Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults.

In chronic neurodegenerative syndromes, neurons progressively die through a generalized retraction pattern triggering retrograde axonal degeneration toward the cell bodies, which molecular mechanisms remain elusive. Recent observations suggest that direct activation of pro-apoptotic signaling in axons triggers local degenerative events associated with early alteration of axonal mitochondrial dynamics. This raises the question of the role of mitochondrial dynamics on both axonal vulnerability stress and their implication in the spreading of damages toward unchallenged parts of the neuron. Here, using microfluidic chambers, we assessed the consequences of interfering with OPA1 and DRP1 proteins on axonal degeneration induced by local application of rotenone. We found that pharmacological inhibition of mitochondrial fission prevented axonal damage induced by rotenone, in low glucose conditions. While alteration of mitochondrial dynamics per se did not lead to spontaneous axonal degeneration, it dramatically enhanced axonal vulnerability to rotenone, which had no effect in normal glucose conditions, and promoted retrograde spreading of axonal degeneration toward the cell body. Altogether, our results suggest a mitochondrial priming effect in axons as a key process of axonal degeneration. In the context of neurodegenerative diseases, like Parkinson's and Alzheimer's, mitochondria fragmentation could hasten neuronal death and initiate spatial dispersion of locally induced degenerative events.

ß-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network.

INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level. RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (µFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied ß-amyloid (Aß) peptides locally to the different cellular compartments. We observed that Aß application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aß deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aß peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aß stress at the network level we developed new µFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aß application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration. CONCLUSION: Thanks to µFD-based reconstructed neuronal networks we evaluated the distant effects of local Aß stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aß production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aß can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

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.

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.

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.

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 in vitro 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 in vivo 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 in vivo, confirm the power and biological relevance of this microfluidic architecture.