Anatomy and Neuroscience - Theses
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ItemPyroglutamate-Aß in the pathogenesis of Alzheimer's disease( 2013)Background: Amyloid-beta (Aβ) peptides are central contributors to Alzheimer’s disease (AD) pathogenesis. Although Aβ peptides are present in all human brains, the AD brain is particularly enriched for oligomeric Aβ species and Aβ peptides containing post-translational modifications such as oxidation, amino-truncation and pyroglutamate (pE). Metal interactions are a critical aspect of Aβ-induced neurotoxicity, however the effects of pE formation on Aβ-metal reactions such as nucleated oligomerisation, redox cycling and the production of reactive oxygen species (ROS) have not been investigated. Recent reports have indicated that pE-Aβ peptides are more neurotoxic than full-length Aβ, although a mechanistic difference in the toxic properties of these peptides has yet to be established. Increased levels of glutaminyl cyclase (QC) are thought to be responsible for the abundance of pE-Aβ in the AD brain, via the cyclisation of exposed N-terminal glutamate to pyroglutamate on amino-truncated Aβ. The relative levels of QC protein and mRNA are reported to be elevated in the temporal cortex and peripheral blood of individuals with AD compared to healthy controls. However, there are no published values of QC enzymatic activity in human central nervous system tissues. The involvement of QC in pE-Aβ formation and AD pathogenesis has led to the recent generation of QC inhibitors as a potential therapeutic intervention for AD. For this reason, there is a critical need to establish standardised levels of QC protein and activity in populations of healthy individuals and people with AD. Furthermore, there is a lack of animal models of pE-Aβ expression, thus the generation of simple pE-Aβ expression models may facilitate the study of potential QC inhibitors as an AD therapeutic. Objectives: I aimed to assess potential differences between synthetic pE-Aβ and full-length Aβ peptides in terms of their oligomerisation rate, fibril ultrastructure, cellular life-span and neurotoxicity. I also sought to compare the Aβ variants for their capacity to undergo nucleated polymerisation in the presence of Cu2+ or Zn2+, in addition to the generation of ROS and oxidative modifications such as dityrosine via redox cycling reactions with Cu2+ and ascorbate. To determine whether changes in soluble QC (sQC) expression and activity are a feature of AD pathogenesis, I aimed to establish standardised ranges of sQC protein and activity in the human brain through analysis of post-mortem cortical tissue samples from a cohort of AD and control brains. Finally, I sought to generate a Caenorhabditis elegans nematode model of pE-Aβ expression for in vivo comparisons of Aβ variant cytotoxicity and cellular interactions. Results: Vastly different rates of fibrilisation and fibril ultrastructures were observed for amino-truncated and pE-Aβ peptides compared with full-length peptides. Amino-truncated Aβ showed accelerated fibril seeding compared to full-length Aβ, while further addition of Cu2+ inhibited fibrilisation and produced aggregates of different ultrastructures between the seeded mixtures. In contrast, Zn2+ promoted fibrilisation but was also found to rapidly and reversibly aggregate Aβ peptides in short incubation periods. Redox-cycling reactions of Aβ, Cu2+ and ascorbate demonstrated significant differences between full-length Aβ and pE-Aβ peptides in the profiles of oligomers produced as well as the rate of hydroxyl radical production and dityrosine formation. The reaction of Aβ1-40 with Cu2+ and ascorbate was further found to cause amide-bond hydrolysis and the formation of amino-truncated Aβ peptides. Both the Aβ1-42 and Aβ3pE-42 peptides were toxic to cortical neurons and inhibited hippocampal long-term potentiation, however methodological differences in the preparation of peptides were found to significantly alter the relative Aβ neurotoxicity. Aβ1-42 was the only peptide to significantly increase neuronal ROS levels, suggesting that the toxicity observed for Aβ3pE-42 was ROS-independent. The levels of Aβ3pE-42 were much higher than Aβ1-42 following 48 h treatment of the peptides on cortical neurons, indicating that Aβ3pE-42 is highly resistant to proteolysis in neurons. Mean levels of sQC protein were modestly, though significantly, elevated in the frontal cortex of individuals with AD compared with healthy controls. No significant difference in the mean levels of total sQC activity or specific activity were observed between AD and control subjects. Gel electrophoresis and mass-spectrometry analyses of a C. elegans strain designed to express Aβ1-42 (CL2120) unexpectedly revealed that the predominant peptide expressed was actually Aβ3-42 – a precursor substrate for pE-Aβ formation. Genetic manipulation of this strain to co-express human sQC resulted in production of an additional Aβ species in these worms with hydrophobic properties consistent with Aβ3pE-42. Conclusions: Previous studies have established that amino-truncation and pE formation greatly enhance the oligomerisation and fibrilisation of Aβ peptides. The data presented here demonstrate that these modifications also affect the capacity of Aβ to undergo facile redox cycling with Cu2+, thus altering the relative production of cytotoxic ROS and oxidative protein modifications such as dityrosine. Aβ3pE-42 showed either comparable or enhanced toxicity to cortical neurons compared with Aβ1-42, although different peptide dissolution methods were seen to skew the relative toxicity of each peptide. Aβ1-42 greatly increased cytosolic ROS in neurons, whereas pE-Aβ peptides did not, suggesting that pE-Aβ induced neurotoxicity is ROS-independent. Furthermore, pyroglutamate formation renders Aβ resistant to proteolysis in neurons, indicating that pE-Aβ peptides are both neurotoxic and biologically persistent. The reported abundance of pE-Aβ in AD brains is not however a function of increased QC activity in the frontal cortex, suggesting that the rate of pE-Aβ formation is either due to regional-specific changes in QC activity or increased production of amino-truncated Aβ precursors, or both. The process of Aβ amino-truncation in vivo may also be due to multiple processes such as aminopeptidase activity and the interactions of Aβ with Cu2+. C. elegans models of pE-Aβ expression may facilitate further studies into the biological properties of these amyloidogenic peptides and the screening of potential therapeutics to inhibit their formation.
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ItemAnatomical changes at the axon initial segment in neuronal hyperexcitability( 2013)The axon initial segment (AIS) is an important sub-cellular region in neurons, playing diverse and critical roles in neuronal excitability, the maintenance of neuronal polarity, and the regulation of cytoplasmic trafficking. Previously thought to be a uniform, static structure, it is now apparent that the AIS exhibits greater levels of complexity and plasticity than previously predicted, and is an increasingly interesting and relevant focus of research in neuroscience. A range of proteins are expressed at high densities at the AIS, some exclusively, including structural molecules, ion channels and cell adhesion molecules. The molecular composition and structural characteristics of the AIS vary by neuronal subtype, brain region and developmental stage, resulting in differences in functional phenotypes of these neurons, although the more subtle aspects of this are yet to be elucidated. The important roles played by AIS-localised proteins, along with the potential consequences of disruption to AIS integrity, composition or structure, make this an incredibly important neuronal region to consider in a variety of pathophysiological pathways in the brain. Many AIS proteins have been implicated in CNS disease; in particular a large number of AIS ion channels are implicated in epilepsy. Additionally, the emerging phenomenon of AIS plasticity, by which neuronal excitability is altered as a result of changes in the gross structural anatomy of the AIS, could potentially play a role in epilepsy. In this thesis I explore two aspects of AIS involvement in disorders of neuronal hyperexcitability using immunohistochemistry and high-resolution confocal microscopy. The first study analyses the effects of seizures on AIS structure in two animal models of neuronal hyperexcitability, in which I have identified structural changes in the position of the AIS relative to the soma in animals experiencing seizures. This is the first study to demonstrate plasticity of the AIS in epilepsy, and the results suggest differing roles of this phenomenon in established genetic epilepsy and in the pathogenesis of acquired seizure disorders. The second study describes the AIS localisation of an ion channel subtype – the β1 subunit of the voltage-gated sodium channel – in both health and disease states, using a genetic mouse model of a human epilepsy syndrome. I have demonstrated the endogenous localisation of this subunit to the AIS and revealed its disruption in genetic epilepsy, an important finding complementing functional studies in elucidating the pathogenic mechanisms in this type of epilepsy. These studies reveal the novel involvement of AIS structural plasticity in neuronal hyperexcitability as well as a mechanism of AIS dysfunction in genetic epilepsy, together highlighting the ubiquitous influence of AIS function on neurological health. The linking of genetic mutations, environmental conditions and anatomical AIS phenotypes will further enhance our understanding of the pathophysiological basis of disorders of neuronal hyperexcitability and aid identification of novel therapeutic targets for neurological disease.
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ItemAnalysis of Ndfip1 protein interactions using Bimolecular Fluorescence Complementation (BiFC)( 2013)The Nedd4-family interacting protein 1 (Ndfip1) is a neuroprotective protein, and is strongly up-regulated in neurons following brain injury. Ndfip1 is an adaptor and activator of Nedd4 ubiquitin ligases for the identification and binding of proteins prior to their ubiquitination by Nedd4 ligases. A critical question is where in the cell does this occur and what happens following binding of Ndfip1 to their targets? This thesis aims to address this question by direct visualization of inter-molecular interaction using a method known as Bimolecular Fluorescence Complementation (BiFC). This method is based on the complementation between fragments of the Green Fluorescent Protein (GFP) linked to interacting partners. Positive binding between partners lead to reconstitution of the GFP signal that can be directly visualized within the cell by confocal microscopy. I used this method to investigate binding of ubiquitin to Ndfip1, and also binding of Ndfip1 to other proteins. These proteins include (1) phosphatase and tensin homolog deleted on chromosome TEN (Pten), (2) β-amyloid precursor protein (APP), and (3) amyloid β (A4) precursor protein-binding, family B, member 1 (Fe65). With respect to the tumour suppressor protein Pten, my work has yielded new insights regarding our understanding of how this protein is moved within the cell. I discovered that ubiquitinated Pten has a different subcellular distribution pattern compared to unmodified Pten. Specifically, ubiquitinated Pten was found to localize mainly in perinuclear and nuclear regions. Ubiquitinated Pten was found to co-localize with early endosomes (containing Rab5) and recycling endosomes (containing Rab11), but not late endosomes (containing Rab7 and 9). Indicating that ubiquitinated Pten may use the endosomal pathway to travel from plasma membrane to the perinuclear and nuclear regions. Rab5 was found also play an important role in Pten ubiquitination and translocation. Dominate negative Rab5 can perturb early endosome trafficking and function, and cause significant reduction of Pten ubiquitination. I found that loss of Ndfip1 combined with dominate negative Rab5 led to total loss of Pten ubiquitination. These findings increase our understanding of Pten trafficking in the cell and have the potential for clinical applications in both tumour suppression by Pten and improving neuronal survival after brain injury. APP is the precursor protein of Aβ, which can accumulate to form plaques leading to Alzheimer’s disease. In this thesis, I showed that Ndfip1 can bind with APP in vitro and promote APP ubiquitination and degradation. Using the BiFC method, ubiquitinated APP was found to co-localize with Rab5, Rab7, Rab9 and Rab11, indicating that ubiquitinated APP could be trafficked throughout the endosomal system in the cell, this included late endosomes that can merge with the lysosome for the degradation of protein. To determine which degradation pathway was used for APP degradation mediated by Ndfip1, I employed a number of lysosome and proteasome inhibitors. The results showed that Ndfip1 promoted APP degradation is through lysosome pathway and not the proteasome. However, in contrast to Ndfip1 interaction with Pten, the association of Ndfip1 with APP appeared to be localized to the cytoplasmic side of vesicles as well as in the lumen. These findings indicate that Ndfip1 interaction with APP inside the cell is highly dynamic and could reflect differential APP regulation by Ndfip1 that leads to either degradation or trafficking inside the cell. Fe65 is known to interact with APP intracellular domain (AICD) and can be translocated into nucleus as a transcription factor. It is also ubiquitinated by Nedd4-2. I performed experiments to test the relationship between Ndfip1 and Fe65, and found that Ndfip1 can bind and ubiquitinate Fe65 in vitro. Fe65 protein abundance was not changed by Ndfip1 expression in vitro and in vivo. Using BiFC, ubiquitinated Fe65 was found to localize to early and recycling endosomes, but not in late endosomes. Combining my findings for APP and Fe65, I hypothesis that Ndfip1, Nedd4-2, Fe65 and APP may form a complex that is required for ubiquitin-mediated trafficking of APP and Fe65. In this context, Ndfip1 is most likely to function as a scaffold on endosomes for the formation of protein complexes. In conclusion, through the use of BiFC I have investigated the trafficking of three proteins, Pten, APP and Fe65, that is mediated by Ndfip1. I have identified that Ndfip1 functions predominantly on endosomes and serves to function as a scaffold for the recruitment of complexes resulting in the ubiquitination of target proteins. This identification of trafficking systems in the cell has important implications for disease states such as brain injury, cancer and Alzheimer’s disease.
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ItemNormal appearing brain tissue changes in optic neuritis patients at risk of MS( 2013)Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which affects white matter (WM), grey matter (GM) and normal appearing brain tissue (NABT). In 85-90% of MS patients, the first manifestation of the disease is an acute, remitting clinically isolated syndrome (CIS). One of the most frequent symptoms of CIS is optic neuritis (ON). The risk of conversion to MS in isolated optic neuritis (ION) patients with abnormal baseline magnetic resonance imaging (MRI) is about 40% one year post onset. The chance of conversion to MS significantly increases based on the number of lesions on baseline MRI. Therefore, ION patients at risk of MS provide an excellent opportunity to study the events occurring at the early stages of the disease. Pathological studies and MRI provide good diagnostic and prognostic information about MS. MRI techniques as in-vivo non-invasive tools have helped to better understand the substrate, pathophysiology and mechanisms underlying the development and evolution of focal lesions and diffuse damage in MS. There is a strong correlation between pathological changes in GM and NABT and future clinical disability of MS patients. Therefore, advanced MRI techniques such as diffusion tensor imaging (DTI) have been widely applied to discover injuries in both GM and NABT with more specificity and sensitivity. Early diagnosis of MS is essential for initiation of early treatment leading to better prognosis and fewer disabilities in future. In this thesis, the main aim was to investigate longitudinal microstructural changes in NABT in the visual pathways and beyond in ON patients at risk of MS using different modalities of MRI including volumetric and diffusion imaging techniques. Twenty three patients (17f/6m; mean ± SD age = 34.6 ± 8.6 yrs) and eleven healthy age matched controls (8f/3m; mean ± SD age = 36.4 ± 4.9 yrs) were recruited for the studies of this thesis. Patients were scanned around two weeks after their first presentation of ON and then at 1, 3, 6 and 12 months, and controls were scanned with one month separation. Following each scan session; multifocal visual evoked potentials (mfVEP) and optical coherence tomography (OCT) were acquired by a neuro-ophthalmologist. After completing the MRI safety questionnaire, all participants were scanned at the Royal Children’s MRI Centre, Melbourne, Australia on a 3T Siemens MRI scanner with a 12 channel receiver head coil. Each MR session consisted of whole brain and optic nerve T1-weighted images, whole brain and optic nerve T2 fluid-attenuated inversion recovery (FLAIR) images, and one DTI study. The baseline MR session also included gadolinium enhanced MRI. For the purpose of this thesis, whole brain T1-weighted, T2-FLAIR, and DTI images were used. In the first study, in order to investigate longitudinal microstructural changes in NABT in the visual pathway following ON, we chose the body of optic radiations (BORs) as the regions of interest (ROIs). BORs are highly indicative of tightly packed and collinear axon bundles, and diffusion findings in these regions in comparison to the other areas of the optic radiations are more reliable. More than half of the participants (52.2%) converted to MS after 12 months which made our participants an ideal sample of patients in the very early stages of MS. By using DTI, we demonstrated longitudinal microstructural changes in NABORs in ION patients during one year follow up. Most of the longitudinal diffusion parameter alterations were observed in the right NABOR. Microstructural changes were found to commence at a very early stage of MS, nearly one month following ON. We also showed that in ION patients there was a progressive longitudinal decrease in retinal nerve fiber layer (RNFL) thickness, and that earlier axonal loss in the optic nerve correlated with later diffusion changes in NABORs. Furthermore, there was a correlation between left visual hemifield amplitude asymmetry and FA value in the right NABOR a year following ON. The results of this study confirmed previous imaging findings that DTI technique allows to explore subtle WM degeneration and to investigate integrity of neuronal bundles in the brain. Furthermore, our results provided evidence that the involvement of NABORs following ON can be the result of the trans-synaptic damages following ON. In the second study, we chose visual cortices (VCs) as the ROIs to investigate atrophy in normal appearing grey matter (NAGM) in the visual pathway following ON during one year in ION patients at risk of MS. Furthermore, to investigate microstructural changes in beyond the secondary order neurons in the visual pathways, we focused on the connection between primary visual cortices (V1s) and the splenium of the corpus callosum (CC), called cortico-splenial (CS) tracts. CS tracts were defined using probabilistic tractography. By using volumetric assessment, we demonstrated longitudinal decrease in the volume of the right normal appearing primary visual cortex (NAV1). Atrophy in the right NAV1 was observed to commence at the very early stage, nearly one month following ON. We also revealed a correlation between volume of the right NAV1 and left visual hemifield amplitude in the affected eye at 12 months. By using DTI, we showed that there is a progressive microstructural change in bilateral CS tracts. The commencement of the microstructural changes in the CS tracts was observed to be at the very early stage of MS. We also found a correlation between early axonal loss in the optic nerve and later microstructural changes in the CS tracts. The results of this study supported the potential role of MRI as a biomarker in early diagnosis of the disease. Moreover, this study provided further evidence of the involvement of NABT in the visual pathway and beyond following ON due to trans-synaptic damage. In our first two studies we provided evidence supporting the role of trans-synaptic damage in microstructural changes and atrophy in NABT in the visual pathway and beyond following ON. It would therefore be interesting to investigate whether only areas related to the visual pathways are affected in patients with ON, or are there other areas in the brain show microstructural damages? Early recognition of the involvement of the WM or GM and the possible mechanisms of the involvement in ION patients at risk of MS is very important because early diagnosis helps with decisions regarding appropriate treatments and options. Furthermore, early treatment can lead to better prognosis and fewer disabilities. In order to address these questions, we undertook an exploratory study to investigate other WM or GM regions of the brain which have microstructural changes or atrophy following ON. We used tract-based spatial statistics (TBSS) method to investigate areas of the whole brain WM with significant longitudinal diffusion changes during one year follow up in ION patients at risk of MS. The voxel-based morphometry (VBM) was used to investigate longitudinal decrease in the volume of GM areas in whole brain. We found progressive microstructural changes during one year in different WM areas of the brain in ION patients. The regions include ORs (mainly right), all parts of the CC; splenium, body and genu, as well as the left and right thalamus. Moreover, we demonstrated progressive atrophy in the GM areas of the brain including VCs, frontal lobe, and primary motor and somatosensory cortex during one year follow up in ION patients. The results of this study provided evidence for diffuse whole brain longitudinal WM microstructural changes and brain atrophy in ION patients at risk of MS during one year follow up. Pathophysiology underlying the diffuse involvement of the brain WM and GM in ION patients at risk of MS is still unclear. In summary, the results of our studies support DTI as a suitable technique for exploring subtle WM degeneration. Moreover, the findings provide evidence of trans–synaptic neuronal damage as the mechanism involved in the early progressive changes of the NABT in the visual pathways and beyond following ON at the early stage of MS even before the definite diagnosis of MS. Therefore, the results support the potential role of multi-modal MRI as a biomarker in the evaluation of disease evolution and progression, as well as response to treatment. The third study provided evidence for whole brain diffuse longitudinal WM microstructural changes and brain atrophy in ION patients at risk of MS during one year follow up. However, pathophysiology and mechanisms underlying the diffuse involvement of the brain WM and GM in ION patients at risk of MS require further investigations. Our findings suggest that ION patients with abnormal baseline MRI should be considered as an important group of patients with higher risk of conversion to MS and not just treated as patients with simple inflammatory disease. It has been shown that early treatment leads to less disability in future. Therefore, ION patients with abnormal MRI need to be closely monitored for early commencement of MS treatment. We hope that the findings of this thesis will lead to the identification of new outcome measures to monitor the evolution of MS and treatment response and, ultimately to improving disease management in individual patients.
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ItemFunctional analysis of sodium channel gene variation in epilepsy( 2013)A genetic etiology of epilepsy is widely accepted in 50-70% of all epilepsy syndromes. With genome sequencing now increasingly efficient and affordable, more and more novel genes and mutations are being discovered that are associated with epilepsy. However, most of the mutations have been discovered in genes that code for ion channels which has led to the theory that the genetic epilepsies are a family of channelopathies. The voltage-gated sodium channel family have been particularly implicated with over 800 variants discovered in this gene family. Given their critical role in regulating neuronal excitability it is not surprising that genetic variations in sodium channels can have functional and potentially devastating consequences. With a focus on the voltage-gated sodium channels, the three chapters in this thesis used high-throughput automated planar patch-clamp technology to try and develop a deeper understanding of genetic risk in epilepsy. Chapter two examines a novel cause in a mouse model of absence epilepsy that harbours a mutation in the Scn8a gene. The phenotype of this mouse is enhanced on the C3H background, as opposed to C57, where the C3H animal also has a mutation in the Scn2a gene. The individual biophysical profiles of these two mutations were examined on the Nanion patchliner, and their potential genetic interaction was investigated in a computer model of a layer 5 pyramidal neuron, to see if this could be explained by a biological interaction at the axon initial segment. The results revealed an overall loss of function of the NaV1.6V752F mutant, and an overall gain of function in the NaV1.2V929F mutant. When these changes were implemented in the computer model, it revealed that the output was dominated by the NaV1.2V929F mutant, which suggests there is not a biological interaction of these two genes at the axon initial segment. Alternative scenarios where there may be an alternative site for biological epistasis will be revealed with future studies using immunohistochemistry and brain slice patch clamp recording in the mice. It may also be the case that the NaV1.2V929F mutant is not a modifier of the NaV1.6V752F mutant, which will be revealed by genetic studies to identify the modifier genes. The third chapter examined the modulation of NaV1.2 and NaV1.1 by the β1 auxiliary subunit. As mutations in the β1-subunit have been detected in patients with epilepsy, understanding their impact on subunits from excitatory and inhibitory neurons is critical for understanding how this variation impacts on risk for epilepsy. There was a differential modulation revealed where β1 had a greater functional effect on the NaV1.2 channel but a greater effect on current density on the NaV1.1 channel. Therefore if a variant in β1 experiences a functional change this suggests differentially altered levels of excitation and inhibition in the brain, which could feasibly result in an epileptic phenotype. The fourth chapter looked at exploiting the high-throughput capabilities of the Nanion patchliner, and examined eight mutations in the β1-subunit co-expressed with NaV1.1 and NaV1.2 that have been associated with epilepsy. With this influx of data we needed to devise a new way to represent this data, and converted all raw measurements to effect size values, and represented them on tornado diagrams. With this measurement we could then more easily directly compare parameters from the individual protocols and calculate averages both across mutations, and across parameters. From this data set it is quite apparent that the β1 mutants modulate the α-subunits quite differently, both comparing α-subunits, and comparing mutations. More importantly however this chapter highlighted a new way of thinking about analysis of high-throughput electrophysiology data. As people continue to look into the genetics of epilepsy and reveal novel genes and novel mutations implicated in the disease, we need to look for new ways to tame the genetic complexity, and look for points of convergence. High-throughput technology allows us to decrease the time lapse between the discovery of the genetic variants and the corresponding functional analysis. And the type of analysis as suggested in chapter four, enables us to start to look for points of convergence in the functional data. This data can then be used to train clustering algorithms to group the variants based on their ‘channelomic’ profile. To do this we need a large volume of functional data obtained from variants that have strong corresponding phenotypic data, and future studies should endeavour to accomplish this.
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ItemGenetic, metabolic and pharmacological modulation of seizure susceptibility in mouse models of genetic epilepsy( 2013)Epilepsy is a common neurological disorder that is poorly understood. A large proportion of epilepsies have a strong familial component. The GABAA γ2 (R43Q) mutation was discovered in an Australian family with genetic epilepsy with febrile seizures + (GEFS+) that predominantly have febrile seizures (FS) and childhood absence epilepsy (CAE). A mouse model based on the mutation recapitulates these seizure types and is sensitive to first-line antiepileptic drugs. The model therefore provides an opportunity to study aspects of the genesis of epilepsy with relevance to the human condition. The work performed in this thesis describes the use of this syndrome specific mouse model to investigate aspects of seizure genesis and modulation. Three research questions are addressed; the genetic mechanisms underlying seizure genesis, metabolic and dietary modulation of seizure activity and pharmacological sensitivity to new anti-epileptic drugs in the GABAA γ2 (R43Q) mouse. Clinical heterogeneity in genetic epilepsy is common and is typically characterized by multiple seizure types and incomplete penetrance for a given protein mutation. However, the molecular and genetic basis of clinical heterogeneity is not well understood. Here, two models, GABAA γ2 (R43Q) knock-in and GABAA γ2 knock-out were used to determine the fundamental molecular mechanisms of the GABAA γ2 (R43Q) mutation underlying individual seizure phenotype. Spike-wave discharges (SWD) recorded on electroencephalogram from the GABAA γ2 (R43Q) mouse are associated with behavioural arrest and model absence epilepsy. A reduced latency to first heat-induced tonic-clonic seizure is consistent with a FS phenotype. Both the knock-in and knock-out models expressed SWDs while only the knock-in had a reduced latency to thermogenic seizures. This comparison demonstrates that two fundamental molecular mechanisms independently cause the two major seizure types in the mouse model. Haploinsufficiency could account for the SWD phenotype while a dominant impact of the mutation must be required for the FS phenotype. Subsequent investigation using mice of varying genetic background showed that the SWD phenotype required additional genetic susceptibility. In contrast, FS phenotype occurred independently of background genetics consistent with its higher penetrance compared to absence epilepsy in the GABAA γ2 (R43Q) family. Environmental modulation of neuronal excitability has been long known to alter seizure susceptibility. Altered metabolism using dietary intervention, such as the ketogenic diet, is a well recognized epilepsy therapy. The ketogenic diet conveys its anticonvulsant effects presumably through the stabilization of blood glucose and/or providing an alternative energy substrate. Here, the impact of a number of metabolic manipulations was investigated in the GABAA γ2 (R43Q) mouse model. Overnight fasting lowered blood glucose levels and increased SWD occurrence suggesting it as a potential seizure precipitant. Low-GI and triheptanoin diets on the other hand reduced SWD activities suggesting that both stabilization of blood glucose levels and provision of additional energy substrates may independently offer anticonvulsant effects. Importantly, these diets have less tolerability issues making them a potential alternative to the poorly tolerated ketogenic diet. In-vivo drug testing is a critical step for drug discovery. Oxcarbazepine (OXC) is a second-generation drug that is typically used to control partial seizures. Like its older generation carbamazepine, OXC is contraindicated in patients with generalized epilepsy. OXC is metabolized to monohydroxy derivatives (MHD) in two enantiomeric-forms, S-(+)-licarbazepine and R-(+)-licarbazepine. The effects of individual metabolites have not been adequately characterized. In this study, OXC increased the frequency of SWDs in the GABAA γ2 (R43Q) model, consistent with clinical observation. Similarly, both MHDs also caused seizure aggravation. However, OXC and MHDs were ineffective at altering the sensitivity of mice to thermogenic seizures. The findings indicate that like OXC, its derivatives may be contraindicated in certain forms of generalized epilepsy.
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ItemMechanisms that increase vascular reactivity following spinal cord injury( 2013)People with spinal cord injury (SCI) can experience episodes of dangerously high blood pressure, termed autonomic dysreflexia, in response to a range of sensory stimuli. While SCI severs bulbospinal inputs to sympathetic preganglionic neurons, the spinal reflex pathways below the lesion remain intact and are unopposed by inhibitory inputs from the brainstem. As a result, somatosympathetic reflexes can produce pronounced constriction of arterial vessels. Studies in man indicate that SCI not only modifies spinal reflexes but also increases neurovascular transmission in arterial vessels. The objective of this thesis was to gain insight into the mechanisms underlying the augmentation of neurovascular transmission that occurs following SCI. In Chapter 2, I investigated the mechanisms that underlie SCI-induced enhancement of neurovascular transmission in the rat tail artery. Isometric contractions of arterial segments from T11 spinal cord-transected and sham-operated rats were compared 6 weeks postoperatively. SCI more than doubled the amplitudes of contractions evoked by nerve stimulation. In arteries from SCI rats, but not those from sham-operated rats, the L-type Ca2+ channel blocker nifedipine reduced nerve-evoked contractions. Furthermore, while the sensitivity to the agonists phenylephrine (α1-adrenoceptor selective) and clonidine (α2-adrenoceptor selective) was unaffected by SCI, nifedipine had a greater inhibitory effect on contractions to both agents in arteries from SCI rats. In arteries from unoperated rats, the L-type Ca2+ channel agonist Bay K8644 mimicked the effects of SCI. These findings demonstrate that the SCI-induced enhancement of neurovascular transmission in rat tail artery can largely be accounted for by an increased contribution of L-type Ca2+ channels to activation of the vascular muscle. In Chapter 3, the mechanisms underlying the enhancement of neurovascular transmission produced SCI and Bay K8644 were further investigated in rat tail artery. In situ electrochemical detection of noradrenaline and electrophysiological monitoring of purinergic transmission were used to assess if Bay K8644 changed neurotransmitter release. In addition, isometric contractions of arterial segments were used to assess if SCI and Bay K8644 similarly changed the contribution of α1-adrenoceptors to nerve-evoked contractions and if interfering with sarcoplamic reticulum (SR) Ca2+ uptake modified the contribution of L-type Ca2+ channels to activation of tail arteries. Bay K8644 did not change noradrenaline-induced oxidation currents or purinergic excitatory junction potentials. Both SCI and Bay K8644 reduced blockade of nerve-evoked contractions by BMY7378 (α1D-adenoceptor antagonist), but did not change that by RS100329 (α1A-adrenoceptor antagonist). Disruption of the SR Ca2+ stores with ryanodine increased both nerve-evoked contractions and blockade of these responses by nifedipine. The findings demonstrate that SCI and Bay K8644 increase the α1A-adrenoceptor-mediated component of nerve-evoked contractions. The findings also suggest that Ca2+ entering smooth muscle via L-type channels is rapidly sequestered by the SR limiting its access to the contractile mechanism. Studies in individuals with SCI suggest the vasculature is hyperreactive to angiotensin II (Ang II). In Chapter 4, the effects of SCI on the reactivity of rat tail and mesenteric arteries to Ang II were investigated. SCI increased contractions of both vessels evoked by Ang II. In tail arteries, the facilitatory effect of Ang II on neurovascular transmission was greatly increased. In contrast, SCI did not change the facilitatory action of Ang II on neurovascular transmission in mesenteric arteries. These findings provide the first direct evidence that SCI increases the reactivity of arterial vessels to Ang II. In addition, in tail artery, the findings indicate that Ang II may contribute to amplifying spinal reflex activation of this vessel.
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ItemThe effects of diabetes on sympathetic neurovascular transmission( 2013)Impaired neural control of arteries is implicated in the etiology of diabetic foot, a major complication of diabetes. The loss of sympathetic nerve-mediated control of blood flow to plantar skin may be an early change that contributes to the later development of microvascular disease in foot skin. The mechanisms that modify sympathetic regulation of arterial vessels are not understood, but are suggested to be due to diabetes-induced neuropathy. Therefore the primary aim of this thesis was to investigate the effects of diabetes on the sympathetic innervation and activation of plantar metatarsal arteries (PMAs) that supply blood to plantar skin of the hind paw digits in rats. The streptozotocin (STZ) rat model of type I diabetes was chosen as it has been widely used to investigate mechanisms that lead to diabetic complications. Eight-week-old male Wistar rats were treated with STZ (60 mg/kg i.p.) or vehicle (citrate-buffer i.p.; controls). STZ-treated rats received no insulin (STZ-NI) or were treated with a low (~1 unit/day; STZ-LI) or a high (~4 units/day; STZ-HI) dose of insulin. The STZ-NI and STZ-LI rats were hyperglycemic (blood glucose >20 mM), whereas STZ-HI rats were normoglycemic (blood glucose <15 mM). Rats were maintained for 12 weeks when arteries were isolated for in vitro studies. In the first study, wire myography was used to assess vascular function. In comparison with PMAs from control rats, those from STZ-NI rats had reduced nerve-evoked contractions. PMAs from STZ-NI rats also had a decreased density of perivascular nerve fibers revealed by immunolabeling for the pan-neuronal marker β-tubulin III. No changes in vascular function and innervation density were observed in PMAs from STZ-LI and STZ-HI rats. However, in PMAs from both STZ-NI and STZ-LI rats, the β-tubulin III immunoreactive (IR) nerve fibers were thickened. The majority of perivascular nerve fibers were tyrosine hydroxylase (TH)-IR (i.e. originated from sympathetic neurons) and the labeling intensity for this protein increased in PMAs from both STZ-NI and STZ-LI rats. The effects of diabetes on mesenteric arteries (MAs) from STZ-NI rats were also determined. Compared to control MAs, nerve-evoked contractions were not changed in MAs from STZ-NI rats. The density of nerve fibers in the perivascular nerve plexus of MAs was reduced but this change could be explained by an increase in vascular dimensions. There was no change in the width or TH immunolabeling of the nerve fibers. These findings suggest PMAs are particularly sensitive to the effects of diabetes. Thickening of the sympathetic nerve fibers in the perivascular nerve plexus of PMAs suggests diabetes may induce axon remodeling. Peripherin and β-tubulin III are structural proteins that are reported to increase in regenerating axons. The second study investigated whether diabetes changed expression of these neuron-specific proteins in PMAs. Western blotting revealed an increase in peripherin protein content of PMAs from STZ-LI rats compared to those from STZ-HI and control rats. The number of fibers in the perivascular nerve plexus that were peripherin-IR also increased in PMAs from STZ-LI rats. Co-labeling with antibodies to peripherin and neuropeptide Y (a marker for sympathetic axons) revealed that peripherin expression increased in sympathetic axons. No changes in β-tubulin III protein content were detected. These findings are consistent with diabetes stimulating remodeling of the sympathetic nerve terminals. No changes in peripherin protein expression were detected in the tail artery, again suggesting that PMAs are selectively affected by diabetes. The third study investigated whether changes in the structure of the perivascular nerve plexus were accompanied by changes in mRNA expression levels (assessed by quantitative RT-PCR) of genes involved in neurotransmission, axon structure, plasticity, neurotrophin signaling and stress. No diabetes-induced changes in mRNA expression were detected in neuronal cell bodies within the L1-L4 sympathetic chain ganglia. In all experiments, changes observed in PMAs from STZ-NI and/or STZ-LI rats were not observed in those from STZ-HI, suggesting they are due to hyperglycemia. The possibility the changes are explained by loss of a direct influence of insulin on the sympathetic neurons/PMAs, however, cannot be excluded. PMAs appear to be particularly vulnerable to the effects of diabetes. This may be explained by these vessels, which are located close to the plantar surface of the hind paw, also being subjected to biomechanical stress from weight-bearing and locomotion. Together the findings indicate that PMAs provide a suitable model for the assessment of treatments for the prevention of diabetes-induced neurovascular dysfunction seen in diabetic humans.
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ItemThe role of chemokines in the retina( 2013)This thesis investigates mice with genetic knock-outs in certain chemokines or their receptors which have shown signs of Age Related Macula Degeneration (AMD) by causing dysfunction to the immune response in the retina. We explored two mechanisms of retinal damage in mice lacking the chemokine, CCL2 or the chemokine receptor, CX3CR1. We tested the underlying retinal function and cellular and structural responses during aging and after light-induced oxidative damage to examine the role of these signaling pathways in the retina.