Neuroinflammation and protein pathology in Parkinson’s disease dementia

Abstract: Parkinson’s disease dementia is neuropathologically characterized by aggregates of α-synuclein (Lewy bodies) in limbic and neocortical areas of the brain with additional involvement of Alzheimer’s disease-type pathology. Whilst immune activation is well-described in Parkinson’s disease (PD), how it links to protein aggregation and its role in PD dementia has not been explored. We hypothesized that neuroinflammatory processes are a critical contributor to the pathology of PDD. To address this hypothesis, we examined 7 brain regions at postmortem from 17 PD patients with no dementia (PDND), 11 patients with PD dementia (PDD), and 14 age and sex-matched neurologically healthy controls. Digital quantification after immunohistochemical staining showed a significant increase in the severity of α-synuclein pathology in the hippocampus, entorhinal and occipitotemporal cortex of PDD compared to PDND cases. In contrast, there was no difference in either tau or amyloid-β pathology between the groups in any of the examined regions. Importantly, we found an increase in activated microglia in the amygdala of demented PD brains compared to controls which correlated significantly with the extent of α-synuclein pathology in this region. Significant infiltration of CD4+ T lymphocytes into the brain parenchyma was commonly observed in PDND and PDD cases compared to controls, in both the substantia nigra and the amygdala. Amongst PDND/PDD cases, CD4+ T cell counts in the amygdala correlated with activated microglia, α-synuclein and tau pathology. Upregulation of the pro-inflammatory cytokine interleukin 1β was also evident in the substantia nigra as well as the frontal cortex in PDND/PDD versus controls with a concomitant upregulation in Toll-like receptor 4 (TLR4) in these regions, as well as the amygdala. The evidence presented in this study show an increased immune response in limbic and cortical brain regions, including increased microglial activation, infiltration of T lymphocytes, upregulation of pro-inflammatory cytokines and TLR gene expression, which has not been previously reported in the postmortem PDD brain

A Systematic Review and Meta-Analysis of Alpha Synuclein Auto-Antibodies in Parkinson's Disease

Immune dysfunction has been associated with Parkinson's disease (PD) and its progression. Antibodies play an important role in both innate and adaptive responses, acting as powerful effector molecules that can propagate inflammation by activating innate immune cells. Alpha synuclein binding antibodies have been described in PD patients with conflicting associations. In this article, we consider the potential mechanistic basis of alpha synuclein auto-antibody development and function in PD. We present a systematic review and meta-analysis of antibody studies in PD cohorts showing that there is weak evidence for an increase in alpha synuclein auto-antibodies in PD patients particularly in early disease. The confidence with which this conclusion can be drawn is limited by the heterogeneity of the clinical cohorts used, inclusion of unmatched controls, inadequate power and assay related variability. We have therefore made some recommendations for the design of future studies

T lymphocyte senescence is attenuated in Parkinson's disease.

BackgroundImmune involvement is well-described in Parkinson's disease (PD), including an adaptive T lymphocyte response. Given the increasing prevalence of Parkinson's disease in older age, age-related dysregulation of T lymphocytes may be relevant in this disorder, and we have previously observed changes in age-associated CD8+ T cell subsets in mid-stage PD. This study aimed to further characterise T cell immunosenescence in newly diagnosed PD patients, including shifts in CD4+ and CD8+ subpopulations, and changes in markers of cellular ageing in CD8+ T lymphocytes.MethodsPeripheral blood mononuclear cells were extracted from the blood of 61 newly diagnosed PD patients and 63 age- and sex-matched controls. Flow cytometric analysis was used for immunophenotyping of CD8+ and CD4+ lymphocyte subsets, and analysis of recent thymic emigrant cells. Telomere length within CD8+ T lymphocytes was assessed, as well as the expression of the telomerase reverse transcriptase enzyme (hTERT), and the cell-ageing markers p16INK4a and p21CIP1/Waf1.ResultsThe number of CD8+ TEMRA T cells was found to be significantly reduced in PD patients compared to controls. The expression of p16INK4a in CD8+ lymphocytes was also lower in patients versus controls. Chronic latent CMV infection was associated with increased senescent CD8+ lymphocytes in healthy controls, but this shift was less apparent in PD patients.ConclusionsTaken together, our data demonstrate a reduction in CD8+ T cell replicative senescence which is present at the earliest stages of Parkinson's disease

Imaging protein aggregates in Parkinson’s Disease serum using aptamer-assisted single-molecule pull-down

The formation of soluble α-synuclein (α-syn) and amyloid-β (Aβ) aggregates is associated with the development of Parkinson’s disease (PD). Current methods mainly focus on the measurement of the aggregate concentration and are unable to determine their heterogeneous size and shape, which potentially also change during the development of PD due to increased protein aggregation. In this work, we introduce aptamer-assisted single-molecule pull-down (APSiMPull) combined with super-resolution fluorescence imaging of α-syn and Aβ aggregates in human serum from early PD patients and age-matched controls. Our diffraction-limited imaging results indicate that the proportion of α-syn aggregates (α-syn/(α-syn+Aβ)) can be used to distinguish PD and control groups with an area under the curve (AUC) of 0.85. Further, super resolution fluorescence imaging reveals that PD serums have a higher portion of larger and rounder α-syn aggregates than controls. Little difference was observed for Aβ aggregates. Combining these two metrics, we constructed a new biomarker and achieved an AUC of 0.90. The combination of the aggregate number and morphology provides a new approach to early PD diagnosis

Association Between Toll‐Like Receptor 4 ( TLR4 ) and Triggering Receptor Expressed on Myeloid Cells 2 ( TREM2 ) Genetic Variants and Clinical Progression of Huntington's Disease

Background: Although Huntington's disease (HD) is caused by a single dominant gene, it is clear that there are genetic modifiers that may influence the age of onset and disease progression. Objectives: We sought to investigate whether new inflammation‐related genetic variants may contribute to the onset and progression of HD. Methods: We first used postmortem brain material from patients at different stages of HD to look at the protein expression of toll‐like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells 2 (TREM2). We then genotyped the TREM2 R47H gene variant and 3 TLR4 single nucleotide polymorphisms in a large cohort of HD patients from the European Huntington's Disease Network REGISTRY. Results: We found an increase in the number of cells expressing TREM2 and TLR4 in postmortem brain samples from patients dying with HD. We also found that the TREM2 R47H gene variant was associated with changes in cognitive decline in the large cohort of HD patients, whereas 2 of 3 TLR4 single nucleotide polymorphisms assessed were associated with changes in motor progression in this same group. Conclusions: These findings identify TREM2 and TLR4 as potential genetic modifiers for HD and suggest that inflammation influences disease progression in this condition. © 2019 International Parkinson and Movement Disorder Societ

In vivo 18F-flortaucipir PET does not accurately support the staging of progressive supranuclear palsy

Progressive Supranuclear Palsy (PSP) is a neurodegenerative disorder characterised by neuro-glial tau pathology. A new staging system for PSP pathology at post-mortem has been described and validated. We used a data-driven approach to test whether post-mortem pathological staging in PSP can be reproduced in vivo with 18F-flortaucipir PET. Methods: N=42 patients with probable PSP and N=39 controls underwent 18F-flortaucipir PET. Conditional inference tree analyses on regional binding potential values identified absent/present pathology thresholds to define in vivo staging. Following the staging system for PSP pathology, the combination of absent/present values across all regions was evaluated to assign each participant to in vivo stages. Analysis of variance was applied to analyse differences among means of disease severity between stages. In vivo staging was compared with post-mortem staging in N=9 patients who also had post-mortem confirmation of the diagnosis and stage. Results: Stage assignment was estimable in 41 patients: N=10 patients were classified in stage I/II, N=26 in stage III/IV, N=5 in stage V/VI, while N=1 was not classifiable. An explorative sub-staging identified N=2 patients in stage I, N=8 in stage II, N=9 in stage III, N=17 in stage IV and N=5 in stage V. However, the nominal 18F-flortaucipir derived stage was not associated with clinical severity and was not indicative of pathology staging at post-mortem. Conclusion: 18F-flortaucipir PET in vivo does not correspond to neuropathological staging in PSP. This analytic approach, seeking to mirror in vivo the neuropathology staging with PET-to-autopsy correlational analyses might enable in vivo staging with next-generation PET tracers for tau, but further evidence and comparison with post-mortem data are needed.This study was co-funded by the Cambridge University Centre for Parkinson-Plus (RG95450); the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (BRC-1215-20014), including their financial support for the Cambridge Brain Bank; the PSP Association (“MAPT-PSP” award); the Alzheimer’s Research UK East-Network pump priming grant; the Wellcome trust (220258); the Medical Research Council (MR/P01271X/1; G1100464); the Association of British Neurologists, Patrick Berthoud Charitable Trust (RG99368); Alzheimer’s Society (443 AS JF 18017); the Evelyn Trust (RG84654), and RCUK/UKRI (via a Research Innovation Fellowship awarded by the Medical Research Council to CHWG - MR/R007446/1); the Guarantors of Brain (G101149). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care

The neuroinflammatory basis of disease progression and cognitive impairment in Parkinson's disease

Parkinson’s disease (PD) is classically defined as a movement disorder, but a large proportion of patients also develop cognitive dysfunction as the disease progresses. Currently, it is not fully understood why some patients experience mainly movement-related features, whilst others suffer from a more aggressive disease course with the development of an early dementia. This latter complication is a key milestone in the progression of the disease; almost half of PD patients will develop dementia within 10 years of diagnosis, which has a profound impact on quality of life and care requirements. Abnormal protein aggregation in the form of cortical Lewy bodies and Alzheimer’s-type pathology are well-described pathological correlates of PD dementia, but what determines the rate of progression of this pathology and the time to dementia is unknown. A strong candidate which may play a key role to the rate of cognitive decline is neuroinflammation. Whilst immune activation is well-described in PD, how it links to protein aggregation and its role in PD dementia and disease progression has not been explored, and this forms the focus of this PhD thesis. I hypothesize that neuroinflammatory changes are a critical and early contributor to the pathology of PD dementia. Chapter 1 describes the background and rationale of this PhD thesis including a literature review of the neuropathology of Parkinson’s and its associated dementia. It also provides an overview of the role of the immune system in PD, with a particular focus on Toll-like receptors which are key molecules involved in the activation of the innate immune response. Chapter 2 describes a detailed neuropathological investigation of the distribution and characteristics of inflammatory change in postmortem PD and control brains. It also explores the correlation between neuroinflammatory change, aberrant forms of α-synuclein, tau and amyloid-β and the rate of cognitive decline during life. This study found an increase in activated microglia in the amygdala and hippocampus of PD brains compared to controls, accompanied by infiltration of T lymphocytes in the brain parenchyma. Increased expression of pro-inflammatory cytokines was also observed, both in the substantia nigra and amygdala, as well as upregulation in Toll-like receptors 2 (TLR2) and 4 (TLR4). Chapter 3 describes a study investigating the relationship between neuroinflammation, tau pathology and disease prognosis, in vivo, using PET neuroimaging in early PD. Patients were stratified according to their risk of rapid progression to dementia in “high” versus “low” risk groups, defined using novel clinical and genetic predictors. This study involved PET neuroimaging with [11C]PK11195, a marker of neuroinflammation, and [18F]AV-1451, a radioligand used to visualize tau accumulation, as well as immunophenotyping and TLR expression analysis in the blood and cerebrospinal fluid. PET imaging with [11C]PK11195 showed an increase in neuroinflammation in both PD groups compared to controls in the temporal and orbitofrontal cortex. Whilst [18F]AV-1451 binding was minimal in healthy individuals, it is markedly increased in both low and high risk PD patients in several brain regions. This study also found changes in lymphocyte and monocyte populations in the blood of PD patients compared to controls, as well as increased expression of TLR2 in monocytes, both in the blood and CSF. Chapter 4 describes a pilot study evaluating the time-course of neuroinflammatory change and Toll-like receptor expression in a novel rat model of α-synuclein. This in vivo model was based on the transvascular delivery of α-synuclein pre-formed fibrils. A secondary aim of this pilot study was to assess the tolerability of two TLR-blocking agents (Candesartan cilexetil and TAK242) and their efficacy in reducing TLR expression and protein levels over a two-month treatment in this model. This chapter showed a gradual increase in microglia in the brainstem, accompanied by an increase in TLR levels, particularly TLR4, at 2- 4- and 6-months post-α-synuclein injection. Both TLR-blocking agents were well-tolerated, but I could not demonstrate significant TLR blockade in this small pilot study. Chapter 5 presents the key conclusions of this PhD thesis. My postmortem work suggests that microglial activation is increased in brain areas associated with cognition, namely the hippocampus and the amygdala, and this is accompanied by increased expression of pro-inflammatory mediators. I also identified TLR2 and TLR4 as particular molecules of interest in postmortem PD brain, in biofluids of PD patients at early stages of the disease, and in a novel α-synuclein rodent model. Furthermore, I present in vivo imaging evidence of increased tau deposition in the brain in early PD. The end of the chapter discusses plans for further development of this work.This PhD was funded by a scholarship from the Onassis Foundation (Scholarship Program for Hellenes) and the Alborada Studentship from Wolfson College, Cambridge. The experimental work was funded by the Rosetrees Trust (M369-F1), the Evelyn Trust (16/08), and the Medical Research Council (MR/R007446/1)

Age-Related Adaptive Immune Changes in Parkinson's Disease.

Ageing is a major risk factor for most neurodegenerative diseases, including Parkinson's disease (PD). Progressive age-related dysregulation of the immune system is termed immunosenescence and is responsible for the weakened response to novel antigens, increased susceptibility to infections and reduced effectiveness of vaccines seen in the elderly. Immune activation, both within the brain and periphery, is heavily implicated in PD but the role of immunosenescence has not been fully explored. Studies to date provide some evidence for an attenuation in immunosenescence in PD, particularly a reduction in senescent CD8 T lymphocytes in PD cases compared to similarly aged controls. Here, we discuss recent evidence of age-related immune abnormalities in PD with a focus on T cell senescence and explore their potential role in disease pathogenesis and development

Timing Is Everything: The T-Cell Response to α-Synuclein Is Maximal in Early Parkinson's.

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Pathological consequences of MICU1 mutations on mitochondrial calcium signalling and bioenergetics

Loss of function mutations of the protein MICU1, a regulator of mitochondrial Ca(2+) uptake, cause a neuronal and muscular disorder characterised by impaired cognition, muscle weakness and an extrapyramidal motor disorder. We have shown previously that MICU1 mutations cause increased resting mitochondrial Ca(2+) concentration ([Ca(2+)]m). We now explore the functional consequences of MICU1 mutations in patient derived fibroblasts in order to clarify the underlying pathophysiology of this disorder. We propose that deregulation of mitochondrial Ca(2+) uptake through loss of MICU1 raises resting [Ca(2+)]m, initiating a futile Ca(2+) cycle, whereby continuous mitochondrial Ca(2+) influx is balanced by Ca(2+) efflux through the sodium calcium exchanger (NLCXm). Thus, inhibition of NCLXm by CGP-37157 caused rapid mitochondrial Ca(2+) accumulation in patient but not control cells. We suggest that increased NCLX activity will increase sodium/proton exchange, potentially undermining oxidative phosphorylation, although this is balanced by dephosphorylation and activation of pyruvate dehydrogenase (PDH) in response to the increased [Ca(2+)]m. Consistent with this model, while ATP content in patient derived or control fibroblasts was not different, ATP increased significantly in response to CGP-37157 in the patient but not the control cells. In addition, EMRE expression levels were altered in MICU1 patient cells compared to the controls. The MICU1 mutations were associated with mitochondrial fragmentation which we show is related to altered DRP1 phosphorylation. Thus, MICU1 serves as a signal-noise discriminator in mitochondrial calcium signalling, limiting the energetic costs of mitochondrial Ca(2+) signalling which may undermine oxidative phosphorylation, especially in tissues with highly dynamic energetic demands. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech