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Satellite glial cells (SGC) are laminar cells that wrap completely around the sensory neuron and are responsible for buffering extracellular K+ after neuronal excitation. A decrease in the potassium buffering capacity of SGC has been associated with neuropathic pain (NP) behavior and apoptosis. This dissertation investigated the role of the potassium buffering capacity and apoptosis of trigeminal satellite glial cells (SGC) in the maintenance and development of orofacial NP in rats using in vivo and in vitro methodologies. In vivo endpoints were evaluated after performing chronic constriction injury (CCI) of the infraorbital nerve (IoN). NP signs and behavior were evaluated at 5, 10, 20 40 and 80 hours after injury. We evaluated the potassium buffering capacity of SGC by measuring the intracellular potassium concentration and protein levels and gene expression of the Kir4.1 and the SK3 potassium channels and gap junction protein connexin 43 (Cx43). We evaluated apoptosis endpoints including protein levels and gene expression of apoptotic related proteins bcl-2, caspase 9, caspase 3 and p53. Results indicate that NP signs developed as early as 5 hours after injury. After PNI, SGC responded by increasing their intracellular potassium concentration and by increasing protein levels of Kir4.1, SK3 and Cx43. Nonetheless, this increase in protein levels was not accompanied by an increase in gene expression. Apoptosis results revealed that SGC decreased protein levels and gene expression of anti-apoptotic protein Bcl-2. Using in vitro methodologies, we developed primary trigeminal SGC cultures and evaluated how a decrease in the intracellular potassium concentration modulates apoptosis induced by the mitochondrial and death receptor pathways. SGC depleted of potassium after hypoosmotic shock showed a significant increase in early apoptosis after incubation with mitochondrial pathway apoptotic inducer staurosporine when compared to SGC with normal intracellular concentration. This research has revealed that SGC respond early to PNI by increasing their potassium buffering capacity. We also determined that the mitochondrial apoptotic pathway might be involved in the trigeminal SGC response to PNI. From our in vitro experiments we have revealed that potassium is an important modulator of apoptosis induced by the mitochondrial pathway in cultured trigeminal SGC. / Ph. D.

Die Arbeit befasst sich mit dem Anschwellen von Neuronen und Gliazellen der Netzhaut, was einen wichtigen pathogenetischen Faktor des Netzhautödems darstellt. Osteopontin ist ein neuroprotektiver Faktor, der durch GDNF-Stimulation (glial cell line-derived neurotrophic factor) aus Müllerzellen ausgeschüttet wird. Die durch Osteopontin vermittelte Inhibition der osmotischen Zellschwellung von Müllerzellen der Ratte in Anwesenheit von Bariumionen oder H2O2 wird beschrieben und es wird dargestellt, dass Osteopontin keinen Einfluss auf die osmotische Zellschwellung der Bipolarzellen hat. Der für Müllerzellen beschriebene Effekt war dosisabhängig mit einer mittleren effektiven Konzentration von ca. 0,6 ng/ml. Durch den Einsatz pharmakologischer Rezeptor- oder Enzymblocker bzw. Antikörper werden die Schritte der Osteopontinwirkung identifiziert. Osteopontin induziert die Freisetzung von VEGF, Glutamat, ATP und Adenosin aus Müllerzellen. Die Osteopontinwirkung wurde verhindert durch die Blockade von spannungsabhängigen Natriumkanälen, T-Typ Calciumkanälen, Kalium- und Chloridkanälen. Der Effekt ist außerdem abhängig von einem intrazellulären Calciumsignal, der Aktivierung der Phospholipase C und der Proteinkinase C und der vesikulären Exozytose von Glutamat. Die Arbeit kommt zu dem Schluss, dass der neuroprotektive Effekt von Osteopontin teilweise durch das Verhindern eines Anschwellens der Müllerzellen und durch die Induktion einer Freisetzung von VEGF und Adenosin vermittelt wird.

The mouse nervous system undergoes a vast remodelling of synaptic connections postnatally, resulting in a reduced number of innervating axons to target cells within the first few weeks of life. This extensive loss of connections is known as synapse elimination and it plays a critical role in sculpting and refining neural connectivity throughout the nervous system, resulting in a finely tuned and well-synchronised network of innervation. This process has been well characterised at the mouse neuromuscular junction (NMJ), where synapse elimination takes place postnatally in all skeletal muscles. It has been well studied for the reasons that it is easily accessible for live imaging and post-mortem experimental analysis. Studies utilising this synapse to uncover regulators of synapse elimination have mainly focused on the importance of glial cell lysosomal activity, nerve conduction and target-derived growth factor supply. It is clear that non-axonal cell types play key roles in the success of developmental axon retraction at the NMJ, however the role of glial cells in the regulation of this process has not been fully explored, as lysosomal activity is thought of as a consequence of axon pruning rather than a molecular driver. Previous studies have shown that signals emanating from myelinating glial cells can modulate neurofilament composition and transport within the underlying axons. We know that these changes in neurofilament composition and transport are underway during developmental synapse elimination at the NMJ, so it seems logical to predict that myelinating glial cells may play a role in the regulation of axonal pruning. Myelinating glial cells are found along the entire length of lower motor neurons and form physical interactions with the underlying axons at regions known as paranodes. At the paranode, Neurofascin155 (Nfasc155: expressed by the myelinating glial cell) interacts with a Caspr/contactin complex (expressed by the axon). This site has been proposed as a likely site for axon-glial signalling due to the close apposition of the cell membranes. The main focus of this PhD project was to study the potential role of myelinating glial cells in the success of synapse elimination at the NMJ, using a mouse model of paranodal disruption (Nfasc155-/-). Chapters 3 and 4 show the results of this work. This work has revealed a novel role for glia in the modulation of synapse elimination at the mouse neuromuscular junction, mediated by Nfasc155 in the myelinating Schwann cell. Synapse elimination was profoundly delayed in Nfasc155-/- mice and was found to be associated with a non-canonical role for Nfasc155, as synapse elimination occurred normally in mice lacking the axonal paranodal protein Caspr. Loss of Nfasc155 was sufficient to disrupt axonal proteins contributing to cytoskeletal organisation and trafficking pathways in peripheral nerve of Nfasc155-/- mice and lower levels of neurofilament light (NF-L) protein in maturing motor axon terminals. Synapse elimination was delayed in mice lacking NF-L, suggesting that Nfasc155 influences neuronal remodelling, at least in part, by modifying cytoskeletal dynamics in motor neurons. This work provides the first clear evidence for myelinating Schwann cells acting as drivers of synapse elimination, with Nfasc155 playing a critical role in glial cell-mediated postnatal sculpting of neuronal connectivity in the peripheral nervous system. A small section of the results within this thesis are devoted to the study of axon-glial interactions in a mouse model of childhood motor neuron disease, otherwise known as spinal muscular atrophy (SMA). In SMA, there are reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein. The NMJ is a particularly vulnerable target in SMA, manifesting as a breakdown of neuromuscular connectivity and progressive motor impairment. Recent studies have begun to shed light on the role of non-neuronal cell types in the onset and progression of the disease, presenting SMA as a multi-system disease rather than a purely neuronal disorder. Recent evidence has highlighted that myelinating glial cells are significantly affected in a mouse model of SMA, manifesting as an impaired ability to produce key myelin proteins, resulting in deficient myelination. The final results chapter of this thesis (Chapter 5) is focussed on further exploring the effects that loss of SMN has in Schwann cells including their interactions with underlying axons. This work reveals a disruption to axon-glial interaction, shown by a delay in the development of paranodes, supporting the idea that non-neuronal cell types are also affected in SMA. The results within this thesis reveal a novel role for a glial cell protein, Nfasc155, in the modulation of synapse elimination at the NMJ. Mechanistic insight in to Nfasc155’s role in this process is also uncovered and likely involves axonal cytoskeletal transport systems and the filamentous protein NF-L, which have not previously been implicated in the process of synapse elimination. This work highlights an important role for axon-glial interactions during normal postnatal development of the mouse NMJ. This work also highlights a role for axon-glial interactions in disease states of the NMJ. Using a mouse model of SMA, axon-glial interaction was assessed with the finding of a delay in paranodal maturation due to loss of SMN.

Primary cilia are ubiquitous sensory organelles found on most cell types including cells of the dorsal root ganglia (DRG). The DRG are groups of peripheral neurons that relay sensory information from the periphery to the CNS. Other cell types in the DRG include a type of glial cell, the satellite glial cells (SGCs). The SGCs surround the DRG neurons and, with the neurons, form functional sensory units. Currently are no reports describing the numbers of DRG cells that have cilia. We found that 26% of the SGCs had primary cilia. The incidence of cilia on neurons varied with neuron size, a property that roughly correlates with physiological characteristics. We found that 29% of the small, 16% of the medium and 5% of the large neurons had primary cilia. Primary cilia have been shown to have a role in cell proliferation in a variety of cell types. In some of the cells the cilia mediate the proliferative effects of Sonic hedgehog (Shh). In the CNS, Shh signaling through primary cilia affects proliferation during development as well as following injury, but no studies have looked at this function in the PNS. The SGCs and neurons of the DRG undergo complex changes following peripheral nerve injury such as axotomy. One marked change seen after axotomy is SGC proliferation and at later stages, neuronal death. We found that following axotomy there is a significant increase in the percentage of SGCs with primary cilia. We also found a significant increase in the percentage of medium-sized neurons with primary cilia. In other experiments we tested the idea that Shh plays a role in SGC proliferation. When Shh signaling was blocked following axotomy we found decreased proliferation of SGCs. This is the first report of a change in the percentage of cells with cilia following injury in the PNS, and the first report of a role for Shh in SGC proliferation following axotomy.

Satellite glial cells (SGCs) surround the cell bodies of neurons of the peripheral nervous system, including those of the sensory ganglia. Their close apposition to the neuronal soma allows for bi-directional communication between neurons and SGCs, which are thought to regulate neuronal activity. After nerve injury, SGCs in the dorsal root ganglia contribute to neuropathic pain. Although the mechanisms are not fully understood, SGCs show increased coupling via gap junctions, and communicate with the neuron via bi-directional purinergic signaling after nerve injury. The increased coupling between SGCs and neurons may have implications for chronic pain following peripheral nerve injury. In vivo studies suggest that injury through the administration of capsaicin to the sensory nerve endings causes SGCs to be activated and proliferate. We have shown that capsaicin treatment in an in vitro co-culture of sensory neurons and SGCs increased the expression of the proliferation marker, Ki-67 in the glia. Here, we examine whether purinergic signaling plays a role in the promotion of SGC proliferation.

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