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The meninges have long been accepted as purely structural, protecting the central nervous system (CNS). Similarly, the choroid plexus (CP) was thought to only produce cerebrospinal fluid. Advances have dispelled these limited views, and the meninges and CP are now known as critical sites of immune activity, housing various immune cells, including B and T cells, monocytes/macrophages, and dendritic cells, and are increasingly implicated in neuroinflammation. The broad aim of this thesis is to characterise the immune profile of the meninges, CP, and secondary lymphoid tissue in animal models of CNS and peripheral neuropathy. New evidence in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggests inflammation of the meninges and CP changes are crucial for disease induction, warranting further investigation. Firstly, we demonstrated significant changes in the meninges and lymphoid tissue in EAE. Adoptive transfer of immunosuppressive activated regulatory T cells (Tregs) resulted in their migration to the pia and CP, and drainage from the CNS back into lymph nodes 2-4 days following delivery. Treg treatment at the peak of EAE showed mild changes in meningeal/lymphatic immunity. Secondly, to study meningeal involvement in peripheral neuropathy, where literature is severely lacking, we utilised an animal model of peripheral nerve injury, sciatic nerve chronic constriction injury (CCI), and 2 animal models of chemotherapy-induced peripheral neuropathy (CIPN). The immune profile of the meninges, CP, and lymphoid tissue was compared across male and female mice in acute stages of CCI and in a chronic model. We found the immune profile of the meninges, CP, and lymphoid tissue at acute stage of injury is different for males and females, is altered at 7 days and 21 days post-injury and appears to resolve by a chronic time point. In CIPN animal models, we demonstrated the immune profile of the meninges and CP is minimally affected, however

The experiments reported in this thesis investigated arousal and motivational drive by chemogenetically evoking cardiovascular and locomotor responses from the tuberal hypothalamus. The first experiment showed that hSyn and CaMKIIα promoted hM3Dq adeno-associated viral vectors (AAVs) express well in the medial tuberal hypothalamus (MH) and the lateral tuberal hypothalamus (LH), and there were differences between the two AAVs in terms of their tropism. The hSyn AAV showed tropism towards GABAergic neurons over glutamatergic neurons, whereas the CaMKIIα AAV showed tropism towards glutamatergic over GABAergic neurons, for both regions. Experiment 2 confirmed that CNO injection (3 mg/kg i.p.) greatly increased neuron activity in a hM3Dq transduced hypothalamus hemisphere, as measured by cFos expression. cFos expressed strongly throughout the MH and LH, including orexin neurons. Experiment 3A demonstrated that chemogenetic activation of the tuberal hypothalamus elicited marked increases in locomotor activity, heart rate (HR), and mean arterial blood pressure (MAP) over vehicle, which persisted for the 2 hr measurement period (and beyond). Changes in locomotor activity depended on which region within the hypothalamus was activated as well as the promoter used. Strong locomotor responses were elicited from the hSyn and CaMKIIα AAVs in the MH, but only from the CaMKIIα AAV in the LH. In contrast, HR and MAP increases were stronger when evoked from the MH than the LH, and this was independent of the promoter used. Experiment 3B showed that the cardiovascular component of these responses was centrally mediated, rather than being caused by locomotor hyperactivity. Experiment 3C showed that orexin neurons are a key output neuron population that contributes to these responses, as blockade of orexin neurotransmission reduced the evoked responses by approximately half. Taken together, these results show that cardiovascular and locomotor responses, which facilitate a wide variety o

Sphingosine 1-phosphate (S1P) is a signalling lipid that mediates biological processes through five G-protein coupled receptors (S1PR1-5). S1P synthesis occurs through the phosphorylation of sphingosine by sphingosine kinase 1 (SphK1) or SphK2. S1P levels and SphK activity are reduced in multiple neurodegenerative paradigms including Alzheimer’s disease (AD). Pharmacological targeting of S1PRs is neuroprotective in animal models of these diseases. This thesis investigates the cellular and molecular mechanisms behind S1P-mediated neuroprotection. S1P up-regulates four neurotrophic genes (BDNF, PDGFB, HBEGF, and LIF) in primary astrocytes, but not neurons. Induction of these genes is mainly driven by S1PR2 signalling, with minor contributions from S1PR1. Phosphoproteomic analysis showed time-dependent activation of canonical pathways such as the mitogen-activated protein kinase and RhoA pathways, and phosphorylation of cJUN and Yes associated protein (YAP). Immediate early genes (IEGs) were also induced with S1P. Transcription start sites of these neurotrophic factors show predicted binding sites for cJUN and YAP, and the IEGs. While distinct phosphosites were regulated by S1PR1 and S1PR2 signalling, a large subset was regulated by both. However, the RhoA-YAP pathway was exclusively activated by S1PR2. The clinical drug Fingolimod (FTY720) is a sphingosine analogue that is phosphorylated in vivo by SphK2 to form the S1P structural analogue FTY720-P. Previous reports detailing the neuroprotective properties of S1PRs used FTY720-P to investigate the neurotrophic response. Unlike S1P, FTY720-P does not activate the S1PR2-RhoA-YAP pathway in astrocytes. Accordingly, S1P was a much more potent inducer of IEGs and neurotrophic gene expression than FTY720-P. Additionally, S1P but not FTY720-P significantly attenuated excitotoxic neuronal cell death in vitro when co-cultured with glia. Neuroprotection was ablated upon incubation with a LIF neutralising antibody, but not with

Obesity is a health problem affecting more than 40% of US adults and 13% of the global population. Anti-obesity treatments including diet, exercise, surgery and pharmacotherapies have so far failed to reverse obesity incidence. Herein, we target obesity with a pharmacotherapeutic approach that decreases caloric efficiency by mitochondrial uncoupling. We show that a recently identified mitochondrial uncoupler BAM15 is orally bioavailable, increases nutrient oxidation, and decreases body fat mass without altering food intake, lean body mass, body temperature, or biochemical and haematological markers of toxicity. BAM15 decreases hepatic fat, decreases inflammatory lipids, and has strong antioxidant effects. Hyperinsulinemic-euglycemic clamp studies show that BAM15 improves insulin sensitivity in multiple tissue types. Collectively, these data demonstrate that pharmacologic mitochondrial uncoupling with BAM15 has powerful anti-obesity and insulin sensitizing effects without compromising lean mass or affecting food intake.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Of the multitude of motor, sensory and cognitive complaints reported by sufferers, pain represents an extremely common and difficult to treat symptom in MS. This is an issue that, at least in part, stems from a lack of reliable and translatable pain outcome measures in animal models of MS. Experimental autoimmune encephalomyelitis (EAE) shares many key pathological characteristics with MS, namely neuroinflammation, demyelination and axonal damage. The broad aim of this thesis was to investigate the effects of immunoregulatory approaches on pain associated with EAE. We show here that in addition to stimulus-evoked pain, mice with EAE also develop spontaneous pain, an important and often overlooked component of MS-associated pain. Comprehensive analysis of immune, glial and neural changes in correspondence with observable pain behaviours showed that distinct stages of EAE are associated with specific pain phenotypes and neuroinflammation in both the peripheral and central nervous systems. We next aimed to test the effects of modulation of regulatory T (Treg) cells, potent suppressors of the immune system, on EAE progression and pain behaviours. We found that both systemic and centrally-delivered Treg cells were effective in reducing pain behaviours in EAE mice, and centrally-delivered Treg cells appeared associated with a reduction in astrogliosis and increased myelination in key regions comprising the facial nociceptive pathway. Similarly, intrathecal injection of interleukin (IL)-35, a novel anti-inflammatory cytokine produced by Treg cells, significantly reduced stimulus-evoked and spontaneous pain behaviours in EAE mice. This effect was Treg cell-dependent and appeared associated with an upregulation of IL-10 expression in CNS-infiltrating T and B cells, a reduction in the number of monocytes accumulated within the brain stem trigeminal nuclei, and the inhi