Potential sources of interference with the highly sensitive detection and quantification of alpha-synuclein seeds by qRT-QuIC.

Parkinson's disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha-synuclein (aSyn) in the brain. The detection of aSyn in well accessible fluids has been one of the central approaches in the development of biomarkers for PD. Recently, real-time quaking-induced conversion (RT-QuIC) has been successfully adapted for use with aSyn seeds. Here, we systematically analysed parameters potentially impacting the reliability of this assay by using quantitative real-time quaking-induced conversion (qRT-QuIC) with in vitro-formed aSyn seeds. Seeds diluted in cerebrospinal fluid (CSF) accelerated the seeding reaction and slightly increased the sensitivity without affecting specificity. Repeated freeze-thaw cycles decreased the apparent lag times of seeds diluted in ddH2 O but did not alter the seeding activity of seeds diluted in CSF. High levels of artificial contamination with blood resulted in prolonged apparent lag times, while sensitivity and specificity were unaffected. Altogether, qRT-QuIC with aSyn seems to be robust concerning sensitivity and specificity in our model system, but quantitative interpretation might be limited under certain conditions.

Different Effects of α-Synuclein Mutants on Lipid Binding and Aggregation Detected by Single Molecule Fluorescence Spectroscopy and ThT Fluorescence-Based Measurements.

Six α-synuclein (aSyn) point mutations are currently known to be associated with familial parkinsonism: A30P, E46K, H50Q, G51D, A53E, and A53T. We performed a comprehensive in vitro analysis to study the impact of all aSyn mutations on lipid binding and aggregation behavior. Markedly reduced lipid binding of A30P, moderately attenuated binding of G51D, and only very slightly reduced binding for the other mutants were observed. A30P was particularly prone to form metal ion induced oligomers, whereas A53T exhibited only weak tendencies to form oligomers. In turn, fibril formation occurred rapidly in H50Q, G51D, and A53T, but only slowly in A30P, suggesting mutants prone to form oligomers tend to form fibrils to a lesser extent. This was supported by the observation that fibril formation of wild type aSyn, A30P, and A53T was impaired in the presence of ferric iron. Additionally, we found the aggregation kinetics of mixtures of A30P or A53T and wt aSyn to be determined by the faster aggregating aSyn variant. Our results implicate differential mechanisms playing a role in aSyn pathology on the molecular level. This might contribute to a better understanding of Parkinson's disease pathogenesis and provide potential links to develop prevention strategies and disease-modifying therapy.

Increased prevalence of bladder and intestinal dysfunction in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is predominantly characterized by a progressive loss of motor function. While autonomic dysfunction has been described in ALS, little is known about the prevalence of lower urinary tract symptoms (LUTS) and intestinal dysfunction. We investigated disease severity, LUTS and intestinal dysfunction in 43 patients with ALS attending our outpatient department applying the ALS functional rating scale, the International Consultation on Incontinence Modular Questionnaire, the Urinary Distress Inventory and the Cleveland Clinic Incontinence Score. Results were compared to the German population of a cross-sectional study assessing LUTS in the healthy population, the EPIC study. Results showed that urinary incontinence was increased in patients with ALS aged ≥ 60 years compared to the EPIC cohort (female: 50%/19% (ALS/EPIC), p = 0.026; male: 36%/11% (ALS/EPIC), p = 0.002). No difference was seen at 40-59 years of age. Urge incontinence was the predominant presentation (73% of symptoms). A high symptom burden was stated (ICIQ-SF quality of life subscore 5.5/10). Intake of muscle relaxants and anticholinergics was associated with both urinary incontinence and severity of symptoms. Furthermore, a high prevalence of constipation (46%), but not stool incontinence (9%), was noted. In conclusion, the increased prevalence of urge incontinence and high symptom burden imply that in patients with ALS, LUTS should be increasingly investigated for.