Phosphorylation of the M1 muscarinic acetylcholine receptor provides neuroprotection in mouse prion disease

The M1 muscarinic acetylcholine receptor (mAChR) plays a crucial role in learning and memory and is a validated drug target for the treatment of Alzheimer’s disease (AD). Pharmacological activation of the M1 mAChR can not only improve cognitive symptoms in AD patients but has also been proven to slow down disease progression in preclinical mouse models of neurodegeneration. Thus, the M1 mAChR has a promising potential as drug target for diseasemodifying therapies of AD (Scarpa et al., 2020). However, development of clinically effective M1 mAChR-targeted ligands has been challenging due to associated adverse effects, highlighting the need to dissect clinically relevant M1 mAChR-mediated pathways from those leading to undesirable outcomes. By employing a novel transgenic mouse model expressing a phosphorylationdeficient mutant of the M1 mAChR (M1-PD) (Butcher et al., 2016), our group previously found that adverse effects can be minimised through pathways favouring receptor phosphorylation (Bradley et al., 2020). This thesis aimed to extend this study and explore the role of M1 mAChR phosphorylation/arrestindependent pathways in the disease modification potential of the M1 mAChR. I combined M1-PD transgenic mice with prion neurodegenerative disease, a model of terminal neurodegeneration, leading to the discovery that disease is accelerated in M1-PD mice, thereby revealing an inherent neuroprotective property of the M1 mAChR that is dependent on receptor phosphorylation. To provide insight into the potential signalling mechanisms of the M1-PD, in vitro functional assays were performed on cell lines expressing the M1-PD version or wild-type of the M1 mAChR. Lack of receptor phosphorylation significantly impaired agonist-induced receptor internalisation, which is an important process in the desensitisation of G protein-dependent signalling. However, removal of M1 mAChR receptor phosphorylation was shown to have little impact on phosphoinositide accumulation, which is indicative of Gαq protein activation. The mouse prion disease model was then investigated through behavioural observations and histological and biochemical studies to characterise neurodegenerative disease progression through the detection of markers of disease. Mouse prion disease is caused by neurotoxic aggregates of misfolded prion proteins, and shares key hallmarks with human neurodegenerative diseases such as AD. These include memory and hippocampal function decline, disease markers such as APO-E, clusterin and serpinA3N, and widespread neuroinflammation, as indicated by the upregulation of astrocytic and microglial markers GFAP, Vimentin, Iba1 and CD86. Importantly, the appearance of misfolded, neurotoxic prion was shown to occur prior to the start of dosing studies of M1 mAChR-selective ligands (Bradley et al., 2017, Dwomoh et al., 2021), establishing that the therapeutic effects exerted by the M1 mAChR are not due to prevention of disease, but disease-modification. In this thesis, mouse prion disease model was also demonstrated to feature the significant upregulation of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, similar to other neurodegenerative disorders characterised by chronic neuroinflammation. Removal of M1 mAChR phosphorylation in mice caused a significant acceleration of prion neurodegenerative disease progression. This was evident from behavioural changes such as faster hippocampal decline and symptom onset and shorter lifespan compared to wild-type animals, but also significantly elevated accumulation of misfolded prion and upregulation of markers of disease and neuroinflammation. Particularly, the pro-inflammatory cytokine TNF-α was significantly upregulated in prion-infected M1-PD mice with compared to wildtype mice, suggesting the M1 mAChR might be involved in the regulation of TNF-α. These findings unravelled an important neuroprotective property that is inherent to the M1 mAChR and depends on the receptor’s phosphorylation/arrestin-dependent signalling. In addition, these data have important implications for development of new drug treatments for neurodegenerative diseases, especially, M1 mAChR ligands that maintain receptor phosphorylation will more likely deliver neuroprotection that could not only improve memory symptoms but slow disease progression. Given the parallels between mouse prion disease and human proteinopathies, the neuroprotective mechanism observed here mediated by the M1 mAChR, is likely to be relevant to other human neurodegenerative conditions such as AD

Disease progression and genetic risk factors in the primary tauopathies

The primary tauopathies are a group of progressive neurodegenerative diseases within the frontotemporal lobar degeneration spectrum (FTLD) characterised by the accumulation of misfolded, hyperphosphorylated microtubule-associated tau protein (MAPT) within neurons and glial cells. They can be classified according to the underlying ratio of three-repeat (3R) to four-repeat (4R) tau and include Pick’s disease (PiD), which is the only 3R tauopathy, and the 4R tauopathies the most common of which are progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). There are no disease modifying therapies currently available, with research complicated by the wide variability in clinical presentations for each underlying pathology, with presentations often overlapping, as well as the frequent occurrence of atypical presentations that may mimic other non-FTLD pathologies. Although progress has been made in understanding the genetic contribution to disease risk in the more common 4R tauopathies (PSP and CBD), very little is known about the genetics of the 3R tauopathy PiD. There are two broad aims to this thesis; firstly, to use data-driven generative models of disease progression to try and more accurately stage and subtype patients presenting with PSP and corticobasal syndrome (CBS, the most common presentation of CBD), and secondly to identify genetic drivers of disease risk and progression in PiD. Given the rarity of these disorders, as part of this PhD I had to assemble two large cohorts through international collaboration, the 4R tau imaging cohort and the Pick’s disease International Consortium (PIC), to build large enough sample sizes to enable the required analyses. In Chapter 3 I use a probabilistic event-based modelling (EBM) approach applied to structural MRI data to determine the sequence of brain atrophy changes in clinically diagnosed PSP - Richardson syndrome (PSP-RS). The sequence of atrophy predicted by the model broadly mirrors the sequential spread of tau pathology in PSP post-mortem staging studies, and has potential utility to stratify PSP patients on entry into clinical trials based on disease stage, as well as track disease progression. To better characterise the spatiotemporal heterogeneity of the 4R tauopathies, I go on to use Subtype and Stage Inference (SuStaIn), an unsupervised machine algorithm, to identify population subgroups with distinct patterns of atrophy in PSP (Chapter 4) and CBS (Chapter 5). The SuStaIn model provides data-driven evidence for the existence of two spatiotemporal subtypes of atrophy in clinically diagnosed PSP, giving insights into the relationship between pathology and clinical syndrome. In CBS I identify two distinct imaging subtypes that are differentially associated with underlying pathology, and potentially a third subtype that if confirmed in a larger dataset may allow the differentiation of CBD from both PSP and AD pathology using a baseline MRI scan. In Chapter 6 I investigate the association between the MAPT H1/H2 haplotype and PiD, showing for the first time that the H2 haplotype, known to be strongly protective against developing PSP or CBD, is associated with an increased risk of PiD. This is an important finding and has implications for the future development of MAPT isoform-specific therapeutic strategies for the primary tauopathies. In Chapter 7 I perform the first genome wide association study (GWAS) in PiD, identifying five genomic loci that are nominally associated with risk of disease. The top two loci implicate perturbed GABAergic signalling (KCTD8) and dysregulation of the ubiquitin proteosome system (TRIM22) in the pathogenesis of PiD. In the final chapter (Chapter 8) I investigate the genetic determinants of survival in PiD, by carrying out a Cox proportional hazards genome wide survival study (GWSS). I identify a genome-wide significant association with survival on chromosome 3, within the NLGN1 gene. which encodes a synaptic scaffolding protein located at the neuronal pre-synaptic membrane. Loss of synaptic integrity with resulting dysregulation of synaptic transmission leading to increased pathological tau accumulation is a plausible mechanism though which NLGN1 dysfunction could impact on survival in PiD

Neuropsychology, eye tracking and neuroimaging perspectives on Posterior Cortical Atrophy

This thesis describes investigations of the clinico-radiological syndrome Posterior Cortical Atrophy, addressing two broad themes: the consequences of PCA for everyday activities (particularly scene perception) and the heterogeneity of symptoms in PCA. Despite improvements in the recognition and characterisation of PCA, we have little understanding of what the world looks like to someone with PCA. This thesis investigates patients’ perception of real-world stimuli (scenes) using a number of methodologies; characterising their response times when categorising scenes and giving a novel qualitative report of patients’ verbal descriptions (Chapter 2). It is possible that oculomotor behaviour is a contributory factor in these tasks, therefore a study of fixation, saccades and smooth pursuit was carried out. This characterised in detail for the first time the oculomotor abnormalities present in PCA (Chapter 3), facilitating further investigation of patients’ eye movements when viewing scenes (Chapters 4 and 5), revealing a striking impairment in the ability to change fixation patterns in response to task demands. The consequences of PCA for everyday activities are also investigated through a questionnaire given to carers allowing a wide range of symptoms and behaviours to be investigated over different stages of disease severity (Chapter 6). The range of symptoms and severity that this questionnaire measures will eventually allow better characterisation of the heterogeneity within PCA, and the early onset Alzheimer’s Disease spectrum more broadly. One specific manifestation of this heterogeneity is investigated in Chapter 7, demonstrating that a proportion of PCA patients show asymmetric motor symptoms (myoclonus and limb rigidity on the left side) associated with atrophy of motor cortex in the right hemisphere. Together, these studies improve our knowledge of the consequences of PCA for scene perception and more general everyday activities, and address aspects of heterogeneity in the syndrome; with implications for interventions to improve diagnosis and clinical management

Evaluating the neuroprotective effect of curcumin on a PINK1 cell model of Parkinson's disease

Thesis (MSc)--University of Stellenbosch University, 2022.ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of neurons producing the neurotransmitter dopamine. Notably, despite extensive studies that have revealed numerous dysregulated processes associated with PD, fundamental gaps still exist in our knowledge of the disease pathophysiology. Our understanding of the disease includes processes such as mitochondrial dysfunction causing increased oxidative stress and energy failure, as well as misfolded protein accumulation in large inclusions. However, the spatiotemporal sequence of events leading to PD and, importantly, the initial factors that trigger disease onset remain elusive. This challenges the development of effective therapeutic strategies to prevent and cure PD. Moreover, the various side effects associated with conventional pharmaceuticals currently used to treat this disease motivate the search for a natural treatment that can avoid augmenting the suffering associated with an already debilitating disease. It is evident that mitochondrial dysfunction and oxidative stress are likely to be involved in the disease pathogenesis, therefore studying these phenomena in PD may lead to the development of more effective therapeutic strategies. Curcumin is a plant-based polyphenol that has been observed to have antioxidant properties, increase cell viability, and enhance mitochondrial function. Consequently, the aim of the present study was to create a PD cellular model and evaluate the potential protective effects of curcumin. Therefore, we sought to establish an appropriate PD model by transfecting SH-SY5Y cells with wild-type (WT) or G309D mutant PINK1 cDNA, of which the latter has been shown to upregulate dopamine and lead to cytotoxicity. Additionally, since several neurotoxins have been shown to trigger PD, the toxic herbicide paraquat was administered to complement the model with cellular damage and mitochondrial dysfunction. To confirm the model, we performed RT-qPCR to measure gene expression levels of PINK1 and tyrosine hydroxylase (TH), an enzyme in the dopamine synthesis pathway. PINK1 was significantly upregulated in the mutant, however, no significant difference in TH gene expression was observed between groups. We then sought to measure the levels of dopamine in transfected cells using liquid chromatography-mass spectrometry (LC-MS). Although no dopamine was detected using LC-MS, higher levels of phenylalanine, a precursor of dopamine, were observed in the mutant. Using this model, we sought to test the protective effects of curcumin using assays that measure cellular and mitochondrial health. A toxic paraquat concentration of 1.7 mM was chosen to elicit a 50 % decrease in cell viability for the model, while a curcumin concentration of 2.5 μM was chosen as it exhibited no toxic effects. Following the establishment of the model, four treatment groups were established for all experiments thereon: untreated control, curcumin only treatment, paraquat only treatment, and pre-treatment (curcumin treatment followed by paraquat treatment). We found that curcumin was unable to significantly rescue the paraquat-induced reduction in cell viability and mitochondrial membrane potential. The latter was significantly reduced in PINK1 transfected groups, more so in the G309D mutant, indicating the toxic effects of the mutation. Thereafter, the effects of curcumin and polycaprolactone encapsulated nanocurcumin on cell viability were compared. Formulations of curcumin including nanocurcumin are postulated to improve the stability and efficacy of curcumin. Interestingly, curcumin had a greater protective effect, whereas nanocurcumin as well as the empty nanoparticles elicited toxicity. In fact, p re-treatment with the nanocurcumin prior to paraquat treatment caused a 30 % greater loss in cell viability compared to the paraquat treatment alone. Finally, a literature review was published, exploring the potential of consistent dietary consumption of curcumin as an alternative or supplement to existing therapies. We speculate that curcumin binds to α- synuclein protein (found to accumulate in PD) and that this complex is subsequently excreted from the body via the large intestine. In this view, replacing some of the PD drugs in an individual’s treatment regime with a nutraceutical, or ‘functional food’, like curcumin may improve therapeutic benefits with fewer side effects. Considering these results and the published evidence for curcumin as a dietary ‘nutraceutical’, further studies are required to optimize curcumin treatment before advocation of its widespread use as a PD therapeutic agent. Study limitations include the use of an unverified WT plasmid and an undifferentiated cell line, which can be addressed in future work. The findings in this study are of importance as they may contribute to advancing the development of novel plant-based therapies to treat and potentially prevent this detrimental disease.AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is 'n neurodegeneratiewe versteuring wat gekenmerk word deur 'n verlies aan neurone wat die neurotransmitter dopamien produseer. Merkwaardig, ten spyte van omrykende studies wat talle gedisreguleerde prosesse verwant aan PS geopenbaar het, bestaan fundamentele gapings steeds in ons kennis van die siektepatofisiologie. Ons begrip van die siekte sluit prosesse soos mitochondriale disfunksie in wat verhoogde oksidatiewe stres en energiemislukking veroorsaak, asook verkeerde proteïenophoping in groot insluitings. Die tydruimtelike volgorde van gebeure wat tot PS lei en, belangriker, die aanvanklike faktore wat siekte-aanvang veroorsaak, bly egter ontwykend. Dit daag die ontwikkeling van effektiewe terapeutiese strategieë uit om PS te voorkom en te genees. Bowendien, het die huidige konvensionele farmaseutiese behandelinge vir PS tale newe- effekte. Dit dien as motivering vir soektogte na ‘n natuurlike behandeling wat die aanvullende leiding kan verminder van ‘n reeds afbrekende siekte. Dit is duidelik dat mitochondriale disfunksie en oksidatiewe stres betrokke is by die siekte se patologie, daarom kan die bestudering van hierdie verskynsels in PS lei tot die ontwikkeling van meer effektiewe terapeutiese strategieë. Kurkumien is 'n plantgebaseerde polifenol wat waargeneem is om antioksidant eienskappe te toon, verhoog sel lewenskragtigheid en verbeter mitochondriale funksie. Gevolglik was die doel van die huidige studie om 'n PS-sellulêre model te skep en die potensiële beskermende effekte van kurkumien te evalueer. Daarom het ons probeer om 'n toepaslike PS-model te vestig deur SH-SY5Y-selle met wilde-tipe (WT) of G309D muteerde PINK1 cDNA te transreguleer, waarvan laasgenoemde dopamien op gereguleer het wat gelei het tot sitotoksiteit. Daarbenewens, aangesien verskeie neurotoksiene getoon is om PS te aktiveer, is die giftige onkruiddoder, parakwat, toegedien om die model aan te vul met sellulêre skade en mitochondriale disfunksie. Om die model te bevestig, het ons RT-qPCR uitgevoer om geen-uitdrukkingsvlakke van PINK1 en tyrosine hydroxylase (TH), 'n ensiem betrokke by die chemiese samestelling van dopamien, kwantitatief te meet. PINK1 was aansienlik opgereguleer in die muteerde, maar geen beduidende verskil was opgemerk in TH-geenuitdrukking tussen groepe nie. Ons het toe probeer om die vlakke van dopamien in transfekte selle te meet met behulp van vloeibare chromatografie-massaspektrometrie (VC-MS). Alhoewel geen dopamien opgespoor was met behulp van VC-MS nie, was hoër vlakke van fenylalanien, 'n voorloper van dopamien, in die muteerde waargeneem. Met behulp van hierdie model het ons probeer om die beskermende effekte van kurkumien te toets met behulp van eksperimente wat sellulêre en mitochondriale gesondheid kan bepaal. 'n Giftige parakwat konsentrasie van 1.7 mM was gekies om 'n 50% afname in sel lewensvatbaarheid in die model te ontlok, terwyl 'n kurkumien konsentrasie van 2.5 μM gekies was, aangesien dit geen giftige effekte getoon het nie. Na die bevestiging van die model was vier behandelingsgroepe gestig vir alle eksperimente wat volg daarna, dit sluit in: onbehandelde kontrole, kurkumien alleenlik, parakwat alleenlik, en voor-behandeling (kurkumien behandeling gevolg deur parakwat behandeling). Ons het gevind dat kurkumien nie merkwaardig die selle kon beskerm teen die verlaagde sel lewenskragtigheid en mitochondirale membraanpotentiaal wat geïnduseer was deur parakwat nie. Die mitochondriale membraanpotentiaal was aansienlik verminder in die PINK1-getransfekteerde groepe, meer so as in die G309D-muteerde, dit dui die giftige effekte van die mutasie aan. Daarna is die effek van kurkumien en policaprolactone ingeslote nanokurkumien op sel lewensvatbaarheid vergelyk. Formulerings van kurkumien, insluitend nanokurcumin, word gepostuleer om die stabiliteit en doeltreffendheid van kurkumien te verbeter. Interessant genoeg, kurkumien het 'n groter beskermende effek gehad, terwyl nanokurcumin sowel as die leë nanopartikels toksisiteit getoon het. In werklikheid, het die voorbehandeling met nanokurkumien (voor parakwat behandeling) ‘n 30% groter verlies in sel lewenskragtigheid veroorsaak as die selle met slegs parakwat behandeling. Ten slotte is 'n literatuuroorsig gepubliseer, wat die potensiaal van konsekwente dieet verbruik van kurkumien ondersoek as 'n alternatief of aanvulling tot bestaande terapieë. Ons spekuleer dat kurkumien aan α- sinukleien proteien bind (gevind om te versamel in PS) en dat hierdie kompleks daarna uit die liggaam uitgeskei word deur die dikderm. Deur dit in ag te neem, kan die vervanging van sommige van die huidige PS-behandelinge met nutraceutiese produkte, of ‘funksionele kos’, soos kurkumien, voordelig wees en minder newe-effekte hê. Samevattend met hierdie resultate en die gepubliseerde bewyse vir kurkumien as ‘n dieet ‘nutraceutical’, is verdere studies nodig om kurkumien behandeling te optimiseer voordat die wydverspreide gebruik daarvan as 'n PS terapeutiese middel kan gebeur. Studie beperkings sluit in die gebruik van 'n ongeverifieerde WT-plasmied en 'n ongedifferensieerde sellyn, wat in toekomstige werk aangespreek kan word. Die bevindings in hierdie studie is van belang, aangesien dit kan bydra tot die bevordering van die ontwikkeling van nuwe plantgebaseerde terapieë om hierdie nadelige siekte te behandel en moontlik te voorkom

Handbook on clinical neurology and neurosurgery

HANDBOOKNEUROLOGYNEUROSURGERYКЛИНИЧЕСКАЯ НЕВРОЛОГИЯНЕВРОЛОГИЯНЕЙРОХИРУРГИЯThis handbook includes main parts of clinical neurology and neurosurgery

Clinicopathological investigations of the cholinergic basal forebrain in Lewy body disorders and ageing

Cholinergic dysfunction has long been associated with cognitive impairment in Alzheimer’s disease (AD). However, neuropathological and functional imaging studies have also found significant cortical cholinergic deficit in Lewy body disorders (LBD), but in a different pattern from that in AD. There is topographical cholinerigic innervation to the cortex and the hippocampus from the basal forebrain. In light of differences in cognitive deficits seen in LBD and AD, I hypothesised that cholinergic basal forebrain subregions are differentially affected in these disorders. In this thesis, novel tissue techniques have been developed for the visualisation of pathology in human post-mortem brain tissue in three-dimensions. Based on a thorough review of the literature and my personal observations, I have established a simplified subdivisional scheme of the nucleus basalis of Meynert (nbM) in the human brain. Using this scheme, a quantification of nbM cholinergic neurons and assessment of neuropathological burden were performed in a large cohort of LBD and AD cases. Severe neuronal depletion across the entire nbM was observed in LBD with cognitive impairment and relative sparing of the anterior nbM was found in AD, supporting findings from previous neuropathological and imaging studies. Further investigation was carried out in the more rostral, hippocampal-projecting cholinergic group in the vertical limb of the nucleus of the diagonal band of Broca. Significant neurodegeneration in this area was identified in LBD with cognitive impairment, but not AD, suggesting its possible role in retrieval memory function via projection to the hippocampal CA2 subfield. In the final section, it was demonstrated that lactacystin injection into the rat nbM can replicate certain pathological and clinical features of LBD with dementia and this may be a useful model for the disease. Results from these studies support my initial hypothesis regarding differential susceptibility of the basal forebrain subregions in LBD and AD.Open Acces

Biomarkers in Neurodegenerative Diseases

This book focuses on the recent advancements in both fundamental and clinical research, focusing on identifying, developing, and applying new and improved biological markers for specific neurologic disorders in the future. The original research work and review articles published here highlight some unique mechanisms underlying the most prevalent pathophysiological conditions affecting human health. Other areas covered in the book include emerging treatment options and correct diagnoses using different biochemical and imaging techniques

The role of TREM2 in neurodegeneration

Introduction: Alzheimer’s disease (AD) is the most common neurodegenerative disease and has a high prevalence worldwide. Neuroinflammation has long been known to play a role in AD. However, the findings that several genes associated with inflammation were identified as hits in AD GWAS studies brought closer attention to neuroinflammatory mechanisms in AD. TREM2 was identified as a genetic risk factor for late onset AD with a similar odds ratio to that of APOE4. TREM2 is expressed on microglia, and has been shown to be upregulated on the microglia surrounding amyloid plaques both in human post mortem tissue and AD mouse models. In this thesis, the AD pathology, microglial phenotype, genetic inflammatory profile and proteomic profile of six TREM2 variant cases (5 R47H and 1 D87N variant) were investigated and compared to sporadic AD (SAD), familial AD (FAD) and neurologically normal control cases with the hypothesis that the TREM2 variant cases will differ from both SAD and FAD cases. Materials and Methods: Immunohistochemistry was performed on the frontal cortex, temporal cortex, hippocampus, putamen and cerebellum of SAD (n=19), FAD (n=11), TREM2 variant SAD (n=3), TREM2 variant controls (no AD pathology, n=2) and neurologically normal controls (n=6) using antibodies against Aβ, tau (AT8) and microglia (Iba1, CD68, CR3-43 and P2RY12). Microglial load/area, circularity and perimeter scores were calculated for all microglial markers. The frontal cortex was homogenised from a subset of each group and RNA and protein extracted. Nanostring’s Human Inflammation panel with their nCounter Technology was used to determine the genetic profile. The proteomic profile was assessed using label-free quantitative mass spectrometry. The pathological and proteomic profile of the presubiculum was investigated using immunohistochemistry, matrix-assisted laser desorption ionisation mass spectrometry, laser-capture microdissection and further label free quantitative mass spectrometry and compared to the neighbouring area, the entorhinal cortex to assess whether it has protective properties against neurodegeneration. Results: TREM2 variant AD cases differed from other SAD and FAD cases with a significantly increased CD68 load, more circular Iba1, CR3-43 and CD68 microglial positivity suggesting the microglia were in a phenotype more consistent with phagocytosis. Furthermore, these cases showed an increased upregulation of neuroinflammatory processes and neurodegenerative processes at the genetic and proteomic level than SAD cases. TREM2 variant controls however, showed large levels of downregulation in these processes compared to all groups. APOE genotyping identified the TREM2 variant cases correlated with the presence of the ApoE4 isoform. Investigation of the presubiculum area identified a large non-fibrillar Aβ deposit that contained significantly less NFT’s, activated microglia and N-terminally truncated Aβ peptides than in the entorhinal cortex and had an altered proteomic profile more comparable to the TREM2 variant controls than any other AD cases. Conclusions: Overall, this thesis has shown that TREM2 variant cases posess differences in microglial phenotype, genetic and proteomic expression compared to either sporadic or familial AD cases. TREM2 variant controls show altered pathology and genetic profiles compared to TREM2 variant SAD cases and it can be hypothesised that these cases may use similar mechanisms to the neuroprotection observed in the presubiuclum of AD cases. TREM2’s link to APOE and the fact that the APOE genotype lacks an ApoE4 allele in TREM2 variant controls, indicates that APOE may be exerting this change between TREM2 variants, leading us to question whether the TREM2 R47H variant is acting independently. Further investigations into these pathways and the differences between TREM2 variants that develop disease and those that don’t may lead us to the mechanisms that can be targeted for treatments

Development of biofluid biomarkers for Huntington’s disease

Though no treatments can currently prevent onset or slow progression of Huntington’s disease (HD), many huntingtin-lowering drug candidates targeting the root cause of HD are in the development pipeline. This brings much hope that disease-modifying treatments for HD will be a reality. However, success of potential candidates may be hindered by a lack of sensitive tools to measure biological efficacy over short intervals. Decades of attempts to develop robust biofluid biomarkers of HD progression has yielded little success or replication of results. Cerebrospinal fluid (CSF), fluid that bathes the brain and is enriched for brain-specific proteins, is a plausible target for uncovering neuropathologically relevant markers of HD. However, a lack of standardisation of collection protocols, biological rationale and technological sensitivity has hampered the progress of CSF biomarkers within the field of HD. At the core of this thesis lies the HD-CSF study – a single-site CSF collection study, with a standardised protocol designed to generate high-quality CSF and blood with matched clinical and phenotypic data. It is the first CSF collection prospectively designed for longitudinal sampling and having matching MRI data. Mutant huntingtin protein (mHTT) can be quantified in CSF and has been identified as having high potential as a biomarker of HD progression. Further, the interpretation of drug-induced lowering of mHTT in the CNS relies upon elucidation of the natural history of CSF mHTT in HD gene carriers. Neurofilament light (NfL) has emerged as a promising marker of neuronal damage that can be measured in CSF and blood. This thesis includes the first reports of blood NfL in HD, head to head comparison of NfL and mHTT, and assessment of longitudinal alterations in mHTT and NfL, in addition to proposed biomarkers of specific pathogenic pathways. The work in this thesis will have significant implications for the use of NfL and mHTT as pharmacodynamic markers of HD

The Shared Genetic Architecture of Modifiable Risk for Dementia and its Influence on Brain Health

Targeting modifiable risk factors for dementia may prevent or delay dementia. However, the mechanisms by which risk factors influence dementia remain unclear and current research often ignores commonality between risk factors. Therefore, my thesis aimed to model the shared genetic architecture of modifiable risk for dementia and explored how these shared pathways may influence dementia and brain health. I used linkage disequilibrium score regression and genomic structural equation modelling (SEM) to create a multivariate model of the shared genetics between Alzheimer’s disease (AD) and its modifiable risk factors. Although AD was genetically distinct, there was widespread genetic overlap between most of its risk factors. This genetic overlap formed an overarching Common Factor of general modifiable dementia risk, in addition to 3 subclusters of distinct sets of risk factors. Next, I performed two multivariate genome-wide association studies (GWASs) to identify the risk variants that underpinned the Common Factor and the 3 subclusters of risk factors. Together, these uncovered 590 genome-wide significant loci for the four latent factors, 34 of which were novel findings. Using post-GWAS analyses I found evidence that the shared genetics between risk factors influence a range of neuronal functions, which were highly expressed in brain regions that degenerate in dementia. Pathway analysis indicated that shared genetics between risk factors may impact dementia pathogenesis directly at specific loci. Finally, I used Mendelian randomisation to test whether the shared genetic pathways between modifiable dementia risk factors were causal for AD. I found evidence of a causal effect of the Common Factor on AD risk. Taken together, my thesis provides new insights into how modifiable risk factors for dementia interrelate on a genetic level. Although the shared genetics between modifiable risk factors for dementia seem to be distinct from dementia pathways on a genome-wide level, I provide evidence that they influence general brain health, and so they may increase dementia risk indirectly by altering cognitive reserve. However, I also found that shared genetics risk between risk factors in certain genomic regions may directly influence dementia pathogenesis, which should be explored in future work to determine whether these regions represent targets to prevent dementia