In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences ...of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson's disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson's disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson's disease.
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”.
α‐Synuclein (α‐syn) under physiological and pathological conditions: Under physiological conditions, α‐syn is an unstructured soluble monomer bound to membranes with two α‐helices. Under pathological conditions, α‐syn dimerizes and subsequently aggregates into oligomers/protofibrils, which ultimately form into mature fibrils. Until now it still remains unclear as to which strain of α‐syn is most toxic. We suggest that there is a pressing need for further characterization of the diversity and clinico‐pathological relevance of distinct strains of α‐syn in Lewy body disease. Hence antibody‐based approaches, targeting different strains of α‐syn, could accelerate the understanding of the complexities in synucleinopathies.
This article is part of the Special Issue “Synuclein”.
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).
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.
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.
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.
AD is the most common neurodegenerative disorder characterized by progressive memory impairment and cognitive deficits. The pathology of AD is still unclear; however, several studies have shown that ...the aggregation of the Aβ peptide in the CNS is an exclusively pathological process involved in AD. Currently, there is no proven medication to cure or prevent the disease progression. Nevertheless, various therapeutic approaches for AD show only relief of symptoms and mostly work on cognitive recovery. However, one of the promising approaches for therapeutic intervention is to use inhibitors for blocking the Aβ peptide aggregation process. Recently, herbal phenolic compounds have been shown to have a therapeutic property for treatment of AD due to their multifaceted action. In this study, we investigated the effectiveness of SA, Gn Rb1, and DMyr on inhibiting the aggregation and toxicity of Aβ40 and Aβ42 using different biochemical and cell-based assays. Our results showed that SA and DMyr inhibit Aβ40 and Aβ42 fibrillation, seeded aggregation, and toxicity. Gn Rb1 did not have any effect on the aggregation or toxicity induced by Aβ40 and Aβ42. Moreover, SA and DMyr were able to disaggregate the preformed fibrils. Overall, these compounds may be used alone or synergistically and could be considered as a lead for designing new compounds that could be used as effective treatment of AD and related disorders.
Despite decades of intensive research, to date, there is no accepted diagnosis for Parkinson's disease (PD) based on biochemical analysis of blood or CSF. However, neurodegeneration in the brains of ...PD patients begins several years before the manifestation of the clinical symptoms, pointing to serious flaw/limitations in this approach.
To explore the potential use of alpha-synuclein (α-syn) species as candidate biomarkers for PD, we generated specific antibodies directed against wide array of α-syn species, namely total-, oligomeric- and phosphorylated-Ser129-α-syn (t-, o- and p-S129-α-syn). Next we sought to employ our antibodies to develop highly specific ELISA assays to quantify α-syn species in biological samples. Finally we verified the usefulness of our assays in CSF samples from 46 PD patients and 48 age-matched healthy controls. We also assessed the discriminating power of combining multiple CSF α-syn species with classical Alzheimer's disease biomarkers. The combination of CSF o-/t-α-syn, p-S129-α-syn and p-tau provided the best fitting predictive model for discriminating PD patients from controls. Moreover, CSF o-α-syn levels correlated significantly with the severity of PD motor symptoms (r = -0.37).
Our new ELISA assays can serve as research tools to address the unmet need for reliable CSF biomarkers for PD and related disorders.
Abstract α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinson’s disease (PD), dementia with ...Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including β-syn, γ-syn, β-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimer’s disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies.
We aimed to characterize in vivo α-synuclein (α-syn) aggregates in skin nerves to ascertain: 1) the optimal marker to identify them; 2) possible differences between synucleinopathies that may justify ...the clinical variability. We studied multiple skin nerve α-syn deposits in 44 patients with synucleinopathy: 15 idiopathic Parkinson's disease (IPD), 12 dementia with Lewy Bodies (DLB), 5 pure autonomic failure (PAF) and 12 multiple system atrophy (MSA). Ten healthy subjects were used as controls. Antibodies against native α-syn, C-terminal α-syn epitopes such as phosphorylation at serine 129 (p-syn) and to conformation-specific for α-syn mature amyloid fibrils (syn-F1) were used. We found that p-syn showed the highest sensitivity and specificity in disclosing skin α-syn deposits. In MSA abnormal deposits were only found in somatic fibers mainly at distal sites differently from PAF, IPD and DLB displaying α-syn deposits in autonomic fibers mainly at proximal sites. PAF and DLB showed the highest p-syn load with a widespread involvement of autonomic skin nerve fibers.
1) p-syn in skin nerves was the optimal marker for the in vivo diagnosis of synucleinopathies; 2) the localization and load differences of aggregates may help to identify specific diagnostic traits and support a different pathogenesis among synucleinopathies.
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.
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.
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