Neuroinflammation is linked to dementia risk in Parkinson's disease.

The development of dementia is a devastating aspect of Parkinson's disease (PD), affecting nearly half of patients within 10 years post-diagnosis. For effective therapies to prevent and slow progression to PD dementia (PDD), the key mechanisms that determine why some people with PD develop early dementia, while others remain cognitively unaffected, need to be understood. Neuroinflammation and tau protein accumulation have been demonstrated in post-mortem PD brains, and in many other neurodegenerative disorders leading to dementia. However, whether these processes mediate dementia risk early on in the PD disease course is not established. To this end, we used PET neuroimaging with 11C-PK11195 to index neuroinflammation and 18F-AV-1451 for misfolded tau in early PD patients, stratified according to dementia risk in our 'Neuroinflammation and Tau Accumulation in Parkinson's Disease Dementia' (NET-PDD) study. The NET-PDD study longitudinally assesses newly-diagnosed PD patients in two subgroups at low and high dementia risk (stratified based on pentagon copying, semantic fluency, MAPT genotype), with comparison to age- and sex-matched controls. Non-displaceable binding potential (BPND) in 43 brain regions (Hammers' parcellation) was compared between groups (pairwise t-tests), and associations between BPND of the tracers tested (linear-mixed-effect models). We hypothesized that people with higher dementia risk have greater inflammation and/or tau accumulation in advance of significant cognitive decline. We found significantly elevated neuroinflammation (11C-PK11195 BPND) in multiple subcortical and restricted cortical regions in the high dementia risk group compared with controls, while in the low-risk group this was limited to two cortical areas. The high dementia risk group also showed significantly greater neuroinflammation than the low-risk group concentrated on subcortical and basal ganglia regions. Neuroinflammation in most of these regions was associated with worse cognitive performance (Addenbrooke's Cognitive Examination-III score). Overall neuroinflammation burden also correlated with serum levels of pro-inflammatory cytokines. In contrast, increases in 18F-AV-1451 (tau) BPND in PD versus controls were restricted to subcortical regions where off-target binding is typically seen, with no relationship to cognition found. Whole-brain 18F-AV-1451 burden correlated with serum phosphorylated tau181 levels. Although there was minimal regional tau accumulation in PD, regional neuroinflammation and tau burden correlated in PD participants, with the strongest association in the high dementia risk group, suggesting possible co-localization of these pathologies. In conclusion, our findings suggest that significant regional neuroinflammation in early PD might underpin higher risk for PDD development, indicating neuroinflammation as a putative early modifiable aetiopathological disease factor to prevent or slow dementia development using immunomodulatory strategies.

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-ß (Aß) 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 Aß 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+Aß)) 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 Aß 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.

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson's disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis. We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular ß-sheet structure, present in the CSF and serum of 15 early stage Parkinson's disease patients compared to 10 healthy age-matched controls. We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson's disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson's disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-ß immunodepletion in an independent cohort of 11 patients with early stage Parkinson's disease and 10 control subjects. ß-Sheet aggregates in the serum of Parkinson's disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-ß in contrast to 30% α-synuclein and 70% amyloid-ß in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10-5 for each species)]. The ratio of the number of ß-sheet α-synuclein aggregates to ß-sheet amyloid-ß aggregates in serum extracted using our super-resolution method discriminated Parkinson's disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10-5). Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson's disease and warrants further investigation as a potential biomarker of disease.

T lymphocyte senescence is attenuated in Parkinson's disease.

BACKGROUND: Immune 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. METHODS: Peripheral 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. RESULTS: The 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. CONCLUSIONS: Taken together, our data demonstrate a reduction in CD8+ T cell replicative senescence which is present at the earliest stages of Parkinson's disease.

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 Society.

Toll-like receptors and their therapeutic potential in Parkinson's disease and α-synucleinopathies.

Toll-like receptors (TLRs) are pattern recognition receptors which mediate an inflammatory response upon the detection of specific molecular patterns found on foreign organisms and on endogenous damage-related molecules. These receptors play a major role in the activation of microglia, the innate immune cells of the CNS, and are also expressed in peripheral tissues, including blood mononuclear cells and the gut. It is well established that immune activation, in both the brain and periphery, is a feature of Parkinson's disease as well as other α-synucleinopathies. Aggregated forms of α-synuclein can act as ligands for TLRs (particularly TLR2 and TLR4), and hence these receptors may play a critical role in mediating a detrimental immune response to this protein, as well as other inflammatory signals in Parkinson's and related α-synucleinopathies. In this review, the potential role of TLRs in contributing to the progression of these disorders is discussed. Existing evidence comes predominantly from studies in in vitro and in vivo models, as well as analyses of postmortem human brain tissue and pre-clinical studies of TLR inhibitors. This evidence is evaluated in detail, and the potential for therapeutic intervention in α-synucleinopathies through TLR inhibition is discussed.

Pathological consequences of MICU1 mutations on mitochondrial calcium signalling and bioenergetics.

Loss of function mutations of the protein MICU1, a regulator of mitochondrial Ca2+ 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 Ca2+ concentration ([Ca2+]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 Ca2+ uptake through loss of MICU1 raises resting [Ca2+]m, initiating a futile Ca2+ cycle, whereby continuous mitochondrial Ca2+ influx is balanced by Ca2+ efflux through the sodium calcium exchanger (NLCXm). Thus, inhibition of NCLXm by CGP-37157 caused rapid mitochondrial Ca2+ 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 [Ca2+]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 Ca2+ 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.

The annotation of GBA1 has been concealed by its protein-coding pseudogene GBAP1.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification.

The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

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.

An antibody scanning method for the detection of α-synuclein oligomers in the serum of Parkinson's disease patients.

Misfolded α-synuclein oligomers are closely implicated in the pathology of Parkinson's disease and related synucleinopathies. The elusive nature of these aberrant assemblies makes it challenging to develop quantitative methods to detect them and modify their behavior. Existing detection methods use antibodies to bind α-synuclein aggregates in biofluids, although it remains challenging to raise antibodies against α-synuclein oligomers. To address this problem, we used an antibody scanning approach in which we designed a panel of 9 single-domain epitope-specific antibodies against α-synuclein. We screened these antibodies for their ability to inhibit the aggregation process of α-synuclein, finding that they affected the generation of α-synuclein oligomers to different extents. We then used these antibodies to investigate the size distribution and morphology of soluble α-synuclein aggregates in serum and cerebrospinal fluid samples from Parkinson's disease patients. Our results indicate that the approach that we present offers a promising route for the development of antibodies to characterize soluble α-synuclein aggregates in biofluids.

In Vivo 18F-Flortaucipir PET Does Not Accurately Support the Staging of Progressive Supranuclear Palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by neuroglial tau pathology. A new staging system for PSP pathology postmortem has been described and validated. We used a data-driven approach to test whether postmortem pathologic staging in PSP can be reproduced in vivo with 18F-flortaucipir PET. Methods: Forty-two patients with probable PSP and 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 postmortem staging approach for PSP pathology, we evaluated the combinations of absent/present pathology (or abnormal/normal PET signal) across all regions to assign each participant to in vivo stages. ANOVA was applied to analyze differences among means of disease severity between stages. In vivo staging was compared with postmortem staging in 9 patients who also had postmortem confirmation of the diagnosis and stage. Results: Stage assignment was estimable in 41 patients: 10, 26, and 5 patients were classified in stage I/II, stage III/IV, and stage V/VI, respectively, whereas 1 patient was not classifiable. Explorative substaging identified 2 patients in stage I, 8 in stage II, 9 in stage III, 17 in stage IV, and 5 in stage V. However, the nominal 18F-flortaucipir--derived stage was not associated with clinical severity and was not indicative of pathology staging postmortem. Conclusion:18F-flortaucipir PET in vivo does not correspond to neuropathologic staging in PSP. This analytic approach, seeking to mirror in vivo neuropathology staging with PET-to-autopsy correlational analyses, might enable in vivo staging with next-generation tau PET tracers; however, further evidence and comparisons with postmortem data are needed.

Small soluble α-synuclein aggregates are the toxic species in Parkinson's disease.

Soluble α-synuclein aggregates varying in size, structure, and morphology have been closely linked to neuronal death in Parkinson's disease. However, the heterogeneity of different co-existing aggregate species makes it hard to isolate and study their individual toxic properties. Here, we show a reliable non-perturbative method to separate a heterogeneous mixture of protein aggregates by size. We find that aggregates of wild-type α-synuclein smaller than 200 nm in length, formed during an in vitro aggregation reaction, cause inflammation and permeabilization of single-liposome membranes and that larger aggregates are less toxic. Studying soluble aggregates extracted from post-mortem human brains also reveals that these aggregates are similar in size and structure to the smaller aggregates formed in aggregation reactions in the test tube. Furthermore, we find that the soluble aggregates present in Parkinson's disease brains are smaller, largely less than 100 nm, and more inflammatory compared to the larger aggregates present in control brains. This study suggests that the small non-fibrillar α-synuclein aggregates are the critical species driving neuroinflammation and disease progression.

[11C]PK11195-PET Brain Imaging of the Mitochondrial Translocator Protein in Mitochondrial Disease.

OBJECTIVE: To explore the possibilities of radioligands against the mitochondrial outer membrane protein TSPO as biomarkers for mitochondrial disease, we performed PET (PET)-MR brain imaging with [11C]PK11195 in 14 patients with genetically confirmed mitochondrial disease and 33 matched controls. METHODS: A case-control study of PET-MR imaging with the TSPO radioligand [11C]PK11195. RESULTS: Forty-six percent of symptomatic patients had volumes of abnormal radiotracer binding greater than the 95th percentile in controls. [11C]PK11195 binding was generally greater in grey matter and significantly decreased in white matter. This was most striking in patients with nuclear TYMP or mitochondrial m.3243A>G MT-TL1 mutations, in keeping with differences in mitochondrial density seen post mortem. Some regional binding patterns corresponded to clinical presentation and underlying mutation, even in the absence of structural changes on MRI. This was most obvious for the cerebellum, where patients with ataxia had decreased binding in the cerebellar cortex, but not necessarily volume loss. Overall, there was a positive correlation between aberrant [11C]PK11195 binding and clinical severity. CONCLUSION: These findings endorse the use of PET imaging with TSPO radioligands as a non-invasive in vivo biomarker of mitochondrial pathology. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that PET-MR brain imaging with TSPO radioligands identifies mitochondrial pathology.

[18F]-AV-1451 binding in the substantia nigra as a marker of neuromelanin in Lewy body diseases.

While [18F]-AV-1451 was developed as a PET radiotracer with high affinity for hyperphosphorylated tau, it has been proposed that loss of 'off-target' [18F]-AV-1451 binding to neuromelanin in the substantia nigra could be a surrogate marker of Lewy body diseases. [18F]-AV-1451 binding was measured in the substantia nigra of patients with Parkinson's disease (n = 35), dementia with Lewy bodies (n = 10) and separate control groups (n = 37; n = 14). Associations with motor symptoms, cognition and disease duration were evaluated using linear regression models. The dementia with Lewy bodies group had significantly reduced substantia nigra [18F]-AV-1451 binding compared to controls after adjusting for age (P < 0.05). However, there were no significant differences in substantia nigra [18F]-AV-1451 binding between Parkinson's disease and controls. Substantia nigra [18F]-AV-1451 binding was not associated with age, disease duration, Movement Disorders Society-Unified Parkinson's Disease Rating Scale and cognitive scores in dementia with Lewy bodies and Parkinson's disease groups. Despite the reduction of substantia nigra [18F]-AV-1451 binding in dementia with Lewy bodies, these findings suggest that substantia nigra [18F]-AV-1451 binding has no value as a diagnostic marker in early Parkinson's disease. Further investigations in longitudinal cohorts are warranted.

Neuroinflammation and protein pathology in Parkinson's disease dementia.

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-ß 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 1ß 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.