Florey Department of Neuroscience and Mental Health - Theses
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The role of extracellular ferritin in ferroptosis: Implications for neurodegenerative disease
Abnormal iron elevation and the associated cell death pathway ferroptosis are implicated in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Ferritin, the major iron binding protein of the body, is present in cerebrospinal fluid (CSF) where it has been shown to be associated with Alzheimer’s disease progression, however the function of extracellular ferritin is not known. Ferroptosis involves the degradation of cytosolic ferritin, but whether extracellular ferritin impacts on ferroptosis is unknown. To better understand the role of extracellular ferritin in neurodegenerative diseases, this project aims 1) to characterize extracellular ferritin in response to iron and 2) to investigate the effect of extracellular ferritin on neuronal ferroptotic cell death in vitro. This thesis demonstrated that the expressions of intracellular and extracellular ferritin are associated with intracellular iron levels in both glia and neuronal cell lines. Extracellular vesicles from cell culture and from brain tissue were found to contain ferritin. Though the levels of soluble ferritin secreted by neurons increased with an increase in intracellular iron levels, the levels of vesicular ferritin remained unchanged. The levels of iron in neuronal extracellular vesicles increased with cellular iron burden. Endocytosis of soluble apo-H ferritin protected cultured neurons against ferroptosis. Taken together, changes in biofluid ferritin levels may represent a response to ferroptosis in neurodegenerative diseases.
Antisense oligonucleotide precision therapy in KCNT1 - severe epilepsy
In recent years, the advancement of sequencing technology used in conjunction with thorough clinical evaluation has enabled clinicians to attribute genetic factors to the etiology of epilepsy. A significant number of mutations in genes encoding ion channels have been identified as the cause of the developmental and epileptic encephalopathies (DEE), a group of severe epilepsy syndromes of childhood and infancy characterized by the presence of abundant epileptiform activity, refractory seizures, intellectual disability, developmental regression, movement disorders, and increased mortality. The gene KCNT1 encodes the sodium activated potassium channel subunit KNa1.1. De novo mutations in this gene have been identified in patients with both severe and milder forms of epilepsy. The most commonly associated phenotypes are epilepsy of infancy with migrating focal seizures (EIMFS) and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). EIMFS is situated on the severe end of the spectrum and manifests during the first six months of life with frequent multifocal seizures in addition to developmental plateau or regression. Patients with KCNT1 associated epilepsy typically respond poorly to conventional anti-seizure medications, resulting in an unfavorable prognosis and compromising their quality of life. Biophysical studies in heterologous expression systems have shown that KCNT1 pathogenic variants found in epilepsy result in strongly increased potassium currents. This overall gain of function (GoF) is currently accepted as the primary mechanism of disease in KCNT1 associated epilepsy. To directly address this pathologic mechanism, antisense oligonucleotide (ASO) mediated knockdown was used to test the idea that reducing Kcnt1 expression would be therapeutic in a mouse model of KCNT1 associated epilepsy. This approach was supported by the tolerance for KCNT1 loss of function (LoF) observed in the general population and the mild phenotype found in Kcnt1 knockout mice. Using the CRISPR Cas9 system, an orthologous pathogenic variant found in KCNT1 EIMFS was inserted in Kcnt1 to develop a rodent model. Mice, homozygous for the mutation display frequent spontaneous seizures, abundant interictal activity in the electrocorticogram (ECoG), behavioral abnormalities and early death. Thus, recapitulating key aspects of KCNT1 severe epilepsy and offering a valuable tool for therapeutic screening. Then, an ASO was designed to specifically hybridize with Kcnt1. An additional nontargeting sequence was used as a control. After a single intracerebroventricular bolus injection, homozygous mice showed a marked knockdown of Kcnt1 mRNA and in comparison to control treated animals, displayed almost complete abolition of seizures, prolonged survival, and improved cognition. Exaggerated pharmacology was explored in wild type mice treated with a dose of ASO that produced more than 90 percent knockdown. Here, behavioral measures revealed some anxiety like traits in ASO treated mice, but knockdown was otherwise tolerated. Additional seizure susceptibility in LoF was tested in Kcnt1 knock out mice, which indicated that LoF was not related to a reduction in seizure threshold. To conclude, the preclinical evidence presented in this thesis supports ASO based gene silencing as a therapeutic approach in KCNT1 GoF epilepsies. This body of work provides proof of concept for such approach and encourages the translation of ASO based therapies for genetic epilepsies, in particular for those with an underlying GoF pathomechanism.
Multielectrode Arrays: A tool for modelling genetic epilepsy
Background: Dravet Syndrome (DS) is classified by severe seizures and progressive developmental delay. In 80% of cases, DS is caused by mutations in the SCN1A gene. Despite a growing understanding of the underlying pathomechanisms of DS thanks to animal and heterologous expression models, clinical outcomes for DS remain poor. This has led to the investigation of new high throughput models for diagnosis and drug discovery as an avenue for precision medicine. Multielectrode arrays (MEAs) allow dissociated neurons to form networks on a surface embedded with microelectrodes that record their extracellular activity. This model is promising in the field of precision medicine due to a greater complexity than heterologous expression systems and potential for higher throughput drug screening than can be achieved using animal model. Following this premise, this thesis addresses the following aims. Aim 1: Establish a workflow for culturing primary cortical neurons on MEAs and determine the ideal culture conditions to rapidly generate stable networks for further high-throughput disease modelling and drug screening. Aim 2: Evaluate the potential of the MEA system in modelling DS. Sensitivity of the system will be determined by comparing activity from networks sourced from animal models that exhibit a severe or mild phenotype as a result of SCN1A haploinsufficiency due to their genetic background. Aim 3: Determine the predictive validity of MEAs for screening therapeutic compounds by applying known anti-epileptic drugs to the DS model networks. Method: Primary cortical mouse neurons were cultured on multiwell MEA plates. Various seeding densities and lengths of maturation were tested to determine the optimal parameters for generating stable and reproducible networks. Stability was assessed by comparing a variety of features describing spiking, bursting, and network connectivity. Genetic epilepsy was assessed by culturing neurons derived from mouse models with SCN1A haploinsufficiency. These mice are of different strains and have genetic backgrounds that influence the severity of the SCN1A+/- phenotype (severe or mild expression). Once again spiking, bursting and network connectivity parameters were compared. Finally, stiripentol, lamotrigine and cannabidiol were applied to DS networks and the discriminatory effects between the DS and normal networks were compared. Results: A stable baseline was generated at DIV21 using seeding density 11,646 cells/mm2. SCN1A haploinsufficiency in the C57BL/6 and SV129 backgrounds mimicked what is observed in vivo, with a significant epilepsy phenotype observed in the C57BL/6 background that was not seen in the SV129 background. While stiripentol, lamotrigine and cannabidiol all had significant effects on the overall activity profile of the networks, the response to the drugs was largely indiscriminate between normal and SCN1A+/- networks. Conclusion: A workflow to generate stable networks was achieved and was successfully implemented when characterizing the genetic DS network models. The MEAs captured the DS phenotype and discriminated between severe and mild phenotypic expression. While there were occasional significant differences between normal and SCN1A+/- network responses to known AEDs, these small differences alone were not enough to confirm that this assay showed predictive validity for drug screening. Further studies examining different drugs and concentrations on the networks, as well as more sensitive analysis methods should be performed to further understand the potential of this assay.
A novel viral based strategy to modulate specific cellular inputs in the rat brain
Communication between neurons is essential for brain function and is reliant on neural circuits that work collectively and specifically during behaviour. Several approaches, including, more recently, chemo- and opto- genetics, have been employed to unravel complex neuronal interactions. These existing approaches have allowed precise exploration of the involvement of specific neurons in modulating particular behaviours and physiology. However, both opto- and chemo- genetics lack the ability to define the input to a cell which induces the behaviour. This is required to form an understanding of circuit dynamics. The aim of this thesis was to develop an approach that enables understanding of relevant inputs to specific neurons, to further elucidate neural circuitry. To develop a novel tool that capably inhibits specific inputs to cells, a well-established chemogenetic approach, the allatostatin receptor (AstR) system, was modified. The AstR is an inhibitory insect receptor activated by Ast - both the ligand and receptor are inactive in mammals. Presently, the AstR can be expressed in particular cell types, which can then be inactivated following local application of allatostatin-3 (Ast-3). A prospective advantage, that hasn’t been realised within this approach, was to genetically encode Ast-3 in vivo, thereby targeting Ast-3 to vesicular release pathways and enabling the characterisation of inputs to neurons and association to behaviour or physiology. In order for mammalian neurons to express Ast-3, a viral gene transfer strategy was adopted to express and release Ast-3 from neurons in an activity-dependent manner. Initially, the activity of synthetic Ast-3 and an Ast-3 with a hemagglutinin tag fused to it (HA-Ast-3) was compared, as the precursor was designed to produce HA-Ast-3 due to current limitations in tracking Ast-3 expression reliably. Both peptides were confirmed to have similar functionality at AstR in a cAMP activity assay. A novel viral vector encoding an Ast-precursor which can direct the expression, processing, amidation and trafficking of HA-Ast-3 was then designed and cloned. This viral vector was characterised in vitro and confirmed to produce bioactive HA-Ast-3 although the amount of HA-Ast-3 produced could not be determined. Once validated in vitro, the Ast-3 expressing plasmid vector (pAM-DCA-Ast-IRES-mCherry) was used to produce a recombinant adeno-associated virus (rAAV), which was purified and confirmed to be of high viral titre, qualitatively and quantitatively in vitro. Infective virus was subsequently confirmed in vivo following rAAV injections into the rat brainstem and observing transduced neurons colocalising with the neurochemical marker, tyrosine hydroxylase. Precursor processing and production of Ast-3 were also established by the detection of the HA-tag and neurophysin I, a carrier protein produced and secreted alongside HA-Ast-3. Prior to validating our approach in a behavioural model, viral transduction efficiency in the sensory cell bodies of the nodose ganglion (NDG) was successfully optimised in rats. Simultaneously, peptide trafficking was also inferred by observing transduced vagal afferent axons in the nucleus tractus solitarius (NTS). To validate the novel tool in vivo, it was functionally tested in the visceral sensory afferent system. Sensory information from visceral organs is transmitted by sensory neurons that send their central projections to the nucleus of the solitary tract in the medulla oblongata. The somata of these sensory afferent neurons are located in the NDG. It is known that lesions of the commissural (c) NTS decrease bodyweight gain, with similar observations after vagal blockade. This well-established phenotype and the visceral afferent sensory system were ideal to validate the novel tool, due to the anatomical separation of the NDG and NTS. Initially, rAAV expressing the Ast precursor was injected bilaterally into NDG of male Sprague Dawley (SD) rats. Two weeks post NDG injections, a control and experimental group was formed by injecting lentiviruses expressing either AstR and GFP, or GFP only, into the cNTS. At day 7 post cNTS injection, experimental rats began to have reduced bodyweight gain compared to controls – this deficit in bodyweight continued for the 1-month observation period. This sustained decrease in weight gain resulted in experimental rats weighing significantly less than controls by the completion of the study, presumably due to the inhibition of GI input to NTS neurons that likely affected feeding behaviour. The success of validating the novel tool was followed by an additional behavioural study assessing the integration of visceral sensory afferents that modulate blood pressure. Spontaneously hypertensive rats (SHRs) underwent a similar experimental protocol to SD rats, but were implanted with a telemetry blood pressure (BP) recording device prior to cNTS injections. Comparable to SD rats, I observed significantly reduced weight gain in experimental SHRs, whilst controls had unaltered bodyweight gain throughout the study following NTS injections. In parallel, experimental SHRs exhibited a temporary increase in BP and sustained baroreceptor dysfunction. Baroreceptor sensitivity (BRS) was identified to be causal for elevated BP in experimental SHRs, as BRS was significantly diminished compared to controls throughout the study. These phenotypic observations further confirm peptide/receptor interactions and extension of the existing AstR system to enable characterisation and correlation of inputs to behaviour and physiology. The studies described in this thesis have led to the development of a novel viral based method for chronically inhibiting specific inputs to specific neurons. Inputs can be defined to recognize their role in behaviour and their place in the neural circuit, thereby providing a more coherent depiction of connectivity among neurons. Moreover, a novel method for examining bodyweight and BP homeostasis has been revealed and a potential underlying mechanism involving vagal afferents has been identified. This represents a basis to further explore disease states involving these two phenotypes. Future experiments will involve investigating and characterising specific inputs to other defined cell types throughout the brain. The novel tool can also be refined to enable reversible input inhibition and thereby increase temporal resolution. Overall, my novel method adds a new layer of function to the chemogenetic toolbox for dissection of neuronal pathways which will ultimately lead to better understanding of neural circuits.
Characterising Gene-Environment Interaction Effects on Cognition: Touchscreen Assessment of a Metabotropic Glutamate Receptor 5 Knockout Mouse Model of Cognitive Impairment Subject to Environmental Enrichment
Cognitive abilities are critical for human functioning and flourishing yet unfortunately dysfunction of cognition is a common symptom in developmental, psychiatric and neurodegenerative disorders. Observational studies in humans have demonstrated that variation in cognitive abilities is associated with genetic and environmental factors but are unable to easily examine gene-environment interactions or make causal claims regarding the contribution of each factor. Rodent models enable the investigation of causal effects in cognition as they make feasible the necessary genetic and environmental manipulations. This thesis examined a genetic mouse model of cognitive impairment housed under differing environmental conditions. These animals were cognitively phenotyped using touchscreen operant chambers (touchscreens), a testing method that offers a broader and more consistent assay of cognitive testing in rodents than traditional behavioural tasks. Environmental factors such as exercise and education are known to be associated with improved cognitive performance in humans. A plausibly analogous environmental manipulation to examine these factors is environmental enrichment (EE), which entails any positive modification of the ‘standard housing’ (SH) conditions in which laboratory animals are typically held, usually involving increased opportunity for cognitive stimulation and physical activity. EE has been reported to enhance baseline performance of wild-type animals on traditional cognitive behavioural tasks. We used touchscreens to analyse the effects of EE on a battery of cognitive tasks, hypothesising that EE would enhance the performance of mice. Our hypothesis was partially supported in that EE induced enhancements in cognitive flexibility as observed in pairwise discrimination and reversal learning improvements. However, no other significant effects of EE on cognitive performance were observed. Genetic variation was examined using metabotropic glutamate receptor 5 knockout (mGlu5 KO) mice, which have previously been shown to have deficits of synaptic plasticity and cognitive function with relevance to schizophrenia. While schizophrenia presents with generalized cognitive impairments, the cognitive phenotype of mice lacking mGlu5 has so far only been explored using largely hippocampal-dependent spatial and contextual memory tasks. To address this, we assessed mGlu5 KO mice on a battery of touchscreen cognitive tasks. mGlu5 KO mice were found to have impaired basic discrimination learning, perseverative deficits and motivational alterations. We then combined these two approaches to examine the causal effects of gene-environment interactions on cognition using touchscreen testing of mGlu5 KOs and their WT littermates under SH and EE conditions. Unfortunately, unforeseen health issues with our mice prevented successful completion of the experiment and limits possible interpretation. This research has implications for both understanding the mechanisms of EE as well as informing translational approaches using the mGlu5 KO preclinical model of schizophrenia-like cognitive impairment. Continuing to develop and behaviourally characterise rodent models of genetic and environmental variation should help guide our understanding of the impact of these factors with regards to human cognition in health and disease.
The Role of the Piriform Cortex in Absence Epilepsy
The piriform cortex is critically involved in our sense of smell and engages with the olfactory network via specific neural oscillations. It is also considered to be common node of the focal epilepsy network however it is unknown if it plays a role in absence epilepsy. The main focus of this thesis was to determine whether there is any pre-seizure involvement of the piriform cortex in absence epilepsy. Using a rat model of absence epilepsy and non-epileptic controls, multi-channel microelectrode arrays were implanted in the piriform cortex and mediodorsal thalamus, the piriform cortex’s direct link to thalamo-cortical networks underlying absence epilepsy, to record multiunit clusters and field potentials. Three distinct signal processing analyses were developed and applied to the study of the pre-seizure onset connectivity changes in the piriform cortex which encompass four distinct but interrelated research aims. The first aim was to determine the transient firing patterns of multiunit clusters within and between the piriform cortex and mediodorsal thalamus around the onset of absence seizures. The phase locking between multiunit cluster spikes and neural oscillations (spike-LFP phase locking) which govern olfactory network communication was shown to change within the piriform cortex prior to seizure onset, suggesting it contributes to seizure initiation. Early changes in spike-LFP phase locking suggested possible pre-seizure onset changes in local and long range functional connectivity of the piriform cortex and mediodorsal thalamus. A critical issue of analysing functional connectivity using microelectrode arrays is overlapping effect of neighbouring electrodes. Hence the second aim of this thesis was determining the optimal method for resolving the effect of neighbouring electrodes to accurately estimate functional connectivity. Band-amplitude fluctuation orthogonalization followed by inter-site phase clustering estimates was shown to be the optimal method. This finding has important implications for future medical bionics consisting of electrodes with small inter-electrode distances. Following the optimal method of functional connectivity being derived, the third aim of this thesis, determining the local and long range functional connectivity of piriform cortex and mediodorsal thalamus, could be addressed. Results demonstrated pre-seizure onset increase in local functional connectivity within the piriform cortex and increases in long range connectivity with the mediodorsal thalamus in neural oscillatory bands of the olfactory network. The final aim of this thesis was to determine effective connectivity of the piriform cortex to mediodorsal thalamus in order to determine changes in information flow from the piriform cortex to the mediodorsal thalamus in absence epilepsy. A novel method of effective connectivity was developed. The results demonstrated pre-seizure onset increases in effective connectivity of a similar frequency band to where absence seizures localize. This suggests the piriform cortex to mediodorsal thalamus pathway is predisposed to propagating absence seizure activity. Overall, the piriform cortex was discovered to contribute to the initiation of absence seizures and the piriform cortex to mediodorsal thalamus pathway is recruited as part of the absence epilepsy network. The brain connectivity methods developed in this thesis can be applied to the study of brain regions and neural pathways of epilepsy and other neurological disorders.
Exploring the interaction between iron and tau in neurodegeneration
There is an emerging link between the accumulation of iron in the brain and abnormal tau pathology in a number of neurodegenerative disorders, such as Alzheimer’s disease (AD). In brains of AD clinical cases, high levels of iron are reportedly co-localized with tau in neurofibrillary tangles (NFTs) – the neuropathological hallmark of a class of disorders referred to as tauopathies. Furthermore, iron is reported to act as a cofactor for NFT formation through several mechanisms that include regulating tau phosphorylation and inducing conformational changes of tau via a putative iron binding motif, potentially leading to tau aggregation. These data, together with our own work showing that tau has a role in mediating cellular iron efflux, provide evidence supporting a critical tau:iron interaction that may impact both the symptomatic presentation and the progression of disease. Importantly, this may also have relevance for therapeutic directions, and indeed, the use of iron chelators such as deferiprone (DFP) and deferoxamine have been reported to alleviate the phenotypes, reduce phosphorylated tau levels and stabilise iron regulation in various animal models. The work outlined in this thesis focused on gaining further insight into the putative tau:iron interaction in neurodegeneration using a mouse model of tauopathy (rTg(tauP301L)4510). Spatial and temporal analysis of the brain iron profile in rTg(tauP301L)4510 and wild-type (WT) littermates revealed significant age-related elevations in brain iron levels, with rTg(tauP301L)4510 mice accumulating significantly more iron (250%) in the brain compared to WT mice. Based on these findings, it was hypothesized that by chelating iron with DFP in rTg(tauP301L)4510 mice would slow down disease progression and decrease tau pathology. Using two different experimental paradigms, the effect of iron chelation therapy was examined in young rTg(tauP301L)4510 mice (4-months) for 16 weeks and in aged rTg(tauP301L)4510 mice (12-months) for 4 weeks. Treatment with DFP under both paradigms decreased brain iron levels and reduced pathological tau (that includes sarkosyl-insouble tau and tau aggregates). Behavioural analysis revealed a strong trend towards improved cognitive function in young rTg(tauP301L)4510 mice and a significant improvement in short-term spatial reference memory in aged rTg(tauP301L)4510 mice. Interestingly, our results also revealed anxiolytic properties of DFP that was indicated by a decrease in hyperactivity in both young and aged rTg(tauP301L)4510 mice. The findings from this thesis suggest that a disruption in tau and an increase in iron both play an important role in neurodegeneration. This is the first study (to our knowledge) that demonstrates the compounding effect of tau dysregulation (caused by the overexpression of human mutant tau in rTg(tauP301L)4510 mice) and age on brain iron levels in an animal model of tauopathy. Furthermore, this study also reveals the effects of DFP on tau pathology and behaviour in rTg(tauP301L)4510 mice, which has not previously been reported. As this compound is moving towards clinical translation, this thesis demonstrates the potential benefits of chelating iron as a therapeutic strategy for tauopathies such as AD, frontotemporal dementia and progressive supranuclear palsy.
Investigating Brain Repair and Development Using Stem Cells
The directed differentiation of human pluripotent stem cells into neuronal subtypes has generated immense enthusiasm that they could be used therapeutically or to study development in vitro. Two areas of particular interest are the replacement of midbrain dopaminergic neurons that degenerate in Parkinson’s disease and the ability to study the early development of the human cortex. The loss of midbrain dopamine neurons in Parkinson’s disease leads to a breakdown in basal ganglia circuitry and motor dysfunction. Previous clinical trials utilizing fetal dopamine tissue have provided proof-of-principle that transplantation of new dopamine neurons can relieve motor symptoms for up to two decades. However, the widespread use of fetal tissue in the clinic presents multiple ethical and logistical hurdles. As a result, the generation of human pluripotent stem cell-derived dopamine neurons has been an area of intense focus in recent years. Current protocols are not amenable to clinical translation and generate dopamine neurons that poorly reinnervate the striatum following transplantation. Here we develop a fully-defined protocol for midbrain dopamine generation and improve transplantation survival, plasticity and function via overexpression of glial cell line-derived neurotrophic factor. In contrast to dopaminergic differentiation strategies, protocols for deriving neurons of the neocortex are relatively limited, generating heterogenous populations of progenitors, neurons and glia. The human neocortex is arguably the most altered structure during mammalian evolution and underlies the cognitive abilities that define human intelligence. However, the majority of research into cortical development has been carried out in frog, chick or mouse models. Therefore, there is an unmet need to investigate how the relative complexity of the human cortex is developed. In particular, the intrinsic and extrinsic cues that control temporal development and drive the progressive generation of neuronal subtypes that form the six-layered mammalian neocortex remain unknown. Here we investigated the role of FGF-ERK signaling in the development of early-born, deep layer neurons of the neocortex in a reductionist pluripotent stem cell model. We find that FGF-ERK signaling, in part, modulates the timing and temporal progression of neocortical progenitors. Together, these studies advance the use of pluripotent stem cell tools to model neural development and to aid in neuroregeneration.
Role of relaxin-3/RXFP3 forebrain networks in descending control of pain in the mouse
Pain is a complex biological phenomenon that is beneficial and necessary for our survival, warning of changes and hazards in the environment that compromise optimal function; and it is a highly regulated biological process. However, continuous activation of this key peripheral and central nervous system signalling system results in maladaptive changes characterized by altered tissue and organ structure and activity. Clinically, pain lasting more than 3 months is termed chronic pain and it is under these conditions that it can become a major burden for affected individuals. It is estimated that ~20% of the European population suffers from moderate-to-severe chronic pain, and ~15% of US citizens suffers from some form of chronic pain Chronic pain is also accompanied by serious social and economic burdens, making research in this field a high priority globally. Chapter 1 provides a general overview of pain and nociceptive signalling, detailing the most important concepts in the pain research field, including the main neural pain pathways, for understanding the research covered elsewhere. Chapter 2 describes the role of neuropeptide signalling in nociception, at the different levels of pain transmission, and introduces the relaxin-3/RXFP3 system. The Chapter details the role of different opioid and non-opioid peptides in modulating the nociceptive response, and includes research I performed that was included in a recently published original research article, as well as a the text of a review article, which focuses on the role of non-opioid neuropeptide systems in nociceptive signalling. Nociceptive signals are heavily modulated by an abundance of neurochemical signals, including neuropeptides. Neuropeptides are usually expressed in prepro-forms, and undergo processing (e.g., cleavage) to produce their mature form. Many neuropeptides were first described in the second half of the 20th Century, but as new biochemical and cellular assays were developed, more neuropeptides were discovered. Most neuropeptides signal with high specificity via G-protein-coupled receptors (GPCRs) linked to different G-proteins. The presence of neuropeptides and/or their receptors in central nervous system (CNS) areas linked to nociceptive processing and transmission suggests putative roles for these systems in the control of nociception. Relaxin-3 is a neuropeptide that is synthesized by populations of neurons in a brainstem region known as the nucleus incertus (NI) and some other hindbrain regions. Since its discovery 18 years ago, relaxin-3 has been linked to the control of a wide range of behaviours such as anxiety, arousal, and reward-seeking behaviours, through activation of the Gi/o-protein-coupled receptor, RXFP3. These putative roles of relaxin-3/RXFP3 signalling, as well as its shared functions with other neuropeptide systems that modulate nociception, suggested a possible link between RXFP3 activation and modulation of pain sensitivity. Chapter 3 describes current knowledge of relaxin-3/RXFP3 signalling and function, and provides a context for my research, describing the relevant literature and pilot data that led to the development of the project. Specifically, this Chapter describes behavioural data that implicate the relaxin-3/RXFP3 system in the modulation of nociception. My initial studies assessed the effect of RXFP3 activation and inhibition on mechanical and thermal pain sensitivity in normal and persistent pain conditions in male, adult mice. These studies demonstrated that central administration of the RXFP3 agonist peptide, RXFP3-A2, via intracerebroventricular (icv) injection, produced relief of mechanical, but not thermal, pain sensation. Moreover, icv injection of the RXFP3 antagonist peptide, R3(B1-22)R, augmented mechanical and thermal pain sensitivity. These data suggest that relaxin-3/RXFP3 signalling has a tonic action in maintaining mechanical and thermal pain thresholds, and the potential for activation of RXFP3 to produce pain relief. Chapter 4 describes neuroanatomical aspects of descending control pathways and the overlap with relaxin-3/RXFP3 networks, including previous findings regarding the distribution of these networks in the brain of mice and rats. These data provide a context for my findings, which include the assessment of levels of neuronal activation in different brain areas following RXFP3 activation, and neurochemical phenotyping of RXFP3 mRNA-expressing neurons in these areas, using a combination of immuno- and in situ hybridization histochemistry. Using established immunohistochemical methods, I examined the possible involvement of different brain areas in these effects, by assessing the number of c-Fos-positive cell nuclei and the intensity of Fos-immunostaining under different conditions. These studies revealed no significant differences in the number of c-Fos-positive cell nuclei or staining intensity in vehicle and RXFP3 agonist treated mice. Additionally, I determined the level of correlation between the numbers of nuclei counted in different areas under the same conditions, but found no correlation between brain areas, including the anterior cingulate cortex (ACC), bed nucleus of the stria terminalis (BNST), and the amygdala, regardless of the condition analysed. Further characterization of relevant brain areas using multiplex fluorescent in situ hybridization revealed that RXFP3 mRNA is expressed by discrete populations of neurons in areas such as the ACC, BNST, thalamus, and amygdala. I evaluated the co-expression of Rxfp3 mRNA with mRNA encoding the specific neuronal markers, parvalbumin and somatostatin, and determined the relative proportion of RXFP3 mRNA positive neurons that express one, both, or neither of these transcripts. Chapter 5 describes the comorbid states that influence nociceptive signalling in the context of chronic pain, and the relevance of synaptic plasticity for the expression of these comorbid disorders. Additionally, I describe experimental data on the possible expression of pain-induced anxiety-like behaviours. In these studies, I examined the possible expression of anxiety-like behaviour in mice subjected to the same persistent pain model used to assess the effects of RXFP3 modulation on mechanical and thermal sensitivity. In two separate behavioural tests, anxiety-like behaviours were not altered in mice with persistent hindlimb pain relative to control, suggesting this model may not be sufficient to induce increased anxiety in mice, and that effects of RXFP3 modulation observed specifically targeted nociceptive transmission. Overall, my findings implicate the forebrain relaxin-3/RXFP3 system in the control of pain transmission, providing new opportunities for the development of therapeutic tools for pain management, by targeting a neuropeptide system that impacts several behaviours that are altered under chronic pain conditions. Chapter 6 will discuss my findings in relation to the relevant literature, and describe the future directions of these research studies, and their translational potential for human therapeutics.
The nature of memory decline and learning dysfunction in preclinical Alzheimer’s disease
Background: In cognitively normal (CN) older adults, abnormal levels of amyloid-beta indicates that the pathophysiological process of Alzheimer’s disease (AD) has begun, although it may be up to 20 years before these individuals meet clinical criteria for dementia Measurement of episodic memory is a cornerstone of neuropsychological assessment in AD, which is consistent with clinicopathological studies showing that the earliest neuronal loss begins within the medial temporal lobe (MTL), a brain region crucial for learning and memory. Neuropsychological compendia detail many tests of episodic memory well validated for use in AD, however, there are fewer standardized neuropsychological tests of learning validated for use in AD. The overarching aim of this thesis was to investigate the relationship between AD pathological markers such as beta-amyloid, and trajectories of neuropsychological performance on memory and learning tasks in CN older adults. Methods: The nature and magnitude of cognitive impairment and decline associated with abnormal levels of amyloid beta in CN older adults was determined via meta-analysis of studies published from 2012 to 2016. Indices of memory and learning on computerised cognitive tests were examined in amyloid negative and amyloid positive CN older adults, with estimates of impairment and decline reported over periods of 18 and 36 months. Finally, a novel web-based learning task was designed and validated to enable the modelling of learning and memory in amyloid negative and amyloid positive CN older adults. Results: Meta-analytic estimates showed significant impairments of small to moderate magnitude (Cohen’s d’s 0.15-0.32) associated with abnormal amyloid in CN older adults in the domains of visuospatial function, processing speed, episodic memory, executive function, and global cognition. Significant decline of small-to-moderate magnitude associated with abnormal amyloid in CN older adults were found for semantic memory, visuospatial function, episodic memory, and global cognition (Cohen’s d’s 0.24-0.30). Rates of learning on the computerised cognitive tests at baseline were equivalent between the amyloid negative and amyloid positive CN groups, while significant differences in longitudinal trajectory of performance was evident. The amyloid negative group showed significant practice effects over time, which were absent in the amyloid positive group, suggesting an inability to learn from repeated exposure. Daily measurement of cognition via a remote, online assessment was sensitive to both age and pathology related changes in ability to learn new information over one week in CN older adults. The magnitude of this effect was very large (Cohen’s d = 1.50), approximately three times larger than current longitudinal estimates of cognitive decline in amyloid positive CN individuals over a year or more. Conclusions: Results indicated that the presence of abnormal amyloid in CN older adults has a significant, negative effect on a range of cognitive domains, although the magnitude of this effect is only small-to-moderate and is largest for memory. Furthermore, amyloid positive CN older adults display aberrant performance on memory and learning tasks over time in the form of a lack of practice effects. This suggests that in amyloid positive CN individuals, the processes by which learning occurs as a function of repeated exposure and presentation to stimuli may be compromised. The large magnitude of impairment in daily learning on the web-based task highlights that the ability to learn new information is dysfunctional in the preclinical stage of AD, and that a sensitive way of measuring cognition in very early disease stages is via repeatable learning assessments. This suggests the need for a paradigm shift towards understanding cognitive dysfunction in preclinical AD as dysfunctional learning, rather than memory.
Advanced technologies for neural transplantation: new approaches for neural repair and modelling diseases
Many neurological diseases affecting the central nervous system are still incurable because of the scarce and incomplete knowledge of disease mechanisms and the lack of effective treatments. Despite the effort, these disorders represent a challenge for medical science. The poorly representative disease models and technical limitation in deriving and characterising neural cells after transplantation into animal model hindered the development of new therapies. Cell therapy has become a promising therapeutic approach for diseases characterised by cell loss, although significant hurdles need to be overcome before it can be progressed into mainstream therapies. Today, brain tumours remain incurable and there are no therapies able to cure these diseases. Well-defined GBM in vivo models and cellular technologies might be useful for the investigation of pathophysiological mechanisms regulating brain tumours, helping the development of new therapeutic strategies. This thesis explores the potential and the limitations of using human gene manipulated cell lines into different disease contexts: cell therapy for motor neuron disease (MND) and brain tumours. Using a differentiation protocol, we investigated the possibility to derive and characterise spinal motor neurons (spMNs) derived from human pluripotent stem cells (hPSCs). We demonstrated that the specification of different spMN subtypes can be regulated using a combination of small molecules for defined timing. These results contribute to clarify important human developmental aspects and to propose an in vitro source of spMNs useful to study disease mechanisms and propose a cell-based therapy for MND. Accordingly, we conducted a systematic evaluation of the abilities and properties of spMNs derived from different reporter hPSC lines after transplantation into the rat spinal cord. This research highlight that grafted cells require a period of 6 months to mature and integrate into the host spinal motor circuitry. This study demonstrates that stem cell therapy might be a promising approach for treating diseases affecting the central nervous system and characterised by cell loss. Two reporter tumour cell lines derived from patient biopsies were grafted into the brain of athymic mice to establish, study and characterise an animal model of GBM. The results highlight the utility of tagged cells in brain tumour research, which allow us to better understand not only the tumour generation and progression but also evaluate the efficacy of new treatments. Furthermore, we presented a new tool for studying the role of ions in GBM, the chemogenic platform DREADD. GBM cells were engineered to express Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and used to chronically manipulate calcium and potassium homeostasis in order to evaluate their effect on cell metabolism and cell behaviour in vivo. Our results show that these ions are involved in key tumour mechanisms such as cell proliferation and migration. This work contributes to a mixed literature where there has been conjecture as to whether the dysregulation of ions homeostasis is responsible of key tumour mechanisms. Taken together, these studies offer new tools that might be useful for the understanding and comprehension of biological events related to a huge number of diseases affecting the central nervous system and will have implications in clinical approaches for treating MND and GBM.
The role of copper in neurodegenerative ubiquitinopathy
Ubiquitin (Ub) is one of the most stable and soluble intracellular proteins. Intriguingly, the accumulation of intracellular Ub is associated to protein aggregates that are characteristic features of neurodegenerative diseases. Nevertheless, the factors that promote Ub aggregation and/or accumulation are unknown. Conversely, in vitro studies have shown that the interaction between Ub and cupric copper (Cu(II)) promotes the formation of high molecular weight (HMW), SDS and trypsin resistant, named as Ubres species. There is a general consensus that bioavailable copper only exists as Cu(I) in the cytosol, and there is limited evidence to support the presence of Cu(II) in this compartment. However, in vivo Ub denaturation may require a pool of labile Cu(II) that, to date, has not been identified. Cu(I) is maintained in the cytosol by a complex network of copper ligands and chaperones, and by reducing agents, predominantly glutathione (GSH). Brain glutathione depletion as well as copper dyshomeostasis are features of neurodegeneration. Therefore, we hypothesized that intracellular Cu(II) is elevated by the loss of GSH in these diseases. In this thesis, using XANES and a fluorescent Cu(II)-probe (CBS), we demonstrated that labile Cu(II) pools are present in the cytosol. Moreover, our results showed that decreasing GSH levels in cells, increases intracellular Cu(II) levels. Loss of Cu(I) and the emergence of Cu(II) may exacerbate the metabolic brain lesions in these neurodegenerative disorders. In fact, the increase in intracellular Cu(II) concurred with an increase in the levels of HMW Ub species in a SDS-insoluble cellular fraction, which were resistant to enzymatic proteolysis, characteristic features of Ubres species. We also found the presence of these Ubres species in brain tissue from Alzheimer’s disease and Lewy body dementia diagnosed patients. The outcomes of this research shed lights on the mechanism that may explain the ubiquinopathy of these neurogenerative disorders and may help to develop novel therapeutic strategies for these diseases.