Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of α-synuclein protein implicated in Parkinson's disease

Abstractα-Synuclein (α-syn) protein has been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the α-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We report here that human wild-type α-syn, PD-linked mutant α-syn(Ala30Pro) and mutant α-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. The mutant α-syn forms more β-sheet and mature filaments than the wild-type protein. These findings suggest that accumulation of α-syn as insoluble deposits of amyloid may play a major role in the pathogenesis of these neurodegenerative diseases

Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulates human alpha-synuclein in transgenic mouse brain.

Alpha-synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently, it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. The expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism with which its effect is mediated by remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous mouse and transgenic human) expressed in each brain, the expanded risk allele contributed 2.6-fold more to the SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk

Nigrostriatal overabundance of α-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity

α-Synuclein (α-syn) is a presynaptic protein present at most nerve terminals, but its function remains largely unknown. The familial forms of Parkinson's disease associated with multiplications of the α-syn gene locus indicate that overabundance of this protein might have a detrimental effect on dopaminergic transmission. To investigate this hypothesis, we use adeno-associated viral (AAV) vectors to overexpress human α-syn in the rat substantia nigra. Moderate overexpression of either wild-type (WT) or A30P α-syn differs in the motor phenotypes induced, with only the WT form generating hemiparkinsonian impairments. Wild-type α-syn causes a reduction of dopamine release in the striatum that exceeds the loss of dopaminergic neurons, axonal fibers, and the reduction in total dopamine. At the ultrastructural level, the reduced dopamine release corresponds to a decreased density of dopaminergic vesicles and synaptic contacts in striatal terminals. Interestingly, the membrane-binding-deficient A30P mutant does neither notably reduce dopamine release nor it cause ultrastructural changes in dopaminergic axons, showing that α-syn's membrane-binding properties are critically involved in the presynaptic defects. To further determine if the affinity of the protein for membranes determines the extent of motor defects, we compare three forms of α-syn in conditions leading to pronounced degeneration. While membrane-binding α-syns (wild-type and A53T) induce severe motor impairments, an N-terminal deleted form with attenuated affinity for membranes is inefficient in inducing motor defects. Overall, these results demonstrate that α-syn overabundance is detrimental to dopamine neurotransmission at early stages of the degeneration of nigrostriatal dopaminergic axon

Rotenone and elevated extracellular potassium concentration induce cell-specific fibrillation of α-synuclein in axons of cholinergic enteric neurons in the guinea-pig ileum.

Under embargo until 7 Nov 2017, as per publisher policy.Background: Parkinson’s disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson’s disease are Lewy bodies and neurites, of which α-synuclein fibrils are the major component. α-Synuclein aggregation has been reported in the gut of Parkinson’s disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α-synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α-synuclein in cholinergic neurons in the gut (1), suggesting they may be particularly vulnerable to degeneration in Parkinson’s disease. Methods: In this study, we used immunohistochemistry to detect α-synuclein oligomers and fibrils via conformation-specific antibodies after rotenone treatment or prolonged exposure to high [K+] in ex vivo segments of guinea-pig ileum maintained in organotypic culture. Key Results: Rotenone and prolonged raising of [K+] caused accumulation of α-synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time- and, in the case of rotenone, concentration-dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT-immunoreactivity, also in a concentration-dependent manner. Conclusions & Inferences: To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson’s disease

Structure and neurotoxicity of novel amyloids derived from the BRI gene

Abstract A number of human neurodegenerative diseases involve aggregated amyloid proteins in the brain, e.g. Alzheimer's disease (β-amyloid) and Parkinson's disease (α-synuclein). Other examples are rare familial dementias which involve the BRI gene. In a British family, mutation of the termination codon extends the reading frame of BRI to yield a furin-processed 34-residue peptide (Abri; British dementia peptide), 11 residues longer than the wild-type (WT). In a Danish family, a ten-base insertion also yields a 34-residue peptide (Adan; Danish dementia peptide). To explore the roles of Abri and Adan in neurodegeneration, we synthesized Abri and Adan in oxidized and reduced forms and generated transgenic mice colonies expressing the WT and mutated forms of BRI. We have generated transgenic mice colonies bearing the genes coding for WT-BRI, Adan and Abri under the control of the Thy1 promoter. Whereas WT-BRI transgenic mice express full-length WT-BRI protein in their brains, Adan protein is fully processed to small peptides. FBD (familial British dementia) and FDD (familial Danish dementia) are rare autosomal dominant neurodegenerative disorders that share features of AD (Alzheimer's disease), including amyloid plaques surrounded by astrocytes and microglia, neurofibrillary tangles, neuronal loss and progressive dementi

Inhibition of alpha-synuclein seeded fibril formation and toxicity by herbal medicinal extracts.

Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process. Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking β-sheet content after subjecting them to proteinase K digestion. Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.The work conducted by Dr. El-Agnaf laboratory was supported by Qatar Biomedical Research Institute under the Start-up Fund SF 2017–007. Funding for this work was provided in part by NIH/NIA grant R37AG019391 to D.E. This study was made possible by NPRP grant 4–1371–1-223 from the Qatar National Research Fund (a member of Qatar Foundation). The funding bodies provided financial support for this study; they had no role in the study design, performance, data collection and analysis, decision to publish and preparation/writing of the manuscript

Synthesis, Conformation, and Activity of Human Insulin-Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) was first identified through searches of the expressed sequence tags (EST) databases. Primary sequence analysis showed it to be a prepropeptide that was predicted to be processed in vivo to yield a two-chain sequence (A and B) that contained the insulin-like disulfide cross-links. The high affinity interaction between INSL5 and the receptor RXFP4 (GPCR142) coupled with their apparent coevolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for RXFP4. Given that the primary function of the INSL5–RXFP4 pair remains unknown, an effective means of producing sufficient quantities of this peptide and its analogues is needed to systematically investigate its structural and biological properties. A combination of solid-phase peptide synthesis methods together with regioselective disulfide bond formation were used to obtain INSL5. Both chains were unusually resistant to standard synthesis protocols and required highly optimized conditions for their acquisition. In particular, the use of a strong tertiary amidine, DBU, as Nα-deprotection base was required for the successful assembly of the B chain; this highlights the need to consider incomplete deprotection rather than acylation as a cause of failed synthesis. Following sequential disulfide bond formation and chain combination, the resulting synthetic INSL5, which was obtained in good overall yield, was shown to possess a similar secondary structure to human relaxin-3 (H3 relaxin). The peptide was able to inhibit cAMP activity in SK-N-MC cells that expressed the human RXFP4 receptor with a similar activity to H3 relaxin. In contrast, it had no activity on the human RXFP3 receptor. Synthetic INSL5 demonstrates equivalent activity to the recombinant-derived peptide, and will be an important tool for the determination of its biological function

Cerebrospinal fluid α-synuclein species in cognitive and movements disorders

Total CSF α-synuclein (t-α-syn), phosphorylated α-syn (pS129-α-syn) and α-syn oligomers (o-α-syn) have been studied as candidate biomarkers for synucleinopathies, with suboptimal specificity and sensitivity in the differentiation from healthy controls. Studies of α-syn species in patients with other underlying pathologies are lacking. The aim of this study was to investigate possible alterations in CSF α-syn species in a cohort of patients with diverse underlying pathologies. A total of 135 patients were included, comprising Parkinson’s disease (PD; n = 13), multiple system atrophy (MSA; n = 9), progressive supranuclear palsy (PSP; n = 13), corticobasal degeneration (CBD; n = 9), Alzheimer’s disease (AD; n = 51), frontotemporal degeneration (FTD; n = 26) and vascular dementia patients (VD; n = 14). PD patients exhibited higher pS129-α-syn/α-syn ratios compared to FTD (p = 0.045), after exclusion of samples with CSF blood contamination. When comparing movement disorders (i.e., MSA vs. PD vs. PSP vs. CBD), MSA patients had lower α-syn levels compared to CBD (p = 0.024). Patients with a synucleinopathy (PD and MSA) exhibited lower t-α-syn levels (p = 0.002; cut-off value: ≤865 pg/mL; sensitivity: 95%, specificity: 69%) and higher pS129-/t-α-syn ratios (p = 0.020; cut-off value: ≥0.122; sensitivity: 71%, specificity: 77%) compared to patients with tauopathies (PSP and CBD). There are no significant α-syn species alterations in non-synucleinopathies.This study was supported by Strat-up Funding to OE from Qatar Biomedical Research Institute (SF 2007–007) and Qatar National Research Fund (NPRPNo.: 8–517–3-112)

Uncovering a neurological protein signature for severe COVID-19

Coronavirus disease of 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has sparked a global pandemic with severe complications and high morbidity rate. Neurological symptoms in COVID-19 patients, and neurological sequelae post COVID-19 recovery have been extensively reported. Yet, neurological molecular signature and signaling pathways that are affected in the central nervous system (CNS) of COVID-19 severe patients remain still unknown and need to be identified. Plasma samples from 49 severe COVID-19 patients, 50 mild COVID-19 patients, and 40 healthy controls were subjected to Olink proteomics analysis of 184 CNS-enriched proteins. By using a multi-approach bioinformatics analysis, we identified a 34-neurological protein signature for COVID-19 severity and unveiled dysregulated neurological pathways in severe cases. Here, we identified a new neurological protein signature for severe COVID-19 that was validated in different independent cohorts using blood and postmortem brain samples and shown to correlate with neurological diseases and pharmacological drugs. This protein signature could potentially aid the development of prognostic and diagnostic tools for neurological complications in post-COVID-19 convalescent patients with long term neurological sequelae