Welcome to the AD/PD™ 2022 Interactive Program
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PATHOLOGICAL OLIGOMERISATION OF PHOSPHORYLATED NUCLEAR ASYN IS ASSOCIATED WITH ELEVATED DNA DAMAGE IN DEMENTIA WITH LEWY BODIES.
Abstract
Aims
Dementia with Lewy bodies (DLB) is defined by the cytoplasmic accumulation of alpha-synuclein (aSyn) within neurons. Although predominately pre-synaptic, nuclear aSyn (aSynNuc) has been frequently observed in-vitro where it is associated with altered DNA integrity. Yet the presence and role of aSynNuc in the human brain remains controversial and its relevance to DLB pathology unclear, here we set out to resolve the existence and relevance of aSynNuc.
Methods
aSynNuc was investigated via immunohistochemical analysis of fixed postmortem brain tissue and in isolated nuclear preparations from frozen brain tissue. Nuclear preparations were additionally subject to western-blot and label-free mass spectrometry.
Results
Immunohistochemical analysis demonstrated intra-nuclear puncta reactive to a variety of pan-aSyn antibodies as well as to the disease associated phosphorylation specific pS129-aSyn antibody. The presence of aSynNuc was further confirmed via mass spectrometry and biochemical analysis. In all cases, monomeric aSynNuc was detected at ~10 fold lower levels compared to cytoplasmic aSyn. Critically, only in DLB cases were aSynNuc oligomers evident, with both monomers and oligomers being highly phosphorylated. Pathological aSynNuc was observed alongside elevated levels of DNA double strand breaks (DSBs). Elevated DNA damage was observed in the absence of changes to upstream DNA damage signalling kinases (ATM/ATR) suggesting impairments in DSB repair as opposed to an increase in the occurrence of DSB.
Conclusions
Our data support emerging roles for aSyn in DNA damage repair and highlights a novel disease mechanism underlying the neurodegenerative processes of DLB and implicates genomic homeostasis as a potential future therapeutic target.
TAU PATHOLOGY-ASSOCIATED ALTERATIONS IN MICRORNAS IN ALZHEIMER’S DISEASE ENTORHINAL CORTEX
Abstract
Aims
In the present study we performed high-throughput small RNA sequencing in post-mortem entorhinal cortex brain tissue from 85 individuals with either low (Braak 0-II) or high (Braak V-VI) levels of neurofibrillary tangle pathology, with the aim of identifying differentially expressed microRNAs (miRNAs) associated with Alzheimer’s disease.
Methods
We quantified the expression of 490 highly expressed miRNAs, utilizing weighted gene correlation network analysis (WGCNA) to identify co-expressed miRNAs. Subsequently we performed in silico identification of miRNA-regulated protein-protein interaction (PPI) networks to determine potential downstream dysregulated pathways that result from these tau-associated miRNA alterations.
Results
We identified 49 nominally-significant miRNAs, of which 28 were upregulated and 21 were downregulated. Of these, two of the down-regulated miRNAs passed the Bonferroni threshold for multiple testing correction: miR-212-3p and miR-132-3p. Using WGCNA we identified 14 co-ecpressed miRNA modules, with one being significantly associated with pathology, and containing 17 miRNAs, including miR-212-3p and miR-132-3p. To explore the miRNA regulation of PPI networks in AD we identified the mRNA targets of the 49 nominally-significant Braak-associated miRNAs, showing enrichment for ubiquitin-related pathways.
Conclusions
Alterations in miRNA expression levels have been previously reported in AD in multiple brain regions, blood and CSF. However, the use of different tissue types, small sample sixes and various different assay techniques has resulted in inconsistent findings to date. We have quantified genome-wide miRNA expression levels in post-mortem entorhinal cortex and found down-regulation of miR-212-3p and miR-132-3p after Bonferroni adjustment for multiple testing. Pathway analysis highlighted that mRNAs targeted by the nominally-significant miRNAs, were enriched in ubiquitin-related pathways.
NOT JUST LEWY BODIES: A NANOSCALE VIEW OF PARKINSON'S PATHOLOGY IN POST-MORTEM HUMAN BRAIN
Abstract
Aims
Parkinson’s disease (PD) is characterised by the presence of Lewy bodies - large, fibrillar intra-neuronal accumulations of alpha-synuclein (aSyn). It is currently thought aSyn monomers misfold and oligomerize, forming amyloid fibrils which accumulate into these fibrillar Lewy bodies. However, using electron microscopy, we have recently demonstrated that non-hallmark aSyn pathology in post-mortem human brain consists of accumulated membranes, cellular vesicles and organelles in which fibrils were not always detected. It is unknown whether these membranous aSyn accumulations represent an earlier stage in the formation of fibrillar Lewy bodies, or if they arise through a separate mechanism. In this study we developed a new correlative light and electron microscopy pipeline to investigate the ultrastructure of intra-neuronal aSyn accumulation in brain sections from PD patients. The incorporation of confocal light microscopy allowed us to efficiently target specific morphologies of aSyn accumulation thought to represent earlier stages of Lewy body formation.
Methods
We used correlative confocal light and electron microscopy to localise aSyn pathology in chemically fixed post-mortem human brain of five PD patients. Free-floating brain sections were immunolabelled and imaged using confocal laser scanning microscopy. The same sections were then processed for electron microscopy using en bloc staining and resin-embedding for ultrastructural investigation.
Results
We localized and imaged over 280 examples of intra-neuronal aSyn accumulation, showing the ultrastructure of different morphologies of aSyn pathology thought to represent earlier stages of Lewy body formation.
Conclusions
Our data provides key insight into the mechanisms behind accumulation of aSyn and the formation of Lewy bodies in human PD brain.
COMPLEMENT FACTOR CSMD1 IS ASSOCIATED WITH NEOCORTICAL LEWY BODY COUNT IN A JAPANESE-AMERICAN POPULATION-BASE AUTOPSY SERIES: THE HONOLULU-ASIA AGING STUDY
Abstract
Aims
To test the hypothesis that genome wide association studies (GWASs) of the lesions underlying clinical neurological syndromes are a powerful way to uncover the molecular etiology of disease.
Methods
Case material are 308 autopsies of Japanese-American men (mean age at death 87.5 years; 11% with mild, and 30% with severe cognitive impairment prior to death). Lewy bodies were assessed by manual count of alpha-synuclein stained neocortical sections from the frontal, temporal, parietal, and occipital lobes. Genetic association was tested by linear models controlling for age at death assuming an additive genotypic effect.
Results
Top hits include a locus on chromosome 1q32 previously associated with amyotrophic lateral sclerosis (p=5.8e-8), intronic variants on the dopaminergic neuron axonal guidance gene EPHB1 previously associated with Parkinson’s disease (PD) (p=8.4e-6), and a locus containing two independent intron 1 variants on CSMD1, a known risk factor for autosomal dominant PD (p=1.5e-6). Complement inhibitor protein CSMD1 is a type 1 transmembrane cell adhesion molecule implicated in axonal guidance and the development, maintenance, and pruning of synapses in the adult brain. Intermediate hits include genes encoding cell adhesion molecules and a second complement inhibitor implicated in the regulation of synapses (TRAPPC9).
Conclusions
This converging evidence points to cell adhesion proteins and complement mediated synaptic regulation in the etiology of Lewy body lesions. The clear relevance of genes identified confirms the hypothesis that GWAS of neuropathologic endophenotypes is a valid and statistically powerful approach to discovering risk factor genes that moreover directly informs our understanding of the molecular pathways underlying disease.
EXAMINATION OF WHITE MATTER TRACTS IN PROGRESSIVE SUPRANUCLEAR PALSY VARIANTS BY DIFFUSION TENSOR IMAGING PARAMETERS, CONNECTOMICS, AND BRAIN NETWORK ANALYTICS.
Abstract
Aims
We aimed to apply different diffusion MRI (dMRI) techniques to characterize specific changes in white matter (WM) tracts from patients with progressive supranuclear palsy (PSP). We hypothesized that microstructural, connectomics, and network techniques, representing the superior cerebellar peduncle (SCP) and dentatorubrothalamic tract (DRTT) fibers, can differentiate autopsy-confirmed PSP Richardson’s syndrome (PSP-RS) and PSP with predominant speech and language disorder (PSP-SL).
Methods
Twenty-two autopsy-confirmed patients with PSP (12 PSP-RS and 10 PSP-SL) had undergone antemortem 3T head MRI, including diffusion tensor imaging (DTI). Deterministic tractography was used to identify the SCP and DRTT in each patient and fractional anisotropy (FA) and mean diffusivity (MD) were calculated for each tract. A hierarchical linear statistical model analysis was applied to the DTI dataset. Mean connectivity matrix strength was analyzed using Spearman’s correlation coefficients. Graph theory-based global and local network metrics such as network efficiency and small-wordness were also studied.
Results
Greater MD in SCP, as well as lower FA in DRTT fibers, was observed in PSP-RS compared to PSP-SL. DRTT connectivity analysis showed a greater decrease in general connectivity strength and infra-thalamic network circuitry in PSP-RS compared to PSP-SL. While both PSP subtypes showed similar reductions in network complexity measured as small-worldness, lower global network density and efficiency were seen in PSP-RS.
Conclusions
Our outcomes demonstrate that is possible to increase diagnostic discrimination between PSP subtypes by combining diverse structural dMRI techniques. These findings could help physicians improve the assessment and follow-up in this patient population.
WHITE MATTER HYPERINTENSITIES ASSOCIATE WITH PATHOLOGICAL HALLMARKS IN ALZHEIMER’S AND PARKINSON’S DISEASE.
Abstract
Aims
White matter hyper-intensities (WMH) are observed on FLAIR MRI, increase with age and are more prominent in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The aim of this study was to assess the association between WMH and neuropathological hallmarks in clinically-defined and pathologically-confirmed AD, PD and non-neurological control brain donors.
Methods
In-situ 3T-MRI data were collected for 19 AD, 16 PD and 21 controls, and included 3DT1-weighted and 3D FLAIR sequences. WMH were segmented on FLAIR using an in-house developed multiview convolutional neural network, followed by manual editing, and volume obtained with FSL. Fifteen brain regions were dissected at autopsy, immunostained and evaluated for neuropathological diagnosis, including Thal phase, and Braak stages for neurofibrillary tangles (NFT) and α-synuclein (α-syn). Group differences were assessed with linear models (corrected for age, gender, post-mortem delay) and associations with Pearson and Spearman correlations.
Results
WMH load was higher in AD (p=0.003) and borderline higher in PD than controls p=0.055). In the whole cohort, WMH was associated with increased age (r=0.29,p=0.033), normalized brain volume (NBV; r=-0.29,p=0.034), gray matter volume (NGMV; r=-0.31,p=0.021) and Braak NFT stage (r=0.33,p=0.014). Across clinically defined controls and AD, WMH associated with NBV (r=-0.38,p=0.016), NGMV (r=-0.39,p=0.014), left and right hippocampal volume (r=-0.33,p=0.037 and r=-0.33,p=0.041), Braak NFT (r=0.44,p=0.005) and Thal phase (r=0.34,p=0.031). Across clinically defined controls and PD, WMH associated with age (r=0.45,p=0.005), NGMV (r=-0.39,p=0.018), and Braak α-syn stage (r=0.44,p=0.006).
Conclusions
WMH associate with pathological hallmarks of AD and PD and contribute to MRI atrophy.
NEUROPATHOLOGICAL HALLMARKS OF NEURODEGENERATIVE DISEASES DO NOT ASSOCIATE WITH COGNITIVE PERFORMANCE IN CENTENARIANS
Abstract
Aims
Hallmarks of neurodegenerative diseases accumulate with age in the brains of non-demented individuals, which has implications for diagnosis and interpretation of the pathological role of these hallmarks in extreme aging. To investigate the separability of pathological hallmarks of Alzheimer’s disease (AD), we assessed AD-related pathology in an age-continuum of AD and non-demented subjects up to extreme aging. Furthermore, we determined to what extent neuropathology loads discriminate between cognitive performance in centenarians.
Methods
NIA-Reagan amyloid phases, Braak neurofibrillary tangle stages, and CERAD neuritic plaque scores were analyzed in an age continuum comprising 849 AD (aged 37-102), 653 non-demented (aged 16-99) and 86 centenarian (aged 100-115) donors. Centenarian brain tissue was additionally scored for the load of TAR DNA-binding protein 43, Lewy bodies and granulovacuolar degeneration and divided in demented (MMSE <=24, n=39) and non-demented (MMSE>24, n=47), based on MMSE assessment 8.8 (±6.97) months prior to donation.
Results
With increasing age-at-death, AD related neuropathology load increased in non-demented individuals, and decreased in AD cases, converging at around 100 years of age. In centenarians, neuritic plaque load associated with cognitive decline (p=0.02), but all other assessed neuropathologies did not associate with cognitive performance.
Conclusions
The ability of the classic AD-related neuropathological hallmarks to distinguish between health and disease decreases with age until at extreme ages. This suggests that: (1) with age, the ability to maintain cognitive health increasingly depends on being resilient against the toxic effects associated with these pathologies; and/or (2) that neuropathological hallmarks accumulated in older individuals may be a harmless consequence of aging.