Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.

Immune and stress factors in the pathophysiology of the mdx mouse model of Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal multi-system neuromuscular disease caused by loss of dystrophin. The loss of dystrophin from membranes of contractile muscle cells and the dysregulation of the DAPC, induces chronic inflammation due to tissue necrosis and eventual replacement with collagen which weakens muscular force and strength. Dystrophin deficiency may cause under-diagnosed features of DMD include mood disorders such as depression and anxiety and dysfunction of the gastrointestinal tract. The first study in the thesis examined mood in the dystrophin-deficient mdx mouse model of DMD and examined the effects of the tri-cyclic antidepressant, amitriptyline on behaviours. Amitriptyline had anti-depressant and anxiolytic effects in the mdx mice possibly through effects on stress factors such as corticotrophin-releasing factor (CRF). This antidepressant also reduced skeletal muscle inflammation and caused a reduction in circulating interleukin (IL)-6 levels. In the second and third studies, we specifically blocked IL-6 signalling and used Urocortin 2, CRFR2 agonist to investigate their potential as therapeutic targets in mdx mice pathophysiology. Isometric and isotonic contractile properties of the diaphragm, were compared in mdx mice treated with anti IL-6 receptor antibodies (anti IL-6R) and/or Urocortin 2. Deficits in force production, work and power detected in mdx mice were improved with treatment. In study three I investigated contractile properties in gastrointestinal smooth muscle. As compared to wild type mice, mdx mice had slower faecal transit times, shorter colons with thickened muscle layers and increased contractile activity in response to recombinant IL-6. Blocking IL-6 signalling resulted in an increase in colon length, normalised faecal output times and a reduction in IL-6-evoked contractile activity. The findings from these studies indicate that for both diaphragm and gastrointestinal function in a dystrophin-deficient model, targeting of IL-6 and CRFR2 signalling has beneficial therapeutic effects.

Development and characterization of novel transgenic techniques for the study of neuronal circuitry

Modern neuroscience relies heavily on sophisticated tools that allow us to visualize and manipulate cells with precise spatial and temporal control. Transgenic mouse models, for example, can be used to manipulate cellular activity in order to draw conclusions about the molecular events responsible for the development, maintenance and refinement of healthy and/or diseased neuronal circuits. Although it is fairly well established that circuits respond to activity-dependent competition between neurons, we have yet to understand either the mechanisms underlying these events or the higher-order plasticity that synchronizes entire circuits. In this thesis we aimed to develop and characterize transgenic mouse models that can be used to directly address these outstanding biological questions in different ways. We present SLICK-H, a Cre-expressing mouse line that can achieve drug-inducible, widespread, neuron-specific manipulations in vivo. This model is a clear improvement over existing models because of its particularly strong, widespread, and even distribution pattern that can be tightly controlled in the absence of drug induction. We also present SLICK-V::Ptox, a mouse line that, through expression of the tetanus toxin light chain, allows long-term inhibition of neurotransmission in a small subset (<1%) of fluorescently labeled pyramidal cells. This model, which can be used to study how a silenced cell performs in a wildtype environment, greatly facilitates the in vivo study of activity-dependent competition in the mammalian brain. As an initial application we used this model to show that tetanus toxin-expressing CA1 neurons experience a 15% - 19% decrease in apical dendritic spine density. Finally, we also describe the attempt to create additional Cre-driven mouse lines that would allow conditional alteration of neuronal activity either by hyperpolarization or inhibition of neurotransmission. Overall, the models characterized in this thesis expand upon the wealth of tools available that aim to dissect neuronal circuitry by genetically manipulating neurons in vivo.

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

P-glycoprotein inhibition as a strategy to increase drug delivery across the blood-brain barrier: focus on antidepressants

Depression is among the leading causes of disability worldwide. Currently available antidepressant drugs have unsatisfactory efficacy, with up to 60% of depressed patients failing to respond adequately to treatment. Emerging evidence has highlighted a potential role for the efflux transporter P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), in the aetiology of treatment-resistant depression. In this thesis, the potential of P-gp inhibition as a strategy to enhance the brain distribution and pharmacodynamic effects of antidepressant drugs was investigated. Pharmacokinetic studies demonstrated that administration of the P-gp inhibitors verapamil or cyclosporin A (CsA) enhanced the BBB transport of the antidepressants imipramine and escitalopram in vivo. Furthermore, both imipramine and escitalopram were identified as transported substrates of human P-gp in vitro. Contrastingly, human P-gp exerted no effect on the transport of four other antidepressants (amitriptyline, duloxetine, fluoxetine and mirtazapine) in vitro. Pharmacodynamic studies revealed that pre-treatment with verapamil augmented the behavioural effects of escitalopram in the tail suspension test (TST) of antidepressant-like activity in mice. Moreover, pre-treatment with CsA exacerbated the behavioural manifestation of an escitalopram-induced mouse model of serotonin syndrome, a serious adverse reaction associated with serotonergic drugs. This finding highlights the potential for unwanted side-effects which may occur due to increasing brain levels of antidepressants by P-gp inhibition, although further studies are needed to fully elucidate the mechanism(s) at play. Taken together, the research outlined in this thesis indicates that P-gp may restrict brain concentrations of escitalopram and imipramine in patients. Moreover, we show that increasing the brain distribution of an antidepressant by P-gp inhibition can result in an augmentation of antidepressant-like activity in vivo. These findings raise the possibility that P-gp inhibition may represent a potentially beneficial strategy to augment antidepressant treatment in clinical practice. Further studies are now warranted to evaluate the safety and efficacy of this approach.

Novel roles for the GABAB receptor in anxiety and cocaine addiction

The GABAB receptor has been postulated as a possible drug target in the treatment of anxiety disorders and cocaine addiction. Indeed, a wealth of preclinical data is emerging that has shown that mice lacking functional GABAB receptors display a highly anxious behaviour across a range of behavioural models of anxiety. Additionally, novel compounds that act by altering the allosteric conformation of the GABAB receptor to a more active state; the GABAB receptor positive modulators, have been repeatedly demonstrated to have anxiolytic effects in animals. In addition to being a putative anxiolytic drug target, the GABAB receptor has been identified as a novel target for antiaddictive therapies. Indeed GABAB receptor positive modulators have been demonstrated to have anti-addictive properties across a broad variety of behavioural paradigms. Despite these findings, several gaps in our knowledge of the role played by the GABAB receptor in both anxiety and drug abuse disorder exist. The aim of this thesis was to use preclinical animal models in an effort to further probe the role played by the GABAB receptor in anxiety and addiction. Our studies initially examined the role played by the GABAB receptor in the neurodevelopmental processes underpinning of anxiety. Our studies demonstrated that treating mouse pups in early life with the GABAB receptor agonist baclofen produced an anxious phenotype in adult life, whereas treatment with the GABAB receptor antagonist CGP52432 produced no effects on adult behaviour. Further to this, we examined whether the anxious behaviour induced by early life blockade of the serotonin reuptake transporter was dependant on alterations in GABAB receptor function. Our studies however revealed no effect of early life selective serotonin reuptake inhibitor treatment on adult life baclofen sensitivity. The next issue addressed in this thesis is the characterization of the effects of a GABAB receptor positive modulator and a GABAB receptor antagonist in a behavioural model of conditioned fear behaviour. These novel classes of GABAB receptor ligands have been considerably less well characterized in this facet of preclinical anxiety behaviour than in terms of innate anxiety behaviour. Our study however revealed that the GABAB receptor positive modulator GS39783 and the GABAB receptor antagonist CGP52432 were without effect on the acquisition, expression or extinction of conditioned fear in our model. The next element of this thesis dealt with the characterization of a novel mouse model, the GABAB(2)- S892A mouse. This mouse has been engineered to express a form of the GABAB(2) receptor subunit wherein the function determining serine phosphorylation site cannot be phosphorylated. We initially tested this mouse in terms of its GABAB receptor function in adult life, followed by testing it in a battery of tests of unconditioned and learned anxiety behaviour. We also examined the behavioural and molecular responses of the GABAB(2)-S892A mouse to cocaine. All of our studies appear to show that the GABAB(2)-S892A mouse is indistinguishable from wildtype controls. The final aim of the thesis was to investigate the behavioural and molecular sensitivity of the GABAB(1) subunit isoform null mice, the GABAB(1a) -/- and GABAB(1b) -/- mice to cocaine. Our studies revealed that these mice display differing behavioural responses to cocaine, with the GABAB(1a) -/- mouse displaying a hypersensitivity to the acute locomotor effects of cocaine, while the GABAB(1b) -/- displayed blunted locomotor sensitisation to cocaine.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Stress-induced visceral pain in rodents: neurochemical, hormonal, immune and epigenetic mechanisms

Visceral pain is a debilitating disorder which affects up to 25% of the population at any one time. It is a global term used to describe pain originating from the internal organs, which is distinct from somatic pain. Currently the treatment strategies are unsatisfactory, with development of novel therapeutics hindered by a lack of detailed knowledge of the underlying mechanisms. The work presented in this thesis aimed to redress this issue and look in more detail at the molecular mechanisms of visceral pain in preclinical models. Stress has long been implicated in the pathophysiology of visceral pain in both preclinical and clinical studies. Here a mouse model of early-life stress-induced visceral hypersensitivity was validated. Moreover, mouse strain differences were also apparent in visceral sensitivity suggesting a possible genetic component to the underlying pathophysiology. Furthermore, gender and sex hormones were also implicated in stress sensitivity and visceral pain. Using the rat model of maternal separation, some of the epigenetic mechanisms underpinning visceral hypersensitivity, specifically the contribution of histone acetylation were unravelled. Glutamate has been well established in somatic pain processing, however, its contribution to visceral pain has not been extensively characterised. It was found that glutamate uptake is impaired in viscerally hypersensitive animals, an effect which could be reversed by treatment with riluzole, a glutamate uptake activator. Moreover, negative modulation of the metabotropic glutamate (mGlu) receptor 7 was sufficient to reverse visceral hypersensitivity in a stress sensitive rat strain, the Wistar Kyoto rat. Furthermore, toll-like receptor 4 (TLR4) was implicated in chronic stress-induced visceral hypersensitivity. Taken together, these findings have furthered our knowledge of the pathophysiology of visceral pain. In addition, we have identified glutamate transporters, mGlu7 receptor, histone acetylation and TLR4 as novel targets, amenable to pharmacological manipulation for the specific treatment of visceral pain.