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The Dementia with Lewy Bodies (DLB) Consortium has refined its recommendations about the clinical and pathologic diagnosis of DLB, updating the previous report, which has been in widespread use for the last decade. The revised DLB consensus criteria now distinguish clearly between clinical features and diagnostic biomarkers, and give guidance about optimal methods to establish and interpret these. Substantial new information has been incorporated about previously reported aspects of DLB, with increased diagnostic weighting given to REM sleep behavior disorder and 123iodine-metaiodobenzylguanidine (MIBG) myocardial scintigraphy. The diagnostic role of other neuroimaging, electrophysiologic, and laboratory investigations is also described. Minor modifications to pathologic methods and criteria are recommended to take account of Alzheimer disease neuropathologic change, to add previously omitted Lewy-related pathology categories, and to include assessments for substantia nigra neuronal loss. Recommendations about clinical management are largely based upon expert opinion since randomized controlled trials in DLB are few. Substantial progress has been made since the previous report in the detection and recognition of DLB as a common and important clinical disorder. During that period it has been incorporated into DSM-5, as major neurocognitive disorder with Lewy bodies. There remains a pressing need to understand the underlying neurobiology and pathophysiology of DLB, to develop and deliver clinical trials with both symptomatic and disease-modifying agents, and to help patients and carers worldwide to inform themselves about the disease, its prognosis, best available treatments, ongoing research, and how to get adequate support.

Krabbe disease is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene that causes accumulation of the toxic sphingolipid psychosine. GALC variants are also associated with Lewy body diseases, an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in Krabbe disease has pathological similarities to that in Lewy body disease, we performed an observational post-mortem study of Krabbe disease brain tissue (n = 4) compared to infant controls (n = 4) and identified widespread accumulations of α-synuclein. To determine whether α-synuclein in Krabbe disease brain displayed disease-associated pathogenic properties we evaluated its seeding capacity using the real-time quaking-induced conversion assay in two cases for which frozen tissue was available and strikingly identified aggregation into fibrils similar to those observed in Lewy body disease, confirming the prion-like capacity of Krabbe disease-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it results from alterations to biological pathways, such as sphingolipid metabolism. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in Lewy body disease. [ABSTRACT FROM AUTHOR]

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs. [ABSTRACT FROM AUTHOR]

Aggregation of the protein α‐synuclein (α‐syn) into insoluble intracellular assemblies termed Lewy bodies (LBs) is thought to be a critical pathogenic event in LB diseases such as Parkinson's disease and dementia with LBs. In LB diseases, the majority of α‐syn is phosphorylated at serine 129 (pS129), suggesting that this is an important disease‐related post‐translational modification (PTM). However, PTMs do not typically occur in isolation and phosphorylation at the proximal tyrosine 125 (pY125) residue has received considerable attention and has been inconsistently reported to be present in LBs. Furthermore, the proximity of Y125 to S129 means that some pS129 antibodies may have epitopes that include Y125, in which case phosphorylation of Y125 will impede recognition of α‐syn. This would potentially lead to underestimating LB pathology burdens if pY125 occurs alongside pS129. To address the apparent controversy in the literature regarding the detection of pY125, we investigated its presence in the LB pathology. We generated pS129 antibodies whose epitope includes or does not include Y125 and compared the extent of α‐syn pathology recognized in mouse models of α‐synucleinopathies, human brain tissue lysates and fixed post‐mortem brain tissues. Our study demonstrated no difference in α‐syn pathology recognized between pS129 antibodies, irrespective of whether Y125 was part of the epitope or not. Furthermore, evaluation with pY125 antibodies whose epitope does not include S129 demonstrated no labeling of LB pathology. This study reconciles disparate results in the literature and demonstrates pY125 is not a key component of LB pathology in murine models or human tissues in idiopathic LB diseases. [ABSTRACT FROM AUTHOR]

Synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities. [ABSTRACT FROM AUTHOR]

Parkinson's disease is one of the most common movement disorders and is characterized by dopaminergic cell loss and the accumulation of pathological α-synuclein, but its precise pathogenetic mechanisms remain elusive. To develop disease-modifying therapies for Parkinson's disease, an animal model that recapitulates the pathology and symptoms of the disease, especially in the prodromal stage, is indispensable. As subjects with α-synuclein gene (SNCA) multiplication as well as point mutations develop familial Parkinson's disease and a genome-wide association study in Parkinson's disease has identified SNCA as a risk gene for Parkinson's disease, the increased expression of α-synuclein is closely associated with the aetiology of Parkinson's disease. In this study we generated bacterial artificial chromosome transgenic mice harbouring SNCA and its gene expression regulatory regions in order to maintain the native expression pattern of α-synuclein. Furthermore, to enhance the pathological properties of α-synuclein, we inserted into SNCA an A53T mutation, two single-nucleotide polymorphisms identified in a genome-wide association study in Parkinson's disease and a Rep1 polymorphism, all of which are causal of familial Parkinson's disease or increase the risk of sporadic Parkinson's disease. These A53T SNCA bacterial artificial chromosome transgenic mice showed an expression pattern of human α-synuclein very similar to that of endogenous mouse α-synuclein. They expressed truncated, oligomeric and proteinase K-resistant phosphorylated forms of α-synuclein in the regions that are specifically affected in Parkinson's disease and/or dementia with Lewy bodies, including the olfactory bulb, cerebral cortex, striatum and substantia nigra. Surprisingly, these mice exhibited rapid eye movement (REM) sleep without atonia, which is a key feature of REM sleep behaviour disorder, at as early as 5 months of age. Consistent with this observation, the REM sleep-regulating neuronal populations in the lower brainstem, including the sublaterodorsal tegmental nucleus, nuclei in the ventromedial medullary reticular formation and the pedunculopontine nuclei, expressed phosphorylated α-synuclein. In addition, they also showed hyposmia at 9 months of age, which is consistent with the significant accumulation of phosphorylated α-synuclein in the olfactory bulb. The dopaminergic neurons in the substantia nigra pars compacta degenerated, and their number was decreased in an age-dependent manner by up to 17.1% at 18 months of age compared to wild-type, although the mice did not show any related locomotor dysfunction. In conclusion, we created a novel mouse model of prodromal Parkinson's disease that showed RBD-like behaviour and hyposmia without motor symptoms. [ABSTRACT FROM AUTHOR]

Synucleinopathies including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are characterized by the abnormal accumulation and propagation of α‐synuclein (α‐syn) pathology in the central and peripheral nervous system as Lewy bodies or glial cytoplasmic inclusions. Several antibodies against α‐syn have been developed since it was first detected as the major component of Lewy bodies and glial cytoplasmic inclusions. Over the years, researchers have generated specific antibodies that alleviate the accumulation of intracellular aggregated α‐syn and associated pathology in cellular and preclinical models of synucleinopathies. So far, antibodies have been the first choice as tools for research and diagnosis and currently, a wide variety of antibody fragments have been developed as an alternative to full‐length antibodies for increasing its therapeutic usefulness. Recently, conformation specific antibody‐based approaches have been found to be promising as therapeutic strategies, both to block α‐syn aggregation and ameliorate the resultant cytotoxicity, and as diagnostic tools. In this review, we summarize different α‐syn specific antibodies and provide their usefulness in tackling synucleinopathies. This article is part of the Special Issue "Synuclein". [ABSTRACT FROM AUTHOR]

As mitochondrial dysfunction is associated with phenotypes of ageing [[7]], one could speculate that increased levels of LB pathology in this population reflect an acceleration of ageing; however, as we did not find increased levels of other age-associated pathologies, it seems unlikely that accelerated ageing alone could explain these findings. Further understanding of LB pathology in this population is important to understand its clinical relevance in the sub-set of mitochondrial diseases cases affected by it, and to determine the role of mitochondrial dysfunction in the genesis of LB pathology. [Extracted from the article]

Background: Alpha-synuclein (asyn) has been shown to play an important role in the neuropathology of Parkinson's disease (PD). In the diseased brain, classic intraneuronal inclusions called Lewy bodies contain abnormal formations of asyn protein which is mostly phosphorylated at serine 129 (pS129 asyn). This suggests that post-translational modifications may play a role in the pathogenic process. To date, several uniplex assays have been developed in order to quantify asyn not only in the brain but also in cerebrospinal fluid and blood samples in order to correlate asyn levels to disease severity and progression. Notably, only four assays have been established to measure pS129 asyn specifically and none provide simultaneous readout of the total and pS129 species. Therefore, we developed a sensitive high-throughput duplex assay quantifying total and pS129 human asyn (h-asyn) in the same well hence improving accuracy as well as saving time, consumables and samples. Results: Using our newly established duplex assay we measured total and pS129 h-asyn in vitro showing that polo-like kinase 2 (PLK2) can phosphorylate asyn up to 41% in HEK293 cells and in vivo the same kinase phosphorylated h-asyn up to 17% in rat ventral midbrain neurons. Interestingly, no increase in phosphorylation was observed when PLK2 and h-asyn were co-expressed in rat striatal neurons. Furthermore, using this assay we investigated h-asyn levels in brain tissue samples from patients with PD as well as PD dementia and found significant differences in pS129 h-asyn levels not only between disease tissue and healthy control samples but also between the two distinct disease states especially in hippocampal tissue samples. Conclusions: These results demonstrate that our duplex assay for simultaneous quantification is a useful tool to study h-asyn phosphorylation events in biospecimens and will be helpful in studies investigating the precise causative link between post-translational modification of h-asyn and PD pathology. [ABSTRACT FROM AUTHOR]

The accumulation and aggregation of α-synuclein (α-syn) is the main pathologic event in Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-Syn-seeded fibril formation and its induced toxicity occupy a major role in PD pathogenesis. Thus, assessing compounds that inhibit this seeding process is considered a key towards the therapeutics of synucleinopathies. Using biophysical and biochemical techniques and seeding-dependent cell viability assays, we screened a total of nine natural compounds of alkaloid origin extracted from Chinese medicinal herbs. Of these compounds, synephrine, trigonelline, cytisine, harmine, koumine, peimisine, and hupehenine exhibited in vitro inhibition of α-syn-seeded fibril formation. Furthermore, using cell viability assays, six of these compounds inhibited α-syn-seeding-dependent toxicity. These six potent inhibitors of amyloid fibril formation and toxicity caused by the seeding process represent a promising therapeutic strategy for the treatment of PD and other synucleinopathies. [ABSTRACT FROM AUTHOR]

Aggregated α-synuclein (αSyn) protein is a core pathological feature of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Both PD and DLB demonstrate the presence of diverse intracellular α-synuclein (αSyn) species, including C-terminally truncated αSyn (C-αSyn), although it is unknown how C-αSyn species contribute to disease progression. Using recombinant C-αSyn and PD and DLB brain lysates as seeds in the real-time quaking-induced conversion (RT-QuIC) assay, we explored how C-αSyn may be involved in disease stratification. Comparing the seeding activity of aqueous-soluble fractions to detergent-soluble fractions, and using αSyn 1-130 as substrate for the RT-QuIC assay, the temporal cortex seeds differentiated PD and DLB from healthy controls. In contrast to the temporal cortex, where PD and DLB could not be distinguished, αSyn 1-130 seeded by the detergent-soluble fractions from the PD frontal cortex demonstrated greater seeding efficiency compared to the DLB frontal cortex. Moreover, proteinase K-resistant (PKres) fragments from the RT-QuIC end products using C-αSyn 1-130 or C-αSyn 1-115 were more obvious in the frontal cortex compared to the temporal cortex. Morphological examinations of RT-QuIC end products showed differences in the size of the fibrils between C-αSyn 1-130 and C-αSyn 1-115, in agreement with the RT-QuIC results. These data show that C-αSyn species can distinguish PD from DLB and suggest diversity in αSyn species across these synucleinopathies, which could play a role in disease progression. [ABSTRACT FROM AUTHOR]

Background and Aim: Toxic oligomeric α-synuclein (αS; O-αS) has been suggested to play a central role in the pathogenesis of Lewy body diseases such as Parkinson's disease (PD). Cerebrospinal fluid (CSF) levels of αS, O-αS, total and phosphorylated tau, and amyloid β 1-42 (Aβ1-42) are thought to reflect the pathophysiology or clinical symptoms in PD. In this study, we examined correlations of the CSF levels of these proteins with the clinical symptoms, and with each other in drug-naïve patients with PD.Methods: Twenty-seven drug-naïve patients with PD were included. Motor and cognitive functions were assessed using the Unified Parkinson's Disease Rating Scale (UPDRS), Montreal Cognitive Assessment (MoCA), and Neurobehavioral Cognitive Status Examination (COGNISTAT). CSF levels of total αS, O-αS, Aβ1-42, total tau and tau phosphorylated at threonine 181 (P-tau181p) were measured. CSF levels of these proteins were compared with clinical assessments from the UPDRS, MoCA and COGNISTAT using Spearman correlation analysis. Spearman correlation coefficients among CSF protein levels were also evaluated.Results: CSF levels of αS were negatively correlated with UPDRS part III (motor score) (p < 0.05) and bradykinesia (p < 0.01), and positively correlated with COGNISTAT subtest of judgement (p < 0.01) and CSF levels of Aβ1-42 (p < 0.001), total tau (p < 0.001) and P-tau181p (p < 0.01). Lower CSF levels of Aβ1-42, total tau and P-tau181p were significantly related to worsening of some motor and/or cognitive functions. The CSF level of O-αS showed no correlation with any motor and cognitive assessments or with CSF levels of the other proteins.Conclusion: CSF levels of αS are correlated with some clinical symptoms and CSF levels of other pathogenic proteins in drug-naïve PD patients. These correlations suggest a central role for interaction and aggregation of αS with Aβ1-42, tau, and phosphorylated tau in the pathogenesis of PD. Although O-αS has been shown to have neurotoxic effects, CSF levels do not reflect clinical symptoms or levels of other proteins in cross-sectional assessment. [ABSTRACT FROM AUTHOR]

Background: Converging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson's disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS).Methods: Male Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis.Results: Intrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~ 35%) of substantia nigra pars compacta dopamine neurons within 5-6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum.Conclusions: Our results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD. [ABSTRACT FROM AUTHOR]

Abstract: Dementia with Lewy bodies (DLB) is characterized by widespread depositions of α-synuclein, which are described as Lewy bodies. Recently, it was shown that neuronal cells in culture constitutively release α-synuclein into the culture medium and that α-synuclein is normally present in human cerebrospinal fluid (CSF). The aim of the present study was to evaluate the diagnostic value of CSF α-synuclein levels in discriminating DLB from Alzheimer''s disease (AD). α-Synuclein was measured in CSF from 16 patients with DLB and 21 patients with AD. Iodine-123 metaiodobenzylguanidine cardiac scintigraphy was also performed to assess Lewy body pathology. CSF α-synuclein levels did not differ significantly between DLB and AD patients. However, the duration of illness was associated with lower α-synuclein levels (p <0.05) in DLB, while no such association was found in AD. The present data show CSF α-synuclein levels are not sensitive diagnostic markers to discriminate DLB from AD. However, the lower α-synuclein levels in DLB patients with longer duration suggest a reduction in CSF α-synuclein in association with increased severity of α-synucleinopathy in the brain. [Copyright &y& Elsevier]

Abstract: Because accumulation of α-synuclein (αS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several αS-derived peptides by mass spectrometry. The concentration of αS amounted to <0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total αS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF αS concentrations ranged from 0.8 to 16.2 pg/μl. Mean CSF αS values were lower in donors with a primary synucleinopathy (PD, DLB: n =57) than in the other two groups (AD, NCO: n =35; p =0.025). By contrast, living Creutzfeldt–Jakob disease patients showed markedly elevated CSF αS levels (n =8; mean, 300 pg/μl; p <0.001). Our results unequivocally confirm the presence of αS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF αS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF αS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies. [Copyright &y& Elsevier]

Abstract: Parkinson''s disease (PD) and dementia with Lewy bodies (DLB) are both characterized by the formation and intraneuronal accumulation of fibrillar aggregates of α-synuclein (α-syn) protein in affected brain regions. α-Syn has biochemical properties and a structural motif characteristic of fatty acid binding proteins. Using the fatty acid binding resin Lipidex-1000, we investigated the capture of α-, β-, and γ-syn proteins as lipid-associated proteins from normal and DLB brain lysates, and from normal human cerebrospinal fluid (CSF). These were eluted from Lipidex-1000 and analyzed by SDS–NuPAGE followed by Western blotting. Using this methodology, we have been able to extract full-length and truncated forms of α-syn from brain lysates. We also extracted low levels of β-syn from DLB brains, but failed to extract any γ-syn. We were able to capture only full-length monomeric α-syn from normal human CSF. Our data confirm the fatty acid binding properties of α-syn, and to a lesser extent β-syn, but suggest that γ-syn does not share this same characteristic. [Copyright &y& Elsevier]