Jean-Michel Peyrin

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of axonal diodes. This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.

PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases

α-secretase–mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-β (Aβ) peptides, and α-cleavage of cellular prion protein (PrPC) prevents its conversion into misfolded, pathogenic prions (PrPSc). The mechanisms leading to decreased α-secretase activity in Alzheimers and prion disease remain unclear. Here, we find that tumor necrosis factor-α–converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrPSc or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide–dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aβ deposition and in the brains of individuals with Alzheimers disease. PDK1 induces phosphorylation and caveolin-1–mediated internalization of TACE. This dysregulation of TACE increases PrPSc and Aβ accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrPC and TNFR1, and attenuates PrPSc- and Aβ-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrPSc infection and in APP-transgenic mice reduces Alzheimers disease-like pathology and memory impairment.

Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip.

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing inxa0vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of inxa0vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments inxa0vivo, confirm the power and biological relevance of this microfluidic architecture.

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.—Magnifico, S., Saias, L., Deleglise, B., Duplus, E., Kilinc, D., Miquel, M.‐C., Viovy, J.‐L., Brugg, B., Peyrin, J.‐M. NAD+ acts on mitochondrial SirT3 to prevent axonal caspase activation and axonal degeneration. FASEB J. 27, 4712–4722 (2013). www.fasebj.org

Synapto-Protective Drugs Evaluation in Reconstructed Neuronal Network

Chronic neurodegenerative syndromes such as Alzheimer’s and Parkinson’s diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile “brain on chip” platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD+ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

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 (PrPC) into abnormally folded scrapie PrP (PrPSc). Prions can propagate both in neurons and astrocytes, yet neurotoxicity mechanisms remain unclear. Recently, PrPC was proposed to mediate neurotoxic signaling of β‐sheet‐rich PrP and non‐PrP conformers independently of conversion. To investigate the role of astrocytes and neuronal PrPC 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 PrPSc to neurons lacking PrPC (prion‐resistant), or expressing a PrPC convertible (sheep) or not (mouse, human). We show that interaction between neuronal PrPC and exogenous PrPSc was not sufficient to induce neuronal death but that efficient PrPC 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, PrPSc accumulation in neurons led to neuritic damages and cell death, both potentiated by glutamate and reactive oxygen species. Thus, conversion of neuronal PrPC rather than PrPC‐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.— Cronier, S., Carimalo, J., Schaeffer, B., Jaumain, E., Béringue, V., Miquel, M.‐C., Laude, H., Peyrin, J.‐M. Endogenous prion protein conversion is required for prion‐induced neuritic alterations and neuronal death. FASEB J. 26, 3854–3861 (2012). www.fasebj.org

A “dry and wet hybrid” lithography technique for multilevel replication templates: Applications to microfluidic neuron culture and two-phase global mixing

A broad range of microfluidic applications, ranging from cell culture to protein crystallization, requires multilevel devices with different heights and feature sizes (from micrometers to millimeters). While state-of-the-art direct-writing techniques have been developed for creating complex three-dimensional shapes, replication molding from a multilevel template is still the preferred method for fast prototyping of microfluidic devices in the laboratory. Here, we report on a dry and wet hybrid technique to fabricate multilevel replication molds by combining SU-8 lithography with a dry film resist (Ordyl). We show that the two lithography protocols are chemically compatible with each other. Finally, we demonstrate the hybrid technique in two different microfluidic applications: (1) a neuron culture device with compartmentalization of different elements of a neuron and (2) a two-phase (gas-liquid) global micromixer for fast mixing of a small amount of a viscous liquid into a larger volume of a less viscous liquid.

Trans-synaptic degeneration following acute axonal severing and beta-amyloid deposits in reconstructed neuronal networks

Background: The brain is a complex structure comprising many different neuronal populations making specific and polarized connections. The establishment of synaptic contacts are key events in brain development, and conversely, synaptic degeneration is an early and seminal process in various acute syndromes and neurodegenerative diseases eg, Alzheimer’s disease (AD). The molecular mechanisms involved in both synaptic and axonal degeneration remain to be elucidated. Importantly, since neurons are highly polarized cells, both chronic and acute brain injuries induce localized insults that targets subparts of neurons and/or neuronal networks. However, the “distal” consequences of such focal aggression on the neuronal integrity are still unknown. Moreover, recent studies reveal that neurodegenerative hallmarks in AD (beta amyloid accumulation and tau phosphorylation) progress both spatially and temporally following specific neuronal pathways. This calls for studies analyzing acute and chronic neurodegenerative processes in neuronal networks. Methods: In this study, we use a new micro-fluidic culture platform that enables the in vitro reconstruction of oriented neuronal networks with two or three different interconnected neuronal populations (cortex, striatum and hippocampus) each one being individually accessible. We used these reconstructed networks to model i) acute cortical axon severing or ii) the effect offocalized Aß deposition on a restricted area of the network to evaluate their consequences on distant axonal and synaptic integrity. Results: Our results show that both, cortical axon severing and cortical amyloid deposition leads to the propagation of an anterograde degenerative signal involving distant axo-dendriticsynaptic disconnection. Interestingly, this synaptic collapse is associated with an altered glutamatergic neurotransmission that culminates in trans-synaptic degenerative events in the connected post-synaptic neurons. Comparing the vulnerability of different post synaptic neurons, we test whether insults restricted to presynaptic cortical neurons leads to the propagation of degeneration along preferentialneur onal pathways.Conclusions: In conclusion our work aims at assessing the cellular and molecular mechanisms involved in the progression of degenerative events along neuro-anatomical pathways.