Optimizing Western Blots for the Detection of Endogenous α-Synuclein in the Enteric Nervous System

Background:Alpha-synuclein containing inclusions in neurons, the characteristic pathological lesions of Parkinson’s disease (PD), are not limited to the central nervous system, but also affect the enteric nervous system (ENS). This suggests that the ENS offer some potential as a surrogate of central nervous system pathology and that it may represent an original source of biomarkers for PD. However, the usefulness of α-synuclein detection in gastrointestinal biopsies as a biomarker for PD is still unclear, as the different immunohistochemical methods employed to date have led to conflicting results. Objective:Our aim is to propose an optimized immunoblotting method for the detection of endogenous α-synuclein in the healthy ENS that may be used to supplement the immunohistochemical analysis. Methods:Primary culture of rat ENS and homogenates of human small intestine were analyzed by Western Blot using seven different α-synuclein and phospho-α-synuclein antibodies along with two methods that increase α-synuclein retention on blot membranes, namely incubation of the membranes with paraformaldehyde (PFA) or treatment of samples with the crosslinker dithiobis[succinimidylpropionate] (DSP). Results:A moderate improvement in the detection of endogenous enteric α-synuclein was observed following membrane fixation with PFA for only two of the seven antibodies we tested. Immunodetection of total and phosphorylated α-synuclein in the ENS was markedly improved when samples were treated with DSP, regardless of the antibody used. Conclusions:Our results demonstrate that the detection of α-synuclein in the gut by Western Blot can be optimized by using methods for enhanced membrane retention of the protein along with the appropriate antibody. Such an optimized protocol opens the way to the development of novel biomarkers for PD that will enable a quantification of α-synuclein in gastrointestinal biopsies

A phosphatase cascade by which rewarding stimuli control nucleosomal response

ArticleInternational audienceDopamine orchestrates motor behaviour and reward-driven learning. Perturbations of dopamine signalling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here we show that drugs of abuse, as well as food reinforcement learning, promote the nuclear accumulation of 32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein (DARPP-32). This accumulation is mediated through a signalling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser 97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases the phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser 97 profoundly alters behavioural effects of drugs of abuse and decreases motivation for food, underlining the functional importance of this signalling cascad

Immunohistochemical Method and Histopathology Judging for the Systemic Synuclein Sampling Study (S4)

Immunohistochemical (IHC) α-synuclein (Asyn) pathology in peripheral biopsies may be a biomarker of Parkinson disease (PD). The multi-center Systemic Synuclein Sampling Study (S4) is evaluating IHC Asyn pathology within skin, colon and submandibular gland biopsies from 60 PD and 20 control subjects. Asyn pathology is being evaluated by a blinded panel of specially trained neuropathologists. Preliminary work assessed 2 candidate immunoperoxidase methods using a set of PD and control autopsy-derived sections from formalin-fixed, paraffin-embedded blocks of the 3 tissues. Both methods had 100% specificity; one, utilizing the 5C12 monoclonal antibody, was more sensitive in skin (67% vs 33%), and was chosen for further use in S4. Four trainee neuropathologists were trained to perform S4 histopathology readings; in subsequent testing, their scoring was compared to that of the trainer neuropathologist on both glass slides and digital images. Specificity and sensitivity were both close to 100% with all readers in all tissue types on both glass slides and digital images except for skin, where sensitivity averaged 75% with digital images and 83.5% with glass slides. Semiquantitative (0-3) density score agreement between trainees and trainer averaged 67% for glass slides and 62% for digital images

Safety and efficacy of continuous subcutaneous levodopa–carbidopa infusion (ND0612) for Parkinson's disease with motor fluctuations (BouNDless): a phase 3, randomised, double-blind, double-dummy, multicentre trial

© 2024 Elsevier Ltd. All rights reserved.Background: Conventional oral levodopa therapy for the treatment of Parkinson's disease can be associated with variations in plasma concentrations. Levodopa infusion strategies might provide more consistent drug delivery and fewer motor fluctuations. We aimed to assess the safety and efficacy of a continuous 24 h/day subcutaneous infusion of ND0612 (a levodopa-carbidopa solution) compared with oral immediate-release levodopa-carbidopa for the treatment of motor fluctuations in people with Parkinson's disease. Methods: We conducted a phase 3, randomised, double-blind, double-dummy, active-controlled, multicentre trial at 117 academic and community neurology sites in 16 countries, including in Europe, Israel, and the USA. Eligible participants were men and women aged 30 years or older with a diagnosis of Parkinson's disease (Hoehn and Yahr stage ≤3 in the on state) who experienced at least 2·5 h/day of off time. Participants underwent an open-label run-in phase (<12 weeks), during which time optimal regimens were established for both oral immediate-release levodopa-carbidopa and for 24 h/day subcutaneous ND0612 infusion (levodopa-carbidopa 60·0/7·5 mg/mL), with supplemental oral levodopa-carbidopa if needed. Participants were then randomly assigned (1:1) to 12 weeks of double-blind treatment with their optimised regimen of either subcutaneous ND0612 or oral levodopa-carbidopa, with matching oral or subcutaneous placebo given as required to maintain blinding. Randomisation was done via an interactive web response system, stratified by region, using a permuted block schedule. Participants, study partners, treating investigators, study site personnel, and the sponsor were masked to treatment group allocation. The primary efficacy endpoint was the change from baseline (ie, time of randomisation, when all patients were receiving an optimised open-label ND0612 regimen) to end of the double-blind phase in total daily on time without troublesome dyskinesia, analysed by intention to treat. This trial is registered with ClinicalTrials.gov, NCT04006210, and is complete. Findings: Between Sept 30, 2019, and April 8, 2022, 381 participants were enrolled, of whom 259 (68%) were randomly assigned, 128 (49%) to subcutaneous ND0612 and 131 (51%) to oral levodopa-carbidopa. 243 (94%) participants completed the study. Treatment with subcutaneous ND0612 provided an additional 1·72 h (95% CI 1·08 to 2·36) of on time without troublesome dyskinesia compared with oral levodopa-carbidopa (change from baseline of -0·48 h [-0·94 to -0·02] with subcutaneous ND0612 vs -2·20 h [-2·65 to -1·74] with oral levodopa-carbidopa; p<0·0001). Significant treatment differences favouring subcutaneous ND0612 were also found in the first four of nine prespecified hierarchical outcomes of daily off time (-1·40 h [95% CI -1·99 to -0·80]), Movement Disorders Society-Unified Parkinson's Disease Rating Scale part II scores (-3·05 [-4·28 to -1·81]), Patients Global Impression of Change (odds ratio [OR] 5·31 [2·67 to 10·58]), and Clinical Global Impression of Improvement (OR 7·23 [3·57 to 14·64]). Hierarchical testing ended after the fourth secondary endpoint. Adverse events were reported by 287 (89%) of 322 participants during open-label ND0612 optimisation, and by 103 (80%) of 128 in the ND0612 group and 97 (74%) of 131 in the oral levodopa-carbidopa group during the double-blind phase. The most common adverse events were infusion-site reactions (266 [83%] participants during open-label ND0612, and 73 [57%] in the ND0612 group vs 56 [43%] in the oral levodopa-carbidopa group during the double-blind phase), most of which were mild. Serious adverse events in four participants in the ND0612 group were related to study treatment (infusion-site cellulitis [n=2], infusion-site abscess and infusion-site ulcer [n=1]; and paraesthesia and peripheral sensorimotor neuropathy [n=1]). One participant in the ND0612 group died during the double-blind phase, but the death was not related to study treatment (fall leading to traumatic brain injury). Interpretation: Results of this phase 3 study showed that subcutaneous ND0612 used in combination with oral immediate-release levodopa-carbidopa increased on time without troublesome dyskinesia and reduced off time, with a favourable benefit-risk profile. ND0612 might offer a safe and efficacious subcutaneous levodopa infusion approach to managing motor fluctuations in people with Parkinson's disease. The ongoing open-label extension phase will provide further information on the long-term efficacy and safety of treatment.info:eu-repo/semantics/publishedVersio