Biopsable Neural Tissues: Toward New Biomarkers for Parkinson's Disease?

Biomarkers for Parkinson's disease (PD) are mainly intended for the early diagnosis of the disease and to monitor its progression, two aspects insufficiently covered by clinical evaluation. In the last 20 years, the search for biomarkers has been supported by technological advances in the fields of molecular genetics and neuroimaging. Nevertheless, no fully validated biomarker is yet available, and there is still a need for biomarkers that will complement those already available. Development of biomarkers for PD has been hampered by the fact that the core pathology lies in the brainstem, hidden from direct study in living patients. In this context, the recent observations that clearly demonstrated the presence of PD pathology in peripheral neural tissues provide new opportunities to develop original histopathological markers of the disease. Some of these peripheral tissues, especially the enteric nervous system, by being assessable using routine biopsies, could represent a window to assess in vivo the neuropathological processes occurring in PD

Top-Down Knowledge Compilation for Counting Modulo Theories

Propositional model counting (#SAT) can be solved efficiently when the input formula is in deterministic decomposable negation normal form (d-DNNF). Translating an arbitrary formula into a representation that allows inference tasks, such as counting, to be performed efficiently, is called knowledge compilation. Top-down knowledge compilation is a state-of-the-art technique for solving #SAT problems that leverages the traces of exhaustive DPLL search to obtain d-DNNF representations. While knowledge compilation is well studied for propositional approaches, knowledge compilation for the (quantifier free) counting modulo theory setting (#SMT) has been studied to a much lesser degree. In this paper, we discuss compilation strategies for #SMT. We specifically advocate for a top-down compiler based on the traces of exhaustive DPLL(T) search.Comment: 9 pages; submitted to Workshop on Counting and Sampling 2023 at SAT202

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

Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples

No abstract availabl

The enteric nervous system and the digestive neuronal-glial-epithelial unit

In spite of its apparent simplicity, the digestive tract is probably one of the most complex organs of the human body. The complexity of the digestive functions requires an extremely fine regulation, to direct nutriments towards sites dedicated to absorption, to control their absorption, and protect our body against adverse environmental factors (bacteria, toxins…). All these functions are controlled by a second brain: the enteric nervous system (ENS). Neurons and enteric glial cells, which form the ENS, regulate gastrointestinal motility as well as intestinal barrier functions. The physical proximity of neurons and of glial and epithelial intestinal cells, and especially their inter-regulation have led to the definition of the new concept of neuronal-glial-epithelial unit. The ENS is a key regulator of digestive functions, and is also involved in the development of digestive disordersEn dépit d’une apparente simplicité, le tube digestif est probablement l’un des organes les plus complexes du corps humain. En effet, la complexité des fonctions digestives nécessite une régulation extrêmement fine, permettant à la fois de diriger les nutriments vers les sites spécialisés d’absorption du tube digestif, de contrôler leur absorption, et de protéger notre corps de l’agression par des facteurs environnementaux délétères (bactéries, toxiques…). L’ensemble de ces fonctions est assuré par un véritable deuxième cerveau : le système nerveux entérique (SNE). Les neurones et les cellules gliales entériques qui forment le SNE régulent la motilité digestive, mais aussi les fonctions de barrière de l’épithélium intestinal. La proximité physique des neurones ainsi que des cellules gliales et épithéliales intestinales, mais aussi et surtout leurs inter-régulations nous a permis de définir le nouveau concept d’unité-neuro-glio-épithéliale. Le SNE est un régulateur clef des fonctions digestives et participe également au développement de pathologies digestive

Diagnostic value of minor salivary glands biopsy for the detection of Lewy pathology

The recent demonstration of the presence of Lewy pathology in the submandibular glands of Parkinson\u27s disease (PD) patients prompted us to evaluate the diagnostic performance of minor salivary gland biopsy for PD. Minor salivary glands were examined for Lewy pathology using phosphorylated alpha-synuclein antibody in 16 patients with clinically diagnosed PD and 11 control subjects with other neurological disorders. Abnormal accumulation of alpha-synuclein was found in 3 out of 16 PD patients. Two control subjects exhibited weak phosphorylated alpha-synuclein immunoreactivity. Our results do not support the use of minor salivary glands biopsy for the detection of Lewy pathology in living subjects

Transglutaminase-dependent RhoA Activation and Depletion by Serotonin in Vascular Smooth Muscle Cells

The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension

AMPK alpha 1-induced RhoA phosphorylation mediates vasoprotective effect of estradiol

OBJECTIVE: Estradiol (E2) mediates numerous beneficial effects assigned to estrogens, but whereas mechanisms have been described at the endothelial level, direct effects on vascular smooth muscle cells (VSMC) are poorly documented. As evidence accumulates regarding the role of RhoA in vascular pathophysiology and the benefit of RhoA-Rho associated protein kinase (Rock) pathway inhibition, we analyzed if E2 could inhibit it in VSMC. METHODS AND RESULTS: We show that in VSMC, E2 inhibits the RhoA-Rock pathway in a time- and concentration-dependent manner. The inhibition of RhoA-Rock pathway results from E2-induced phosphorylation of the Ser188 of RhoA. Using pharmacological, transfection, and in vitro phosphorylation experiments, we demonstrate that AMP-activated protein kinase subunit alpha 1 (AMPKalpha1) is activated by estrogen receptor stimulation and catalyzes RhoA phosphorylation induced by E2. Ex vivo, ovariectomy leads to an increase in the amplitude of phenylephrine- or serotonine-induced contractions of aortic rings in wild-type mice but not in AMPKalpha1-knock-out mice or E2-supplemented animals. These functional effects were correlated with a reduced level of RhoA phosphorylation in the aorta of ovariectomized female, male, and AMPKalpha1 knock-out mice. CONCLUSION: Our work thus defines AMPKalpha1 as (1) a new kinase for RhoA and (2) a new mediator of the vasoprotective effects of estrogen

Evaluation of alpha-synuclein immunohistochemical methods for the detection of Lewy-type synucleinopathy in gastrointestinal biopsies

The observation showing that Lewy type synucleinopathy (LTS), the pathological hallmark of Parkinson’s disease (PD), is found in the gut of almost all PD subjects led to a substantial amount of research to develop a diagnostic procedure in living patients based on endoscopically obtained gastrointestinal biopsies. However, the existing studies have provided conflicting results regarding the sensitivity and specificity of gastrointestinal biopsies for the detection of LTS. We therefore undertook a multi-center staining and blinded judging of a common set of slides from colonic biopsies to determine the optimal protocol for the detection of LTS. Four different immunohistochemical methods, developed in four separate expert laboratories, were evaluated for their sensitivity and specificity to detect enteric LTS. Test sets of formalin-fixed, paraffin-embedded sections from biopsies of 9 PD subjects and 3 controls were stained with the 4 methods and graded by 4 different observers. Four types of staining morphology (granular staining in the lamina propria, perivascular/vascular wall staining in the submucosa, lacy-granular pattern in the submucosa and epithelial cell nuclear staining) were variably observed in the slides stained by the 4 methods. Positive alpha-synuclein staining was observed by all 5 judges in most of the slides from control cases, regardless of the staining methods that were used. Moreover, none of the tested method or staining pattern had a specificity and sensitivity more than 80 % regarding to PD. Overall, our study suggest that the tested methods are not adequate for the prediction of PD using gastrointestinal biopsies. Future studies are warranted to test new immunostaining methods