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Identification of molecular pathways underlying activity-dependent neuron-glia communication using in vitro microfluidic systems

Final Report Summary - CELESTIAL (Identification of molecular pathways underlying activity-dependent neuron-glia communication using in vitro microfluidic systems)

The function of the nervous system critically depends on continuous interactions among its different components, mainly neurons and glial cells. The latter include oligodendrocytes, astrocytes and microglia in the Central Nervous System (CNS; brain and spinal cord), and the Schwann cells (SCs) in the Peripheral Nervous System (PNS, consisting of all nerves residing outside of the CNS, and innervating peripheral tissues). Glial cells reside usually in close contact with neurons and support their activity by:
• producing myelin, which insulates nerve fibers and thus enables fast transport of electric signals along them (oligodendrocytes in the CNS, SCs in the PNS),
• providing trophic support (e.g. astrocytes in the CNS), and
• participating in immune responses (microglia in the CNS, and dedifferentiated SCs in the PNS).
Disruption of the physiological communication between neurons and glia has detrimental consequences leading to nervous system pathologies, such as those occurring during traumatic nerve injury, in hereditary and acquired neuropathies (like multiple sclerosis, amyotrophic lateral sclerosis, Charcot-Marie Tooth diseases (CMT) and diabetic neuropathies), and with ageing. Consequently, elucidation and comprehensive understanding of the molecular mechanisms mediating those interactions would allow us to appropriately manipulate them, in order to develop novel and more efficient therapeutic interventions to combat such diseases.

The goal of the respective Marie Curie funded project was the identification and further investigation of molecular pathways implicated in the communication between neurons and glia, under healthy and more interestingly, under neuropathic conditions, in which neuronal function is usually compromised. Considering that neurons consist of the cell body (soma, that includes the main cytoplasmic area and the nucleus), the neuronal processes (neurites, usually categorized in dendrites and axons) that stem from the soma and transfer messages in the form of electrical signals, and synapses (formations at the end of neurites that connect neurons to other neurons or to peripheral organs, such as muscles), it is evident that glial cells can interact with neurons at three different levels:
• at neuronal cell somata (e.g. satellite glial cells in sensory ganglia),
• at the level of neuritic processes (e.g. oligodendrocytes and SCs ensheathing or myelinating axons; we will refer to that type of inteaction here after as “extrasynaptic” communication), and
• synapses (including astrocytes in the CNS and perisynaptic SCs in the PNS).
Prompted by publications demonstrating that glial cells can sense and respond to neuronal activity at extrasynaptic regions, and by the lack of respective data and knowledge in that field, we particularly focused on the investigation of extrasynaptic neuron-glia interactions. To that end we used the PNS as our model system, which consists of only a single glial cell type (SCs), and therefore is simpler and more tractable than the CNS for the study of neuron-glia interactions.

To investigate the mechanisms underlying extrasynaptic neuron-glia communication, we implemented both in vitro approaches that were based on cultures of SCs in presence (or not) of neurons, and in vivo approaches, in which we analyzed data obtained after manipulations of living animals (Fig. 1).

In vitro approaches:
Our initial in vitro approach was based on the culture of dissociated rat or mouse embryonic Dorsal Root Ganglia (DRG, containing mainly sensory neurons and SCs) in dual-compartment microfluidic cell culture chambers (developed by Taylor et al., 2005) that allow for separation of neuronal somata from neurites and SCs; In particular, when dissociated DRG were seeded in one compartment of the device, only growing neurites and SCs could –due to their small size- migrate along the narrow microchannels connecting the two compartments, and thus occupy the 2nd compartment of the device. By implanting needle electrodes in the compartment harboring the neuronal somata (Fig. 1, left panel), we were able to invoke selective stimulation of DRG neurons, without directly stimulating SCs (as assessed through live cell imaging of Calcium transients developed in the cells). SCs only occasionally responded to the electrical stimulation, with a time delay, which indicated that those were secondary responses of SCs to signals transmitted by active neurites. The electrical stimulation platform that we developed could be used as an easy-to-construct tool for studies on neuronal signal transmission, and on cellular reactions to neuronal signals. In the frame of the funded project, we performed live cell time-lapse imaging of glial mitochondria behavior during neuronal stimulation.
An alternative in vitro approach was realized by screening specific neurotransmitters (molecules secreted by neurons and transmit their activity signals to other cells) for potential effects on the physiology of SCs (proliferation and ability to myelinate axons; Fig.1 middle left panel). Results of that screening pinpointed a specific neuropeptide transmitter as a potential mediator of neuron-to-SC communication. That combined with in vivo data of the host group, showing upregulation of the same neuropeptide in DRG neurons in mouse models of peripheral demyelinating neuropathies, prompted us to proceeded into further experiments (biochemical, histochemical and pharmacological assays) to elucidate its implication in axon-glia interactions. The totality of our observations indicated that the particular neurotransmitter transmits signals to SCs and affects particular aspects of their physiology, by binding to receptors, which are mainly expressed by SCs of neuropathic but not healthy animals. Consequently, manipulations of the particular signaling pathway could be tested for therapeutic interventions in people suffering from inherited peripheral neuropathies (like CMT).

In vivo approaches:
To complement our in vitro studies on extrasynaptic SC responses to neuronal activity, we also implemented in vivo experimental approaches Fig. 1, right panels). In particular, we checked for alterations of gene expression in SC-enriched tissues (endoneuria of sciatic nerves) from animals with enhanced or compromised sciatic nerve activity. To boost sciatic nerve activity, we maintained animals in enriched environments (cages containing running wheels), whereas to investigate SC responses to dysfunctional neurons, we used mouse models of peripheral neuropathy. This latter approach led to the identification of a promising candidate gene, which is upregulated in SCs during neuropathy, potentially contributing to the support of suffering neurons. Confirmation of that assumption requires further investigations. If proven correct, it may lead to the development of novel therapeutical approaches to combat peripheral neuropathies, based on enhancement of the glial cell ability to support neurons under stress.
Furthermore, we analyzed similar (but pre-existing in the host group) gene expression data on wild type and neuropathic animals, in order to identify SC molecules, which either sense neuronal activity or are affected by PNS dysfunctions. Results of the latter analysis were published in the form of a perspective article under the title “Neuronal activity in the hub of extrasynaptic Schwann cell-axon interaction” (Samara C et al., Front Cell Neurosci., 2013 Nov 25;7:228. doi: 10.3389/fncel.2013.00228. eCollection 2013).

In summary, realization of the Marie Curie-funded research project led to:
• Establishment of a simple electrical stimulation cell culture platform.
• Identification of a neuropeptide transmitter as a mediator of neuron-SC communication during peripheral demyelinating neuropathies, and investigation of its physiological role.
• Identification of SC genes regulated during increased or compromised neuronal activity.
Whereas the established platform can be used as an experimental tool for studies related to neuronal function and intercellular responses to it, characterization of NPY effects on SCs during peripheral demyelinating neuropathies, could assist to the development of more efficient therapeutic treatments. Similarly, further investigations on the physiological significance of SC genes and pathways that are regulated by altered neuronal activity, will shed light on the mechanisms of glia-to-neuron support. Enhancement of such protective glial pathways may provide potential novel therapeutic targets for the alleviation of PNS and/or CNS pathologies.
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