Florey Department of Neuroscience and Mental Health - Theses
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ItemMimicking the effects of gene x environment interaction with small non-coding RNAs associated with anxiety and depression( 2023-06)Depression and anxiety are debilitating psychiatric disorders, which significantly depend on genetic predisposition and changing environments. One in three patients is resistant to current antidepressant treatment, highlighting the need for novel therapeutics to improve patient outcomes. Understanding how combination of genetic and environmental factors contribute to the development of depression and anxiety through microRNAs will aid the development of novel therapeutics for affective disorders. During my PhD, I explored how combinations of genetic and environmental factors affect expression of brain miRNAs in a mouse model of treatment-resistant depression (TRD). In the first part of my PhD project, I described miRNA composition in the hippocampus of the serotonin transporter knock-out (5-HTT KO) mice compared to wild-type mice in standard housing conditions. 5-HTT KO mice have increased depression-like behaviour and do not respond to classic antidepressant treatment, which make them a good model of TRD. This is the first study describing miRNA profiles in the brain tissues of a mouse model of TRD. We discovered novel miRNAs, which could be used as markers of TRD. The enrichment analysis of target genes of these miRNAs revealed pathways relevant to stress response, highlighting the importance of changing environments in the development of TRD. Next, I described miRNA profiles in 5-HTT KO mice after stress and exercise, which was the first study on how gene x environment interactions affect miRNA composition in the brain of mice genetically predisposed to TRD. Based on these data, I targeted several miRNAs using novel approaches of miRNA modulation to mimic beneficial effects of exercise and stress-reduction to develop new treatment for TRD. I observed a significant antidepressant-like effect after treatment with miRNA mimic, which proved that miRNAs can not only be a marker of depression but are themselves targets for the treatment of depression. The follow-up bioinformatic analysis revealed that miRNA mimic treatment enhanced neuroplasticity and neurotrophic support by activating cascade of genes, which protect or rescue neurons that are vulnerable after stress or other insults. This study provided the first description of miRNA alteration in a mouse model with serotonergic dysfunction and highlighted the potential of these miRNAs as therapeutic targets for TRD. These results provide further evidence to highlight the importance of considering gene-environment interactions in the management and treatment of affective disorders. Ultimately, these findings will aid in the development of genetically-informed precision medicine for serotonergically-dependent refractory mental illness.
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ItemInterrogating TAM receptor activation for therapeutic benefit in multiple sclerosis( 2023-05)Multiple sclerosis is an autoimmune neurodegenerative disease characterized by demyelination and axonal damage in the central nervous system (CNS). Remyelination plays a crucial role in axonal protection and functional recovery. The GAS6 protein has emerged as a promising candidate for enhancing remyelination. This thesis aims to uncover the underlying mechanisms through which GAS6 exerts its pro-myelinating effect. Additionally, it delves into the pharmacokinetic properties of this protein, emphasizing the significance of gamma-carboxylation in the GAS6 GLA domain in mediating myelination in vitro, along with its residence time in the CNS. The latter part of this doctoral work demonstrates the utilization of the PEGylation approach to extend the brief CNS residence time of the GAS6 protein. The data from this project suggest that the Tyro3 receptor significantly contributes to GAS6's pro-myelinating effect in the CNS after demyelination. This observation appears to be partly driven by mature oligodendrocytes and seems largely independent of the inflammatory response. This research also underscores the importance of gamma-carboxylation within the GAS6 GLA domain for its pro-myelinating effect in vitro. Additionally, it reveals that GAS6 exhibits a short residence time of less than two hours in the murine brain. Using the PEGylation approach, a bioactive variant of this protein with an extended CNS residence time was successfully generated. In summary, this work identifies the Tyro3 receptor as a potential target for the GAS6 pro-myelinating effect and sheds light on some pharmacokinetic properties of the GAS6 protein, particularly highlighting the significance of its post-translational modifications and brief CNS residence time.
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ItemSperm noncoding RNAs as mediators of paternal epigenetic inheritance modulating offspring affective and social behaviours( 2023-06)Background: Studies have shown that paternal environmental conditions prior to conception can influence the innate behaviours of their offspring, and the evolutionary impacts of such intergenerational effects are therefore of considerable interest. Epigenetic mechanisms have been shown to underlie this inheritance, as the microinjection of sperm small noncoding RNAs into fertilised oocytes induces reprogramming of the early embryo, which is thought to be responsible for the differences observed in adult phenotype. Our group previously showed in a mouse model of daily stress that glucocorticoid treatment of adult male breeders prior to conception leads to increased anxiety-related behaviours in male offspring, and this accompanies changes in the paternal sperm small noncoding RNA profile. Additionally, in a model of paternal running wheel voluntary exercise preconception, our group observed lower anxiety levels and a more robust fear extinction memory in the male offspring, as well as changes in paternal sperm small noncoding RNA expression. Aims: In this study, we aimed to understand the transgenerational effects of paternal stress exposure on the social behaviour of the male progeny and its potential influence on reproductive success by analysing its effects on social reward, male attractiveness and social dominance. We also assessed the paternal sperm long noncoding RNA profile following glucocorticoid treatment or running wheel voluntary exercise. We used CaptureSeq, a sequencing technique that is more sensitive than the ones used in other studies in the field. We next sought to determine the role of sperm long noncoding RNAs by microinjecting them into fertilised oocytes. Results: We report that paternal corticosterone-treatment was associated with increased display of subordination towards other male mice. Those mice were unexpectedly more attractive to female mice while expressing reduced levels of the key rodent pheromone Darcin, contrary to its conventional role in driving female attraction. Furthermore, no overt differences of the prefrontal cortex transcriptome were found in the offspring, implying that peripheral mechanisms are likely contributing to the phenotypic differences. No transgenerational differences were observed. Paternal corticosterone exposure led to dysregulation of sperm long noncoding RNA expression, which encompassed lncRNAs, circular RNAs and transposable elements. Although they have poor functional annotation, bioinformatic approaches indicated their expression in the brain, as well as their potential in regulating brain function. Running wheel exercise led to hundreds of downregulated lncRNAs, as well as transposable elements, and bioinformatic strategies predicted their function in biological processes, such as cell adhesion. Lastly, we separated and isolated the sperm long noncoding RNA population after glucocorticoid exposure and performed microinjections into fertilised oocytes. We observed that the resulting adult offspring had lower body weight and altered behavioural responses in the light-dark box and Porsolt swim test. Conclusion: Our findings highlight the potential of paternal stress to affect intergenerational (mal)adaptive responses. They also provide insights into the potential biology of long noncoding RNAs and highlight that efforts to annotate their function are highly necessary for the understanding of the mechanisms underlying the epigenetic inheritance. We are also the first to show that voluntary exercise modulates sperm long noncoding RNAs expression.
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ItemGastrointestinal complications of Parkinson's disease( 2023-07)Parkinson’s disease (PD) is a progressive neurodegenerative disorder that can also affect gastrointestinal (GI) function. External factors, such as chronic stress, have been shown to play a role in the development of PD. GI disorders, include constipation that is a common non-motor symptom of PD that affects around 80% of patients. Defecation is under voluntary control, beginning from centres in the brain, sending signals down the spinal cord to the lumbosacral defecation centre. However, the mechanisms involved in PD-associated constipation remain unknown. The toxin affecting catecholamine neurons, 6-hydroxydopamine (6-OHDA), is commonly used to model PD and causes constipation when injected into the medial forebrain bundle (MFB). In Chapter 3, I have shown that 6-OHDA rats developed motor dysfunction as well as deficits in colon motility, exhibited by a significantly increased bead expulsion time compared with shams. Furthermore, the number of contractions and propulsion of contents by 6-OHDA rats was significantly reduced in comparison to shams after the administration of capromorelin, a colokinetic that acts on the lumbosacral spinal cord, indicating that 6-OHDA animals have reduced responsiveness of the defecation circuits. Enteric neuropathy was observed in the distal colon, revealing that 6-OHDA lesioning of the MFB has downstream effects at the cellular level. Based on the results of Chapter 3, it leaves open two questions that are addressed in Chapter 4: is the responsiveness of neurons downstream from the spinal defecation centres affected by 6-OHDA and could leakage of 6-OHDA into the periphery contribute to the changed responsiveness. In order to investigate the first question, I used prucalopride, another colokinetic which acts on 5-HT4 receptors in the ENS, and found that there was a trend for reduced responsiveness to prucalopride in 6-OHDA rats compared with shams. This suggests there may be a deficiency at the level of the ENS. To address the second question, I peripherally injected 6-OHDA in rats and shown that peripheral injection of 6-OHDA did not induce motor impairments and GI dysfunction in those animals, with no loss of dopaminergic neurons in the nigrostriatal pathway and no change in bead expulsion time. This toxin-induced model is used widely in rats, however the use of 6-OHDA in mice has not been well established. In Chapter 5, I characterised the GI phenotype in the 6-OHDA mouse model. The slowing of bead expulsion from the colorectum and enteric neuron stress exhibited by 6-OHDA rats are also observed in 6-OHDA lesioned mice, providing a clear indication of compromised ENS function. The results suggest there are downstream, trans-synaptic consequences of 6-OHDA lesioning of the MFB. Chronic stress is able to exacerbate motor dysfunction and increase dopaminergic neuron loss in animal models of PD. The aim of Chapter 6 was to investigate whether chronic isolation stress exacerbates GI dysfunction in a genetic, A53T, mouse model of PD. It was shown that A53T mice displayed progressive motor deficits and slowed colonic bead expulsion and whole gut transit, and that chronic stress worsened these PD symptoms. In summary, my studies presented in this thesis have revealed that there are downstream consequences of lesion of the MFB with 6-OHDA in both rats and mice, compromising neuronal functions in the brain, down to the lumbosacral defecation centre and the ENS of the colorectum. Moreover, chronic stress can exacerbate PD-associated GI dysfunction, highlighting the impact of stress on the gut-brain axis in PD.
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ItemNo Preview AvailableNecessary Steps to Advance Stem Cell-Derived Neural Transplantation Therapy to the Clinic for Stroke( 2023-04)Current treatments for ischaemic stroke are restricted to <10% of patients due to time constraints, leaving many patients untreated. Stem cell-based therapies offer a promising alternate treatment for stroke. Recent advancements in human pluripotent stem cell (hPSC) differentiation protocols to generate defined neuronal populations, such as cortical progenitors, suitable for neural transplantation have enabled preclinical studies to investigate the functional efficacy of this therapeutic approach. Despite evidence that these neural grafts provide neuroprotection and/or replacement of lost neural circuits to reverse motor and cognitive deficits in animal models, there are several outstanding requirements that require attention prior to translation into the clinic. While the FDA may not require animal testing for drug development prior to clinical translation, the testing and advancement of stem cell-based therapies critically relies on models that recapitulate the disease or injury. Although a number of ischaemic stroke models exist in rats, the intracerebral delivery of the potent vasoconstrictor endothelin-1 (ET-1) provides a robust focal ischaemic model, inclusive of a primary insult and vulnerable penumbra capable of being salvaged by stem cell therapy, that is highly reproducible, stable, and relatively quick and easy to induce. However, efforts to achieve a similar model using ET-1 in mice has been underwhelming likely due to insufficient vasoconstriction, thereby hindering the utility of transgenic models that enable probing of key mechanisms, and their use in high-throughput studies for vigorous preclinical testing therapies. In Chapter 3 of this thesis, intracerebral co-administration of ET-1 in the mouse brain with the non-selective nitric oxide synthase inhibitor L-NAME and selective antagonist of vasodilatory endothelin subtype-B receptor RES-701-1 were shown to generate a larger infarct, induce an inflammatory response and exhibit greater motor deficit, as assessed by the ladder walk test in comparison to animals administered with ET-1 alone. Here, we advanced current ET-1 mouse models of ischaemic stroke to facilitate the use of mice for transgenic and/or high-throughput studies. Necessary for clinical translation is the validation that cell products, in this case human pluripotent stem cell (hPSC)-derived neural grafts, generated within current Good Manufacturing Practice (cGMP) clinical conditions behave in a comparable manner to the pre-clinical cell product counterpart. Chapter 4 characterises the differentiation and maturation of a cGMP hPSC line into cortical progenitors in vitro, showing comparable efficacy to generate TBR2+ cortical progenitors as our previously characterised cell lines. Noting the necessity to implant neural progenitors (which engraft with higher cell survival than post-mitotic neurons), we examined the optimal cortical progenitor age for transplantation (day of differentiation 25, 30 and 35) and assessed the impact of a NOTCH signalling inhibitor (aimed at forcing progenitor cell-cycle exit) to circumvent risks associated with neural overgrowths. Assessment of resultant grafts revealed that the treatment of NOTCH inhibitor prior to cell implantation prevented tumour formation. Furthermore, younger cortical progenitors generated a higher proportion of deep layer cortical neurons, and superior and appropriate anatomical innervation of the host brain. We also demonstrated that cryopreserved cGMP hPSC-derived cortical progenitors can generate viable grafts, validating cryopreservation procedures (necessary for clinical translation) for the generation of an ‘off-the-shelf’ cell product for transplantation. This study achieved the first steps towards translating cortical progenitor cell transplantation to repair the ischaemic stroke injured brain. One of the greatest concerns of human pluripotent stem cells in regenerative medicine is the risk of tissue overgrowth/tumours. The incorporation of suicide genes (such as the herpes simplex virus-thymidine kinase gene) into hPSCs can improve the safety of resultant neural grafts by eliminating unwanted cells post-transplant using a systemically delivered prodrug (ganciclovir). A challenge of this approach, however, is the short half-life of the drug in vivo, that is required to coincide with S-phase of the cell cycle (in order to ablate dividing cells). To address this, Chapter 5 utilised a self-assembling peptide hydrogel, with mimetics similar to the brain’s extracellular matrix, to encapsulate the prodrug and consequently prolonged its presentation within the brain. This “one-off” prodrug delivery treatment enabled the efficient activation of the suicide gene in more graft-derived cells, consequently ablating a greater number of proliferative cells within a pluripotent stem cell-derived teratoma in comparison to the standard and suboptimal regime of daily systemic prodrug delivery. Furthermore, within neural grafts, the therapeutic neuronal cells were enriched following our hydrogel-based prodrug delivery. Here, we demonstrated efficient and safe activation of the suicide gene within neural grafts, facilitating the translation of neural transplantation to the clinic. These studies collectively provide important steps in the testing and development of hPSC-derived cortical progenitors and contribute to the advancement of stem cell-based therapies for stroke towards the clinic.
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ItemArtefact reduction methods for EEG-fMRI and fMRI( 2023-01)Advances in technology have led to increasing use of neuroimaging methods, such as electroencephalography (EEG), magnetoencephalography (MEG) and functional MRI (fMRI), that allow researchers to study brain function in a non-invasive way. However, functional neuroimaging studies are frequently contaminated by noise, or artefact, present in the data, and therefore, methods to remove or minimise artefact are crucial for obtaining accurate, reproducible results. This thesis examines artefact reduction in two different functional neuroimaging modalities - fMRI, and simultaneous EEG-fMRI. Firstly, a systematic review of artefact reduction for EEG-fMRI is presented, which contains two sub-reviews: a review of artefact reduction methods available, and a review of artefact reduction methods used in contemporary studies. The first review successfully distils all published artefact reduction methods from a twenty-year period into clear recommendations for researchers using this imaging modality. The second review found that from EEG-fMRI papers published over a four-year period, most users were selecting one or two similar methods, with up to 15% of users not adequately describing their methods used. A key finding from the work was that hardware-based methods of recording artefact are preferable to data-driven approaches, yet data-driven approaches are most commonly used. The second part of this thesis looks at motion artefact in fMRI studies - specifically, the utility of novel hardware - carbon wire loops (CWL) - for detecting head motion. We aimed to determine whether CWL could detect sub-volume motion onset and if so, whether CWL would suggest further data censoring when using a commonly used data-driven method, Framewise Displacement (FD). We hypothesised that the volume prior to motion onset in FD may often be motion affected, due to motion occurring part-way through volume acquisition, and that this motion would be detected by CWL. The results showed that CWL successfully detects motion onset at a sub-volume (slice-based) level. In addition, CWL detected motion in the volume prior to FD onset in an average of 42% of cases. It was concluded that censoring the volume prior to FD onset should only be done when rigorous motion rejection is necessary and CWL are not available. Together, both these works extend the current knowledge and methods for reducing artefact in neuroimaging studies. The first work provides guidelines for researchers using EEG-fMRI, to reduce artefact and successfully report their methods when publishing. The second work shows a proof of concept that CWL can detect motion in fMRI studies, thus laying the groundwork for more sophisticated motion removal algorithms for fMRI in the future.
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ItemInvestigating the effects of upright positions on cerebral hemodynamics and clinical outcomes in acute ischaemic stroke( 2023-03)The effects of upright positions (sitting and standing) on cerebral haemodynamics early post an ischaemic stroke are not well understood. Neither is their influence on longer term clinical outcomes. Concerns that early upright activity (sitting, standing and walking activities, often termed early mobilisation) may worsen cerebral perfusion within penumbral tissue early post stroke, particularly in people with occlusive disease, guided the development of the research in this thesis. This thesis is comprised of four studies that collectively aimed to shed light on the uncertainty around the effects of head positioning, particularly upright positions, on cerebral haemodynamics and clinical outcomes in acute ischaemic stroke. The first study, a comprehensive systematic review and meta-analysis, highlighted the dearth of studies assessing changes in cerebral haemodynamics in upright sitting and standing in acute ischaemic stroke. Yet getting out of bed, sitting up and walking, are early activity practices that, while part of routine stroke unit care, are not strongly guided by evidence. This review also showed the dearth of head positioning studies that examined occlusive disease as a patient-related factor that may contribute to further reductions in cerebral perfusion during upright activity. This review highlighted important research gaps to be addressed in the subsequent studies. The second study aimed to explore whether occlusive disease contributed to worse clinical outcomes in people with ischaemic stroke treated with very early mobilisation (more frequent out of bed upright activities, started <24h of stroke onset). This post hoc retrospective substudy of the A Very Early Rehabilitation (AVERT) trial showed a greater proportion of participants with occlusive disease having worse 3-month clinical outcomes (modified Rankin Scale, deaths) treated with very early mobilisation compared to usual care. However, no significant associations between occlusive disease, clinical outcomes and mobilisation group were found. Although the study was likely underpowered with small numbers of important clinical events, the potential for occlusive disease to influence outcome warranted further exploration. The third and main study of this thesis, was a prospective study investigating orthostatic changes in cerebral haemodynamics while people with acute ischaemic stroke (within 48h of symptoms onset) moved from lying-flat (0 degrees) to upright (90 degrees) sitting and standing. This was the first study to assess middle cerebral artery mean velocities using transcranial Doppler at upright positions in acute ischaemic stroke at such an early stage (within 48h of stroke onset), and which also included people with occlusive disease. This study also assessed orthostatic changes in mean velocity over time by comparing assessments <48h to a later time (3-7 days post stroke). Finally, this study explored the association between early changes in mean velocities (<48h) to 30-day functional outcome measured with modified Rankin Scale. Results from this study showed significant reductions in mean velocities at upright sitting and standing in people with ischaemic stroke, with and without occlusive disease. Our study also showed no association between orthostatic changes in mean velocity and 30-day functional outcome. Interrupted by the COVID pandemic, a limitation of this study was that it was underpowered to definitively test our hypothesis. Nevertheless, given the relatively large sample, and acute timing compared with previous studies, we believe our results contribute valuable information on the effects of upright postures to cerebral haemodynamics in the first days after stroke. Finally, given the important protective effect of collateral flow to the ischaemic penumbra, the last study aimed to explore the influence of collateral flow on orthostatic changes in mean velocities in acute stroke. The clinical hypothesis was that people with ischaemic stroke with poor collateral flow, would show greater reduction in mean velocities at upright positions compared to those with good collateral flow. Exploratory post hoc analyses were performed on data from perfusion images collected retrospectively and added to the dataset from Study 3. Although the results did not show a significant association between good or poor collateral flow and orthostatic reductions in mean velocity, this study introduced important novel hypotheses related to cerebral haemodynamics and upright activity. In conclusion, this thesis has contributed new evidence and insights into head positioning, upright activity and occlusive disease in acute ischaemic stroke. Mean velocities do reduce at more upright positions in people with acute ischaemic stroke both with and without occlusive disease in the first two days post stroke. However the clinical significance of this finding remains unknown. Future research should focus on better understanding the factors influencing cerebral autoregulation and haemodynamics as well as further assessing patient-related factors (i.e. presence of occlusion or stenosis, collateral circulation) that may influence how cerebral blood flow changes with head position in acute ischaemic stroke.
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ItemTowards biologics targeting vasopressin family receptors( 2022)G protein-coupled receptors (GPCRs) are integral membrane proteins that play a critical role in transducing information into a cell, and as such are important drug targets. Biophysical characterisation and drug discovery against GPCRs is challenging as they express at low levels in recombinant systems and demonstrate low stability when removed from the native lipid bilayer environment, which together, can mean that the routine generation of purified receptor protein is difficult. The inability to routinely generate purified GPCR protein is a bottleneck to the application of many biophysical techniques. In particular, this thesis focused on the application of anti-GPCR biologics discovery. The oxytocin receptor (OTR) and vasopressin 1A receptor (V1AR) are emerging therapeutic targets for social disorders such as schizophrenia and autism. The native ligands of these GPCRs are closely related nonapeptides oxytocin and arginine vasopressin. Despite therapeutic interest, there is a lack of selective compounds targeting OTR and V1AR, limitted understanding about how these peptides bind and activate their receptors, and a lack of delineation regarding their expression patterns in the brain. There is therefore a need for selective compounds for OTR and V1AR. I propose that OTR and V1AR binding antibodies, and nanobodies are an interesting modality to explore to these ends. Antibodies are an emerging method of targeting GPCRs, both therapeutically, and as tools for biomedical research. Theoretically, antibodies should bind to GPCR subtypes with improved selectivity compared to small molecules, while also providing benefits such as restricted biodistribution, an extended half-life, and additional functionality via the design of antibody-drug conjugates. However, the identification of antibodies against GPCRs has been hindered by the challenges associated with generating GPCR antigens that represent the 3D conformation of the receptor. The ability to generate a sufficient yield of stable, functional, purified GPCR protein for immunisation or panning is central to this challenge. Thus, the development of antibodies targeting GPCRs has lagged behind other protein families. Nanobodies are single VHH domain camelid antibodies, which are encoded by single gene. Domain antibodies such as nanobodies have emerged as a novel alternative for targeting GPCRs, as the smaller size, and extended complement determining region 3 (CDR3) can enable binding to smaller sites and can be advantageous over small molecule, or traditional antibody approaches. In this thesis I developed methodologies that aim to circumvent the main bottlenecks in receptor antibody and nanobody discovery, working towards the discovery of biologics targeting vasopressin family receptors. Firstly, I enhanced receptor protein expression of the OTR, using a novel method of lentivirus assisted mammalian cell directed evolution. Using this method, I discovered the variant OTR(3A) which has 8-fold enhanced expression compared to OTR(WT) by the introduction of only four amino acid point mutations. Then, I optimised the expression and purification of high expressing variants of V1AR and OTR from mammalian cells. Strategies for generating fluorescent receptor protein preparations were also explored. Subsequently, purified V1AR and OTR were used to generate an immune repertoire of anti V1AR and OTR nanobodies following alpaca immunisation. I developed a custom mammalian cell display panning methodology and demonstrated the feasibility of using a mammalian cell display approach for the identification of nanobodies, based on binding to a target antigen. Finally, in a pilot study, FPR1 and cognate antagonistic antibody Fpro0165 were used to demonstrate that a Fab expressed on the cell surface of a mammalian cell can bind to its cognate receptor expressed on the same cell and Fab-GPCR binding was detected using fluorescent ligand competition, and blockage of peptide induced signalling, laying the foundations for further development of this mammalian display method for anti-GPCR antibody discovery. Together, the work in this thesis has made significant contributions towards developing tools and methodologies that will aid in the discovery of selective binders to OTR and V1AR, and furthermore, will contribute to the generation of anti-GPCR biologics more generally.
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ItemThe value of routine clinical imaging in predicting ischaemic stroke outcome( 2022)Research Objective To investigate the impact of ischaemic lesion characteristics and white matter changes – extracted from routine clinical scans – on outcome as a key biomarker for clinical prediction models. Methods Development of a stroke-population specific tool for semi-automated standardised assessment of lesion characteristics in routine clinical computed tomography (CT) and magnetic resonance imaging (MRI) scans; application of this tool to analyse the impact of lesion volume and lesion location together with white matter changes on functional outcome, gross motor outcome and language outcome; and analysis of the influence of very early mobilisation on functional outcome in the setting of large vessel occlusion (LVO). Results A stroke-population specific CT-MRI atlas with a validated robust and reliable CT normalisation pipeline was developed. Total lesion volume and white matter damage impacted functional and gross motor outcome the most. Aphasia, as a function-specific outcome was primarily impacted by infarction in core language areas. There was no evidence that very early mobilisation is especially harmful in patients with LVO. Implications The stroke-population specific CT-MRI atlas is a valuable tool to make routine clinical scans easily accessible for research in the context of lesion location and has the potential to standardise research in this field. Function-specific outcome parameters are necessary to further look into the lesion location – outcome relationship and systematic research using big data will be required to advance our understanding of the impact of lesion characteristics on stroke outcome. These points are crucial when aiming for the development and implementation of a clinical meaningful prediction model.
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ItemInvestigating mechanisms of mutant huntingtin toxicity by spatially mapping lipid metabolites in a mouse model of Huntington’s disease( 2022)Huntington’s disease (HD) features intraneuronal inclusion body formation by polyglutamine-containing fragments of the mutant huntingtin protein in many brain regions, including the hippocampus, neocortex and striatum. To better understand the molecular changes associated with inclusion body formation and associated pathogenesis, we examined the abundances and turnover rates of membrane lipids in the hippocampus, a region of pronounced inclusion formation associated with cognitive deficits, in a transgenic mouse model of HD (R6/1 line) using deuterium labelling in vivo. The R6/1 HD mice lacked inclusions in the hippocampus at six weeks of age, whereas inclusions were extensive by 16 weeks. We assessed one brain hemisphere collected at three timepoints (6, 12 and 16 weeks) by MALDI-mass spectrometry imaging (MALDI-MSI) and the other hemisphere for liquid-chromatography mass spectrometry (LC-MS) analysis. Hippocampal sub-fields (CA1, CA3 and DG) dense with inclusions showed a reduction in the relative abundance of neuronal-enriched lipids with roles in neurotransmission, synaptic plasticity, neurogenesis and ER-stress protection. Conversely, lipids in the phosphatidylinositol, phosphatidic acid and ganglioside class were increased in lipid synthesis in HD mice, relative to WT mice across all the age groups examined. The changes were also detectable in the HD mice at six weeks of age, indicating they arose prior to the formation of the inclusion bodies and disease symptoms. Since elevated synthesis of lipids in the PI, PA and ganglioside classes is a known adaptive response to Endoplasmic reticulum (ER) stress, our findings suggest this molecular mechanism serves as an early-stage adaptive response to ER stress in pre-symptomatic HD mice and may be targetable therapeutically. Additionally, our study has identified progressive changes in neuronal lipid abundances in the pre-symptomatic and symptomatic stages of HD that closely correlate with known hippocampal-dependent cognitive changes in HD, thus providing early lipid biomarkers that may be targeted therapeutically to slow down HD progression. Most importantly, we have spatially monitored disturbances in lipid metabolism at the primary site of inclusions in HD hippocampi, thus illuminating new insight into the cascade of molecular events in brain regions spanning the development of inclusions in HD mice. These findings required the development of a novel in-house bioinformatics software (KineticMSI), which is made available as an R package and will have broad neuroimaging applications.