Single-molecule detection methods to study alpha-synuclein aggregation in postmortem Parkinson's disease brains.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Response to inflammation is a key determinant in many diseases and their outcomes. Diseases that commonly affect older people are frequently associated with altered inflammatory processes. Neuroinflammation has been described in Parkinson's disease (PD) brain. PD is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and at the sub-cellular level, mitochondrial dysfunction is a key feature. However, there is evidence that a different region of the brain, the cerebellum, is involved in the pathophysiology of PD. We report relative levels of 40 pro- and anti-inflammatory cytokines measured in PD and control cerebellar mitochondria. These data were obtained by screening cytokine antibody arrays. In parallel, we present concentrations of 29 oxylipins and 4 endocannabinoids measured in mitochondrial fractions isolated from post-mortem PD cerebellum with age and sex matched controls. Our oxylipin and endocannabinoid data were acquired via quantitation by LC-ESI—MS/MS. The separate sample sets both show there are clearly different inflammatory profiles between the sexes in control samples. Sex specific profiles were not maintained in cerebellar mitochondria isolated from PD brains.

Major histocompatibility complex class II (MHCII) molecules are antigen presentation proteins and increased in post-mortem Parkinson's disease (PD) brain. Attempts to decrease MHCII expression have led to neuroprotection in PD mouse models. Our group reported that a SNP at rs3129882 in the MHCII gene Human leukocyte Antigen (HLA) DRA is associated with increased MHCII transcripts and surface protein and increased risk for late-onset idiopathic PD. We therefore hypothesized that decreased MHCII may mitigate dopaminergic degeneration. During an ongoing α-synuclein lesion, mice with MHCII reduction in systemic and brain innate immune cells (LysMCre+I-Abfl/fl or CRE+) displayed brain T cell repertoire shifts and greater preservation of the dopaminergic phenotype in nigrostriatal terminals. Next, we investigated a human cohort to characterize the immunophenotype of subjects with and without the high-risk GG genotype at the rs3129882 SNP. We confirmed that the high-risk GG genotype is associated with peripheral changes in MHCII inducibility, frequency of CD4+ T cells, and differentially accessible chromatin regions within the MHCII locus. Although our mouse studies indicate that myeloid MHCII reduction coinciding with an intact adaptive immune system is insufficient to fully protect dopamine neurons from α-synuclein-induced degeneration, our data are consistent with the overwhelming evidence implicating antigen presentation in PD pathophysiology.

BackgroundTo establish whether Parkinson’s disease (PD) brains previously described to have decreased glucocerebrosidase activity exhibit accumulation of the lysosomal enzyme’s substrate, glucosylceramide, or other changes in lipid composition.MethodsLipidomic analyses and cholesterol measurements were performed on the putamen (n = 5-7) and cerebellum (n = 7-14) of controls, Parkinson’s disease brains with heterozygote GBA1 mutations (PD+GBA), or sporadic PD.ResultsTotal glucosylceramide levels were unchanged in both PD+GBA and sporadic PD brains when compared with controls. No changes in glucosylsphingosine (deacetylated glucosylceramide), sphingomyelin, gangliosides (GM2, GM3), or total cholesterol were observed in either putamen or cerebellum.ConclusionsThis study did not demonstrate glucocerebrosidase substrate accumulation in PD brains with heterozygote GBA1 mutations in areas of the brain with low α-synuclein pathology.

There are numerous observations confirming that microglia expressing major histocompatibility complex (MHC) class II molecules are associated with the central nervous system (CNS) in aging and pathological conditions. In this study, we investigated the distribution of MHC class II-positive microglia in Parkinson's disease (PD) brains. The number of MHC class II-positive microglia in the substantia nigra (SN) and putamen increased as the neuronal degeneration of the SN proceeded. These cells were also ICAM-1 (CD54) and LFA-1 (CD11a) positive. The number of activated microglia not only in the SN and putamen but also in the hippocampus, transentorhinal cortex, cingulate cortex and temporal cortex in PD was significantly higher than that in the normal control. Most activated microglia persisted regardless of the presence or absence of Lewy bodies. They were frequently associated not only with alpha-synuclein-positive Lewy neurites, but also with TH-16-positive dopaminergic and WH-3-positive serotonergic neurites, as well as MAP-2- and SMI-32-positive neurites. These activated microglia were also positive for TNF-alpha and interleukin-6, which are known to have a neuroprotective function. We conclude that MHC class II-positive microglia are a sensitive index of neuropathological change and are actively associated with damaged neurons and neurites.

Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

Sporadic Parkinson's disease (PD) is a geriatric disorder with unknown etiology, specifically affecting the nigrostriatal dopaminergic (DA-ergic) pathway of the brain. Amongst several contributing factors, nitric oxide (NO•) is considered to inflict injury to DA-ergic neurons, and to influence PD progression. Supportive evidence for this comes from animal models of PD, where inhibitors of NO• synthase (NOS) are found to protect against DA-ergic neuronal death, and NOS-deficient mice are found to be resistant to PD-producing neurotoxins. Presence of nitrated proteins and upregulated levels of NOS in human postmortem PD brain samples have rendered further support to this contention. While NO• from neuronal NOS contributes to neurodegeneration in PD, NO• produced by inducible NOS from proliferating microglia as inflammatory responses to neuronal insults are suggested to mediate the disease progression. Another view that NO• in small doses serves as a neuroprotective agent in the brain is also discussed, in light of experimental evidence available in vitro and in vivo. This view is based on the argument that NO• could form harmless nitrites and nitrates on reaction with endogenously produced reactive oxygen species (ROS) within the cells. This review essentially discusses the possibilities of considering NO• as a secondary response of DA-ergic cell death, while oxidative stress is the primary cause. Once neurons undergo death processes following uncontrolled oxidative insult, the resulting gliosis-mediated NO• accelerates the events as a secondary mediator. Since the time of initiation of DA-ergic cell death cannot be predicted, NO• could be an ideal molecular target to halt the disease progression.

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High‐throughput molecular approaches such as single‐cell RNA‐sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC‐MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three‐dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging‐based approach enables automatic 3D‐morphology characterization and classification of thousands of individual microglia in situ and revealed species‐ and disease‐specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC‐MAC is a precision diagnostic tool that allows a rapid, unbiased, and large‐scale analysis of microglia morphological states in mouse models and patient brain samples.

Parkinson's disease (PD) is characterized by the accumulation of α-synuclein aggregates and degeneration of melanized neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross-linking. In PD brain, tTG-induced cross-links have been identified in α-synuclein monomers, oligomers and α-synuclein aggregates. However, whether tTG and α-synuclein occur together in PD affected neurons remains to be established. Interestingly, using immunohistochemistry, we observed a granular distribution pattern of tTG, characteristic of melanized neurons in PD brain. Apart from tTG, these granules were also positive for typical endoplasmic reticulum (ER)-resident chaperones, that is, protein disulphide isomerase, ERp57 and calreticulin, suggesting a direct link to the ER. Additionally, we observed the presence of phosphorylated pancreatic ER kinase (pPERK), a classical ER stress marker, in tTG granule positive neurons in PD brain, although no subcellular colocalization of tTG and pPERK was found. Our data therefore suggest that tTG localization to granular ER compartments is specific for stressed melanized neurons in PD brain. Moreover, as also α-synuclein aggregates were observed in tTG granule positive neurons, these results provide a clue to the cellular site of interaction between α-synuclein and tTG.

Immune responses in the Parkinson's disease brain

BackgroundThe knowledge regarding the role of reactive astrogliosis in Parkinson’s disease (PD) is still at the surface level, however, a few in vivo PET studies have speculated that reactive astrogliosis could be an early event in PD pathology. Moreover, a direct link between a‐synuclein pathology and astrocytes in PD has been also shown, where astrocytes play a key role in a‐synuclein propagation by shuttling a‐synuclein between neurons and astrocytes. The PET imaging field is advancing very fast and different astrocytic‐tracers are now available, such as 11C‐Deprenyl (DED) (targeting MAO‐B) and 11C‐BU99008 (detect imidazoline‐2‐binding sites (I2Bs)), which we believe is crucial to explore the role of reactive astrogliosis, including astrocytic heterogeneity in PD. Hence, we used a multi‐tracer approach to understand the role of reactive astrogliosis and to validate its reliability as a potential biomarker in PD and related synucleinopathies.MethodWe used specialized postmortem saturation radioligand binding assay and small frozen human brain section autoradiography in PD and controls (CN) brains.ResultThe 3H‐BU99008 and 3H‐DED saturation binding studies in the frontal cortex (FC) brain homogenate (BH) of PD (n = 2‐3) and CN (n = 2) cases demonstrated relatively higher 3H‐DED binding as compared to 3H‐BU99008 with following Bmax (binding‐density) and Kd values: 3H‐BU99008 CNBmax – 59.1fmol/mg, Kd‐ 2.9nM vs. PDBmax – 51.6fmol/mg, 2.3nM; 3H‐DED CNBmax – 190.6fmol/mg, Kd‐ 2.7nM and PDBmax –244.2fmol/mg, Kd‐ 6.7nM. 3H‐BU99008 binding in the Cau in PD was ∼3‐fold higher than in CN. In contrast, the 3H‐DED binding between CN and PD brains in the Cau BH was almost comparable. Interestingly, qualitative, and semi‐quantitative assessment of small frozen brain section autoradiographies showed increased specific binding of 3H‐BU99008 and 3H‐DED in PD (n = 4) frontal &amp; temporal cortices as compared to CN (n = 2‐3). In Cau, 3H‐BU99008 again showed higher binding than CN whereas 3H‐DED binding was comparable to CN, further complementing our saturation data.ConclusionThe first study to assess reactive astrogliosis signature in postmortem PD brains with astroglial tracers BU99008 and DED. The findings clearly highlight regional differences in BU99008 and DED binding in PD brains and suggest increased reactive astrogliosis at the end stages of the disease warrants further investigation.

Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease causing parkinsonian symptoms. Altered DNA methylation of the microtubule-associated protein tau gene correlates with the expression changes in Alzheimer's disease and Parkinson's disease brains. However, few studies examine the sequences beyond the constitutive promoter. Because activating different microtubule-associated protein tau gene control regions via methylation might regulate the differential tau expression constituting the specific signatures of individual tauopathies, we compared methylation of a candidate promoter, intron 0. We assessed DNA methylation in the brains of patients with different tauopathies (35 Alzheimer's disease, 10 corticobasal degeneration, and 18 PSP) and 19 controls by intron 0 pyrosequencing. We also evaluated methylation in an independent cohort of 11 PSP cases and 12 controls. Frontal (affected by tau pathology) and occipital (unaffected) cortices were analyzed. In the initial samples, one CpG island site in intron 0 (CpG1) showed significant hypomethylation in PSP-affected frontal cortices when compared with controls (P = .022). Such hypomethylation was observed in replicate samples, but not in occipital cortices or other tauopathies. PSP and control samples (combining the initial and replicate samples) remained significantly different after adjustment for potential confounding factors (age, H1/H1 diplotype; P = .0005). PSP-affected tissues exhibited microtubule-associated protein tau RNA hyperexpression when compared with controls (P = .004), although no correlation with CpG1 methylation was observed. This exploratory study suggests that regions other than the constitutive promoter may be involved in microtubule-associated protein tau gene regulation in tauopathies and that intron 0 hypomethylation may be a specific epigenetic signature of PSP. These preliminary findings require confirmation. © 2016 International Parkinson and Movement Disorder Society.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, and recent evidence suggests that pathogenesis may be in part mediated by inflammatory processes, the molecular and cellular architectures of which are largely unknown. To identify and characterize selectively vulnerable brain cell populations in PD, we performed single-nucleus transcriptomics and unbiased proteomics to profile the prefrontal cortex from postmortem human brains of six individuals with late-stage PD and six age-matched controls. Analysis of nearly 80,000 nuclei led to the identification of eight major brain cell types, including elevated brain-resident T cells in PD, each with distinct transcriptional changes in agreement with the known genetics of PD. By analyzing Lewy body pathology in the same postmortem brain tissues, we found that α-synuclein pathology was inversely correlated with chaperone expression in excitatory neurons. Examining cell-cell interactions, we found a selective abatement of neuron-astrocyte interactions and enhanced neuroinflammation. Proteomic analyses of the same brains identified synaptic proteins in the prefrontal cortex that were preferentially down-regulated in PD. By comparing this single-cell PD dataset with a published analysis of similar brain regions in Alzheimer's disease (AD), we found no common differentially expressed genes in neurons but identified many shared differentially expressed genes in glial cells, suggesting that the disease etiologies, especially in the context of neuronal vulnerability, in PD and AD are likely distinct.

Altered expression of autophagic genes in the peripheral leukocytes of patients with sporadic Parkinson's disease

Parkinson’s disease (PD) involves motor and cognitive impairment that nicotinic acetylcholine receptors (nAChRs) such as the α7 subtype are responsible for regulating. The hippocampus, abundant in α7 nAChRs, was quantitatively evaluated for [125I]α-bungarotoxin ([125I]α-Bgtx) binding to α7 nAChRs in postmortem human PD (n = 26; 12 male, 14 female) and cognitively normal (CN) (n = 29; 14 male, 15 female) brain slices. Anti-ubiquitin and anti-α-synuclein immunostained adjacent slices were analyzed using QuPath. Autoradiographs of [125I]α-Bgtx radioligand binding were analyzed in OptiQuant. Ubiquitin and α-synuclein distribution generally aligned with the distribution of α7 nAChRs detected by [125I]α-Bgtx. Binding of [125I]α-Bgtx in PD cases was significantly greater than CN with a 32% increase in gray matter binding. A weak positive correlation between age and [125I]α-Bgtx binding was found in both PD and CN. In comparison to Alzheimer’s disease hippocampus, [125I]α-Bgtx binding in PD gray matter was higher by 41%. Differences in nAChR expression imply unique roles depending on the neurodegenerative pathology. PD may experience an increase in α7 nAChRs as a compensatory mechanism to the loss in neurons, highlighting its neuroprotective capabilities. [125I]α-Bgtx shows potential as a radioligand for α7 nAChRs to elucidate the complexities of PD pathology.