Neuronal cell death may be responsible for the pathogenesis of neuropathic pain. Glial cell line-derived neurotrophic factor (GDNF) protects sensory neurons after injury and offers a promising alternative for the management of intractable pain. However, continuous administration of trophic factors into the central nervous system is costly and difficult to maintain. Therefore, we evaluated the potential of intrathecal GDNF gene delivery for the treatment of neuropathic pain. Recombinant adenovirus encoding GDNF (Ad-GDNF) was characterized and shown to enhance viability of neuronal cultures. After intrathecal injection of Ad-GDNF, an elevated GDNF level was observed in spinal cord for four weeks. In rats with sciatic nerve axotomy,intrathecal injection of Ad-GDNF significantly ameliorated the duration of neuropathic pain. However, animals treated with Ad-GDNF developed hyperalgesia in the early stage of treatment. Immunofluorescence analysis indicated that intrathecal GDNF gene delivery prominently attenuated the neuronal loss due to nerve injury. Unexpectedly, varying degrees of hair loss was found in some rats receiving Ad-GDNF. Histological analysis revealed that hair loss resulted from severe degeneration of hair follicles in skin from Ad-GDNF-treated animals. In summary, the present study demonstrate the feasibility and limitations of GDNF gene delivery for the management of neuropathic pain.

Substantial evidence shows that activation of glial cells in the spinal cord may promote central sensitization and enhancement of pain. Descending facilitation from the rostroventromedial medulla (RVM) is also recognized as a critical component in the maintenance of chronic pain states, although the precise mechanisms driving this activity are unclear. Here, we investigated the possibility that glial activation in the RVM could promote descending facilitation from the RVM in states of enhanced pain. Peripheral inflammation was induced with carrageenan injected into the plantar aspect of the hindpaw of male Sprague-Dawley rats and behavioral responses to noxious thermal and light tactile stimuli were determined. Microinjection of the glial inhibitors minocycline or fluorocitrate, or of SB 203580, a p38 MAPK inhibitor, produced a significant and time-related reversal of behavioral hypersensitivity resulting from hindpaw inflammation. Moreover, carrageenan-induced inflammation appeared to produce an increase in immunolabeling for activated microglia and astrocytes in the RVM, as well as for phosphorylated p38 MAPK; the latter was localized to both microglia and neurons of the RVM. Microinjection of the glial inhibitors into the RVM appeared to diminish immunofluorescent labeling for activated RVM microglia and astrocytes. Carrageenan-induced inflammation also increased RVM protein levels of Iba1 and GFAP and administration of minocycline or fluorocitrate into the RVM attenuated this effect. To examine a possible mechanism of glial activation, α, β-methylene-ATP was microinjected into the RVM, inducing thermal hyperalgesia, and pre-treatment with the P2X antagonists, PPADS and TNP-ATP, delayed the initiation of ATP-induced hyperalgesia. Post-treatment with the antagonists had no effect on established ATP-induced or carrageenan-induced hypersensitivity. The activation of P2X receptors initiates a signaling cascade leading to the production and release of nociceptive mediators, including BDNF. The RVM microinjection of an anti- BDNF antibody reversed carrageenan-induced thermal hyperalgesia. A model of morphine-induced paradoxical pain was also used to examine the role of glial activation in the RVM. Sustained morphine administration induced tactile allodynia and RVM microinjection of minocycline, but not fluorocitrate, attenuated the behavioral hypersensitivity. Sustained morphine also induced morphological changes in microglia of the RVM, suggesting microglial activation. A third model of enhanced pain used to study medullary glial activation was the spinal nerve ligation (SNL) model of neuropathic pain. The SNL injury induced astrocyte activation within the RVM and microinjection of the astrocyte inhibitor fluorocitrate attenuated the nerve injury-induced tactile allodynia. Minocycline administered to the RVM did not attenuate the behavioral hypersensitivity, suggesting a role for astrocytes, not microglia, in nerve injury-induced enhanced pain. The data show that inflammatory, opioid-induced and neuropathic pain is associated with glial activation in the RVM which likely participates in driving descending pain facilitation via glial-neuronal communication. These findings reveal a novel site of glial modulation of pain.

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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.

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yes / Abstract Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial: GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.

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.

Propionic acid (PPA) is a short chain fatty acid that is produced by the human gut microbiome. Propionate, butyrate and acetates are the end products of the fermentation of the complex carbohydrates by human gut friendly microbiome and are being used as sources of energy in our body. PPA is used as a food preservative against molds in various daily products and has been implicated in the pathogenesis of autism. In a recent study we showed that PPA in human neuronal stem cell (NSC) culture increases the astrocyte population and decreases the neuronal number and increases the inflammatory cytokines. In this study, we investigated the potential effects of a propionic acid-derivative, Ibuprofen, a member of the non-steroidal anti-inflammatory drugs (NSAIDs) on neural stem cells proliferation and differentiation in vitro. Ibuprofen is an over counter drug that is used for alleviating pain, headache, and fever. To examine the effect of ibuprofen on developing brain we used human NSC in vitro, exposed them to increasing concentrations of ibuprofen, and investigated neural proliferation and differentiation. Here we show that NSAIDs, not at therapeutic, but very high concentrations cause an imbalance in NSC differentiation towards glial cells, therefore causing astrogliosis seen in some cases of autism spectrum disorder (ASD). Furthermore, upon removal of Ibuprofen, inflammatory cytokines; TNF-alpha, IL-6 and IL-10, significantly increase (p < 0.05) in cells previously exposed to NSAIDs compared to control. Therefore, we are speculating that if such drugs were to be taken in the circumstances of a developing child during the early trimesters of pregnancy, this could result in increased glial:neuron ratio leading to lifelong impediments. Based on the current study our recommendation is to avoid high doses of propionic acid derivatives such as ibuprofen during pregnancy.

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