The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. Historically, SMA has been characterised as a disease primarily affecting lower motor neurons. However, recent breakthroughs have revealed defects in other non-neuronal cells and tissues. In vivo analysis of peripheral nerve showed defects in Schwann cells, manifesting as abnormal myelination and delayed maturation of axo-glia interactions. The experiments in this thesis were designed to build on these observations and examine whether Schwann cell defects are intrinsic and occur as a primary result of low levels of SMN in that cell type, or rather represent a secondary consequence of pathology in neighbouring motor neurons. I initially developed a protocol to allow isolation of high-yields of purified, myelination-competent Schwann cells from ‘Taiwanese’ SMA mice. SMA-derived Schwann cells had significantly reduced SMN levels and failed to respond normally to differentiation cues. Increasing SMN levels restored myelin protein expression in Schwann cells from SMA mice. Perturbations in expression of key myelin proteins were likely due to failure of protein translation and/or stability rather than transcriptional defects. Co-cultures of healthy neurons with SMA Schwann cells revealed a significant reduction in myelination compared to cultures where wild-type Schwann cells were used. The presence of SMA Schwann cells also disrupted neurite stability. Perturbations in the expression of key extracellular matrix proteins, such as laminin α2, in SMA-derived Schwann cells suggests that Schwann cells were influencing neurite stability by modulating the composition of the extracellular matrix. Previous studies have demonstrated that low levels of SMN lead to disruption of ubiquitin homeostasis and decreased expression of ubiquitin-like modifier activating enzyme (UBA1) in the neuromuscular system, driving neuromuscular pathology via a beta-catenin dependent pathway. Label-free proteomics analysis of SMA and control Schwann cells identified 195 proteins with modified expression profiles. Bioinformatic analysis of these proteins using Ingenuity Pathway Analysis (IPA) software confirmed that major disruption of protein ubiquitination pathways was also present in Schwann cells from SMA mice. Immunolabeling and proteomics data both revealed that UBA1 levels were significantly reduced in SMA-derived Schwann cells. However, loss of UBA1 in Schwann cells did not lead to downstream modifications in beta-catenin pathways. Pharmacological inhibition of UBA1 in healthy Schwann cells was sufficient to induce defects in myelin protein expression, suggesting that UBA1 defects contribute directly to Schwann cell disruption in SMA. I conclude that low levels of SMN induce intrinsic defects in Schwann cells, mediated at least in part through disruption to ubiquitination pathways.

Oligodendrocytes in the central nervous system (CNS) are responsible for wrapping axons with myelin in order to insulate them and allow for a faster conduction of the nervous impulse. The axonal signals that determine whether an axon is myelinated, and what regulates the number of wraps is still not fully understood. The importance of signals that initiate myelination is significant because they may point to novel therapies for Multiple Sclerosis, where remyelination prevents the axon degeneration that is thought to underlie chronic disease. Neuregulin 1 (Nrg1) has been identified as a key axonal signaling molecule that regulates myelin thickness and glial fate in the peripheral nervous system (PNS). In the PNS, neuregulin I type III is a necessary and sufficient signal that regulates axoglial interaction. The role of neuregulin in the CNS remains unclear and controversial. Integrins, the major family of extracellular matrix (ECM) receptors are involved in the regulation of many fundamental cellular functions. Interaction with a wide range of receptors including growth factor receptors is well described. Our lab showed that α6β1 integrin regulates oligodendrocyte survival signaling by amplification of neuregulin activity. We have found that mice expressing a dominant-negative β1 integrin (that reduces β1 integrin signaling independently of ligand binding) in myelinating oligodendrocytes require a larger axon diameter to initiate myelination. These results suggest that there are other signals in the axon that also contribute to initiation of myelination. We therefore hypothesized that β1 integrin and neuregulin act in concert and play a role in axoglial interactions that sense the axon size and initiate myelination. By crossing the dominant negative β1 integrin mice with heterozygous mice for neuregulin 1 and analyzing myelination, we have found that neuregulin does not enhance the phenotype previously described. This result together with previous reports that mice lacking NRG1, ErbB3 or ErbB4 (the neuregulin receptors expressed on oligodendrocytes) have normal CNS myelin sheaths demonstrates that neuregulin 1 is not required for CNS myelination. Interestingly, neuregulin 1 has been associated as a susceptibility gene in schizophrenia, a disease independently associated with myelin abnormalities (Davis et al., 2003; Hakak et al., 2001). Post-mortem brains of schizophrenic patients showed an increased level of neuregulin 1 type IV. We have analysed mice overexpressing neuregulin 1 type IV (Nrg1 type IV) and show that increased levels of neuregulin 1 type IV does not alter the brain morphology or myelin pattern and integrity. A possible explanation is that since neuregulin 1 type IV is human specific, the mice lack species-specific receptors or other neuregulins have compensatory equilibrium mechanism that are not destabilized by overexpression of neuregulin 1 type IV.

Myelin is an extensive cell membrane produced by oligodendrocytes to ensheath neuronal axons in the central nervous system with the primary goal of maximizing the efficiency of electrochemical impulse transmission. During brain development, oligodendrocytes differentiate into myelin forming cells in a tightly regulated process which makes them vulnerable to multiple insults. Previous results from the laboratory showed that the timing of oligodendrocyte differentiation and rat brain myelination were altered by perinatal exposure to buprenorphine and methadone, opioid analogues used for treating pregnant addicts. The mechanism by which these opioids exerted their effects involved two opioid receptors, the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOR). However, the role of these receptors and their endogenous ligands in controlling the timing of myelination under normal physiological conditions of brain development is not known. In this dissertation, we found that the endogenous MOR ligand endomorphin-1 (EM-1) acts as a strong promoter of rat pre-oligodendrocyte differentiation, but surprisingly, this effect is observed only in cells isolated from female pups. Interestingly, the stimulatory action of EM-1 was abolished upon co-incubation with the endogenous NOR ligand, nociceptin. Moreover, injections of NOR antagonist to 9-day-old female and male rat pups accelerated rat brain myelination in female rat pups with no significant changes in their male counterparts. Interestingly, the lack of major sex-dependent differences in developmental brain levels of EM-1 and nociceptin and the presence of the two receptors MOR and NOR in male and female oligodendrocytes suggested that the observed sex-specific responses may be highly dependent on critical intrinsic sex-dependent differences within these cells. Although nociceptin alone did not exert observable effects on pre-oligodendrocyte maturation, it increased the number of cells expressing Ki-67, a cell proliferation indicator, in oligodendrocyte progenitor cultures. These results suggest that nociceptin may be playing a stage specific role in oligodendrocyte development during brain maturation. The finding of critical functions of EM-1 and nociceptin in the developing female oligodendrocytes and brain myelination highlights the need for considering sexual dimorphism in the design of safer and more effective therapeutic approaches for treating opioid abuse, pain, and demyelinating disease as multiple sclerosis.

The extent of damage in animal models of spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of the second messenger, cyclic adenosine monophosphate (cAMP), via administration of the phosphodiesterase 4 inhibitor, Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following thoracic contusive SCI injury in rat. Rolipram or vehicle control (10% ethanol) was given daily for 2 weeks post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons (CMAs) was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (2 h PI) increased neuronal survival; 0.5 mg- 1.0 mg/kg protected oligodendrocytes, 1.0 mg/kg produced optimal preservation of CMAs. Administration of 1.0 mg/kg Rolipram via different routes (intravenous [i.v.], subcutaneous [s.c.] or oral, 2 h PI) demonstrated that all routes allowed for significant protection following SCI; the i.v. route provided the best clinical translation. Examination of delayed treatment, initiated 1-48 h after SCI, revealed protective efficacy of Rolipram even when administered up to 48 h PI. With the optimal therapeutic protocol (1.0 mg/kg, i.v.), Rolipram reduced the levels of the chemokine, monocyte chemoattractant protein acutely post-injury and elevated the levels of the anti-inflammatory cytokine, interleukin-10, based on Enzyme-Linked ImmunoSorbent Assay (ELISA) results. Rolipram, when delivered within 48 h PI, was also able to significantly reduce the number of ED1-positive mononuclear phagocytes compared to vehicle-treated controls. This work supports the use of Rolipram as an acute neuroprotectant following SCI, defines an administration protocol, and investigates a potential mechanism for Rolipram-mediated protection.

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Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Western Australia, 2007.