouse, human). We show that interaction between neuronal PrP(C) and exogenous PrP(Sc) was not sufficient to induce neuronal death but that efficient PrP(C) conversion was required for prion-associated neurotoxicity. Prion-infected astrocytes markedly accelerated neurodegeneration in homologous cocultures compared to infected single neuronal cultures, despite no detectable neurotoxin release. Finally, PrP(Sc) accumulation in neurons led to neuritic damages and cell death, both potentiated by glutamate and reactive oxygen species. Thus, conversion of neuronal PrP(C) rather than PrP(C)-mediated neurotoxic signaling appears as the main culprit in prion-induced neurodegeneration. We suggest that active prion replication in neurons sensitizes them to environmental stress regulated by neighboring cells, including astrocytes.

Propagation of Distinct α-Synuclein Strains Within Human Reconstructed Neuronal Network and Associated Neuronal Dysfunctions.

Aggregated alpha-synuclein (α-Syn) in neurons is a hallmark of Parkinson's disease (PD) and other synucleinopathies. Recent advances (1) in the production and purification of synthetic assemblies of α-Syn, (2) in the design and production of microfluidic devices allowing the construction of oriented and compartmentalized neuronal network on a chip, and (3) in the differentiation of human pluripotent stem cells (hPSCs) into specific neuronal subtypes now allow the study of cellular and molecular determinants of the prion-like properties of α-Syn in vitro. Here, we described the methods we used to reconstruct a cortico-cortical human neuronal network in microfluidic devices and how to take advantage of this cellular model to characterize (1) the prion-like properties of different α-Syn strains and (2) the neuronal dysfunctions and the alterations associated with the exposure to α-Syn strains or the nucleation of endogenous α-Syn protein in vitro.

Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye.

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE). Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip. We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10-4% BAC applications on the corneal epithelial cells and nerve terminals. In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

Glioblastoma cell motility depends on enhanced oxidative stress coupled with mobilization of a sulfurtransferase.

Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients' glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients' tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients' tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients' tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.

Neuron dysfunction is induced by prion protein with an insertional mutation via a Fyn kinase and reversed by sirtuin activation in Caenorhabditis elegans.

Although prion propagation is well understood, the signaling pathways activated by neurotoxic forms of prion protein (PrP) and those able to mitigate pathological phenotypes remain largely unknown. Here, we identify src-2, a Fyn-related kinase, as a gene required for human PrP with an insertional mutation to be neurotoxic in Caenorhabditis elegans, and the longevity modulator sir-2.1/SIRT1, a sirtuin deacetylase, as a modifier of prion neurotoxicity. The expression of octarepeat-expanded PrP in C. elegans mechanosensory neurons led to a progressive loss of response to touch without causing cell death, whereas wild-type PrP expression did not alter behavior. Transgenic PrP molecules showed expression at the plasma membrane, with protein clusters, partial resistance to proteinase K (PK), and protein insolubility detected for mutant PrP. Loss of function (LOF) of src-2 greatly reduced mutant PrP neurotoxicity without reducing PK-resistant PrP levels. Increased sir-2.1 dosage reversed mutant PrP neurotoxicity, whereas sir-2.1 LOF showed aggravation, and these effects did not alter PK-resistant PrP. Resveratrol, a polyphenol known to act through sirtuins for neuroprotection, reversed mutant PrP neurotoxicity in a sir-2.1-dependent manner. Additionally, resveratrol reversed cell death caused by mutant PrP in cerebellar granule neurons from prnp-null mice. These results suggest that Fyn mediates mutant PrP neurotoxicity in addition to its role in cellular PrP signaling and reveal that sirtuin activation mitigates these neurotoxic effects. Sirtuin activators may thus have therapeutic potential to protect from prion neurotoxicity and its effects on intracellular signaling.

HSPA9/Mortalin mediates axo-protection and modulates mitochondrial dynamics in neurons.

Mortalin is a mitochondrial chaperone protein involved in quality control of proteins imported into the mitochondrial matrix, which was recently described as a sensor of neuronal stress. Mortalin is down-regulated in neurons of patients with neurodegenerative diseases and levels of Mortalin expression are correlated with neuronal fate in animal models of Alzheimer's disease or cerebral ischemia. To date, however, the links between Mortalin levels, its impact on mitochondrial function and morphology and, ultimately, the initiation of neurodegeneration, are still unclear. In the present study, we used lentiviral vectors to over- or under-express Mortalin in primary neuronal cultures. We first analyzed the early events of neurodegeneration in the axonal compartment, using oriented neuronal cultures grown in microfluidic-based devices. We observed that Mortalin down-regulation induced mitochondrial fragmentation and axonal damage, whereas its over-expression conferred protection against axonal degeneration mediated by rotenone exposure. We next demonstrated that Mortalin levels modulated mitochondrial morphology by acting on DRP1 phosphorylation, thereby further illustrating the crucial implication of mitochondrial dynamics on neuronal fate in degenerative diseases.

The expression level of alpha-synuclein in different neuronal populations is the primary determinant of its prion-like seeding.

Alpha-synuclein (aSyn)-rich aggregates propagate in neuronal networks and compromise cellular homeostasis leading to synucleinopathies such as Parkinson's disease. Aggregated aSyn spread follows a conserved spatio-temporal pattern that is not solely dependent on connectivity. Hence, the differential tropism of aSyn-rich aggregates to distinct brain regions, or their ability to amplify within those regions, must contribute to this process. To better understand what underlies aSyn-rich aggregates distribution within the brain, we generated primary neuronal cultures from various brain regions of wild-type mice and mice expressing a reduced level of aSyn, and exposed them to fibrillar aSyn. We then assessed exogenous fibrillar aSyn uptake, endogenous aSyn seeding, and endogenous aSyn physiological expression levels. Despite a similar uptake of exogenous fibrils by neuronal cells from distinct brain regions, the seeded aggregation of endogenous aSyn differed greatly from one neuronal population to another. The different susceptibility of neuronal populations was linked to their aSyn expression level. Our data establish that endogenous aSyn expression level plays a key role in fibrillar aSyn prion-like seeding, supporting that endogenous aSyn expression level participates in selective regional brain vulnerability.

Propagation of α-Synuclein Strains within Human Reconstructed Neuronal Network.

Reappraisal of neuropathological studies suggests that pathological hallmarks of Alzheimer's disease and Parkinson's disease (PD) spread progressively along predictable neuronal pathways in the human brain through unknown mechanisms. Although there is much evidence supporting the prion-like propagation and amplification of α-synuclein (α-Syn) in vitro and in rodent models, whether this scenario occurs in the human brain remains to be substantiated. Here we reconstructed in microfluidic devices corticocortical neuronal networks using human induced pluripotent stem cells derived from a healthy donor. We provide unique experimental evidence that different strains of human α-Syn disseminate in "wild-type" human neuronal networks in a prion-like manner. We show that two distinct α-Syn strains we named fibrils and ribbons are transported, traffic between neurons, and trigger to different extents, in a dose- and structure-dependent manner, the progressive accumulation of PD-like pathological hallmarks. We further demonstrate that seeded aggregation of endogenous soluble α-Syn affects synaptic integrity and mitochondria morphology.

Controlled proteolysis of normal and pathological prion protein in a microfluidic chip.

A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor's performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.

Prion strain- and species-dependent effects of antiprion molecules in primary neuronal cultures.

Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents.

Magnetic proteinase K reactor as a new tool for reproducible limited protein digestion.

As an aid to differentiating between the prion proteins Prp(c) and PrP(Sc), the preparation and use of immobilized Proteinase K (PK) is described. An accumulation of PrP(Sc) in the central nervous system is the one of the causes of neurodegenerative disease. Current routine diagnosis is based on the postmortem detection of the distinct neuropathological lesion profiles of CNS and by the presence of the PK-resistant core of the prion protein isolated from brain lysates. An assay with PK immobilized to magnetic -COOH micro- and nanoparticles can offer a convenient as well as economic method. The individual immobilization steps were verified by measuring the zeta potential of the particles. The stability of the newly developed PK magnetic reactor, observed during kinetics measurements, was highly satisfactory. The calculated values of the apparent Michaelis constant (4.25 mM for native enzyme and 1.28 mM for immobilized enzyme) were determined from Lineweaver-Burk plots. Human growth hormone was digested using the newly prepared magnetic PK reactor and MALDI-TOF-MS analysis of the digests showed satisfactory efficiency. Controlled digestion of PrP(c) from the Mov mouse cell line was demonstrated with Western blot detection.

Reconstruction of directed neuronal networks in a microfluidic device with asymmetric microchannels.

Microfluidic devices for controlling neuronal connectivity in vitro are extremely useful tools for deciphering pathological and physiological processes occurring in neuronal networks. These devices allow the connection between different neuronal populations located into separate culture chambers through axon-selective microchannels. In order to implement specific features of brain connectivity such as directionality, it is necessary to control axonal growth orientation in these devices. Among the various strategies proposed to achieve this goal, one of the most promising and easily reproducible is the use of asymmetric microchannels. We present here a general protocol and several guidelines for the design, production and testing of a new paradigm of asymmetric microchannels geometries based on a "return to sender" strategy. In this method, axons are either allowed to travel between the emitting and receiving chambers within straight microchannels (forward direction), or are rerouted toward their initial location through curved microchannels (reverse direction). We introduce variations of these "arches" microchannels and evaluate their respective axonal filtering capacities. Importantly, one of these variants presents an almost complete filtration of axonal growth in the non-permissive direction while allowing robust axonal invasion in the other one, with a selectivity ratio as high as 99.7%.