Pascal Derkinderen

Colonic Biopsies to Assess the Neuropathology of Parkinson's Disease and Its Relationship with Symptoms

Background The presence of Lewy bodies and Lewy neurites (LN) has been demonstrated in the enteric nervous system (ENS) of Parkinsons disease (PD) patients. The aims of the present research were to use routine colonoscopy biopsies (1) to analyze, in depth, enteric pathology throughout the colonic submucosal plexus (SMP), and (2) to correlate the pathological burden with neurological and gastrointestinal (GI) symptoms. Methodology/Principal Findings A total of 10 control and 29 PD patients divided into 3 groups according to disease duration were included. PD and GI symptoms were assessed using the Unified Parkinsons Disease Rating Scale part III and the Rome III questionnaire, respectively. Four biopsies were taken from the ascending and descending colon during the course of a total colonoscopy. Immunohistochemical analysis was performed using antibodies against phosphorylated alpha-synuclein, neurofilaments NF 220 kDa (NF) and tyrosine hydroxylase (TH). The density of LN, labeled by anti-phosphorylated alpha-synuclein antibodies, was evaluated using a quantitative rating score. Lewy pathology was apparent in the colonic biopsies from 21 patients and in none of the controls. A decreased number of NF-immunoreactive neurons per ganglion was observed in the SMP of PD patients compared to controls. The amount of LN in the ENS was inversely correlated with neuronal count and positively correlated with levodopa-unresponsive features and constipation. Conclusion/Significance Analysis of the ENS by routine colonoscopy biopsies is a useful tool for pre-mortem neuropathological diagnosis of PD, and also provides insight into the progression of motor and non-motor symptoms.

Short-Chain Fatty Acids Regulate the Enteric Neurons and Control Gastrointestinal Motility in Rats

BACKGROUND & AIMS Little is known about the environmental and nutritional regulation of the enteric nervous system (ENS), which controls gastrointestinal motility. Short-chain fatty acids (SCFAs) such as butyrate regulate colonic mucosa homeostasis and can modulate neuronal excitability. We investigated their effects on the ENS and colonic motility. METHODS Effects of butyrate on the ENS were studied in colons of rats given a resistant starch diet (RSD) or intracecal perfusion of SCFAs. Effects of butyrate were also studied in primary cultures of ENS. The neurochemical phenotype of the ENS was analyzed with antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) and by quantitative polymerase chain reaction. Signaling pathways involved were analyzed by pharmacologic and molecular biology methods. Colonic motility was assessed in vivo and ex vivo. RESULTS In vivo and in vitro, RSD and butyrate significantly increased the proportion of ChAT- but not nNOS-immunoreactive myenteric neurons. Acetate and propionate did not reproduce the effects of butyrate. Enteric neurons expressed monocarboxylate transporter 2 (MCT2). Small interfering RNAs silenced MCT2 and prevented the increase in the proportion of ChAT- immunoreactive neurons induced by butyrate. Butyrate and trichostatin A increased histone H3 acetylation in enteric neurons. Effects of butyrate were prevented by inhibitors of the Src signaling pathway. RSD increased colonic transit, and butyrate increased the cholinergic-mediated colonic circular muscle contractile response ex vivo. CONCLUSION Butyrate or histone deacetylase inhibitors might be used, along with nutritional approaches, to treat various gastrointestinal motility disorders associated with inhibition of colonic transit.

Colonic inflammation in Parkinson's disease.

Lewy pathology affects the gastrointestinal tract in Parkinsons disease (PD) and data from recent genetic studies suggest a link between PD and gut inflammation. We therefore undertook the present survey to investigate whether gastrointestinal inflammation occurs in PD patients. Nineteen PD patients and 14 age-matched healthy controls were included. For each PD patients, neurological and gastrointestinal symptoms were assessed using the Unified Parkinsons Disease Rating Scale part III and the Rome III questionnaire, respectively and cumulative lifetime dose of L-dopa was calculated. Four biopsies were taken from the ascending colon during the course of a total colonoscopy in controls and PD patients. The mRNA expression levels of pro-inflammatory cytokines (tumor necrosis factor alpha, interferon gamma, interleukin-6 and interleukin-1 beta) and glial marker (Glial fibrillary acidic protein, Sox-10 and S100-beta) were analyzed using real-time PCR in two-pooled biopsies. Immunohistochemical analysis was performed on the two remaining biopsies using antibodies against phosphorylated alpha-synuclein to detect Lewy pathology. The mRNA expression levels of pro-inflammatory cytokines as well as of two glial markers (Glial fibrillary acidic protein and Sox-10) were significantly elevated in the ascending colon of PD patients with respect to controls. The levels of tumor necrosis factor alpha, interferon gamma, interleukin-6, interleukin-1 beta and Sox-10 were negatively correlated with disease duration. By contrast, no correlations were found between the levels of pro-inflammatory cytokines or glial markers and disease severity, gastrointestinal symptoms or cumulative lifetime dose of L-dopa. There was no significant difference in the expression of pro-inflammatory cytokines or glial marker between patients with and without enteric Lewy pathology. Our findings provide evidence that enteric inflammation occurs in PD and further reinforce the role of peripheral inflammation in the initiation and/or the progression of the disease.

Cannabinoids activate p38 mitogen-activated protein kinases through CB1 receptors in hippocampus

Cannabinoid receptors (CB1‐R) are the target of a novel class of neuromodulators, the endocannabinoids. Yet, their signalling mechanisms in adult brain are poorly understood. We report that, in rat and mouse hippocampal slices, anandamide and 2‐arachidonoylglycerol, synthetic cannabinoids, and Δ9‐tetrahydrocannabinol activated p38 mitogen‐activated protein kinases (MAPK), but not c‐Jun N‐terminal kinase (JNK). In contrast, lysophosphatidic acid (LPA), a lipid messenger acting on different receptors, increased both p38‐MAPK and JNK phosphorylation. The effects of cannabinoids on p38‐MAPK were mediated through activation of CB1‐R because they were blocked in the presence of SR 141716 A and absent in CB1‐R knockout mice, two conditions that did not alter the effects of LPA. The activation of p38‐MAPK by cannabinoids was insensitive to inhibitors of Src. These results provide new insights into the cellular mechanisms by which cannabinoids exert their effects in hippocampus.

Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimers disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimers disease.

The second brain and Parkinson’s disease

Parkinson’s disease is the second most common neurodegenerative disease after Alzheimer’s disease. It has been classically considered that the pathological hallmarks of Parkinson’s disease, namely Lewy bodies and Lewy neurites, affect primarily the substantia nigra. Nevertheless, it has become increasingly evident in recent years that Parkinson’s disease is a multicentric neurodegenerative process that affects several neuronal structures outside the substantia nigra, among which is the enteric nervous system. Remarkably, recent reports have shown that the lesions in the enteric nervous system occurred at a very early stage of the disease, even before the involvement of the central nervous system. This led to the postulate that the enteric nervous system could be critical in the pathophysiology of Parkinson’s disease, as it could represent a route of entry for a putative environmental factor to initiate the pathological process (Braak’s hypothesis). Besides their putative role in the spreading of the pathological process, it has also been suggested that the pathological alterations within the enteric nervous system could be involved in the gastrointestinal dysfunction frequently encountered by parkinsonian patients. The scope of the present article is to review the available studies on the enteric nervous system in Parkinson’s disease patients and in animal models of the disease. We further discuss the strategies that will help in our understanding of the roles of the enteric nervous system, both in the pathophysiology of the disease and in the pathophysiology of the gastrointestinal symptoms.

A comparison between rectal and colonic biopsies to detect Lewy pathology in Parkinson's disease.

We have shown that routine biopsies of the ascending colon obtained at colonoscopy allow the detection of Lewy neurites (LN) in the enteric nervous system (ENS) of Parkinsons disease (PD) patients. Although colonoscopy is a relatively safe procedure, it requires colon preparation and anesthesia. The present study was therefore undertaken to evaluate whether descending colon and rectal biopsies that are obtainable by rectosigmoidoscopy allow the detection of Lewy pathology in the ENS. A total of 9 controls and 26 PD patients were included and analyzed. Two biopsies were taken from the ascending, descending colon and rectum during the course of a total colonoscopy. Immunohistochemical analysis was performed using antibodies against phosphorylated alpha-synuclein to detect LN and neurofilaments 200 kDa to label the neuronal structures. Biopsies from ascending, descending colon and rectum were morphologically comparable. LN were detected in the biopsies of ascending colon in 17 PD patients (65%), of descending colon in 11 patients (42%) and of rectum in only 6 patients (23%). No LN were seen in control biopsies. Our results show that Lewy pathology follows a rostrocaudal distribution in the colon and rectum of PD patients. Therefore, rectal biopsies have substantially lower sensitivity than ascending colon biopsies to detect Lewy pathology in the gut.

Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF

Wound healing of the gastrointestinal mucosa is essential for the maintenance of gut homeostasis and integrity. Enteric glial cells play a major role in regulating intestinal barrier function, but their role in mucosal barrier repair remains unknown. The impact of conditional ablation of enteric glia on dextran sodium sulfate (DSS)-induced mucosal damage and on healing of diclofenac-induced mucosal ulcerations was evaluated in vivo in GFAP-HSVtk transgenic mice. A mechanically induced model of intestinal wound healing was developed to study glial-induced epithelial restitution. Glial-epithelial signaling mechanisms were analyzed by using pharmacological inhibitors, neutralizing antibodies, and genetically engineered intestinal epithelial cells. Enteric glial cells were shown to be abundant in the gut mucosa, where they associate closely with intestinal epithelial cells as a distinct cell population from myofibroblasts. Conditional ablation of enteric glia worsened mucosal damage after DSS treatment and significantly delayed mucosal wound healing following diclofenac-induced small intestinal enteropathy in transgenic mice. Enteric glial cells enhanced epithelial restitution and cell spreading in vitro. These enhanced repair processes were reproduced by use of glial-conditioned media, and soluble proEGF was identified as a secreted glial mediator leading to consecutive activation of epidermal growth factor receptor and focal adhesion kinase signaling pathways in intestinal epithelial cells. Our study shows that enteric glia represent a functionally important cellular component of the intestinal epithelial barrier microenvironment and that the disruption of this cellular network attenuates the mucosal healing process.

Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione

Enteric glial cells (EGCs) are essential in the control of gastrointestinal functions. Although lesions of EGCs are associated with neuronal degeneration in animal models, their direct neuroprotective role remains unknown. Therefore, the aims of this study were to demonstrate the direct neuroprotective effects of EGCs and to identify putative glial mediators involved. First, viral targeted ablation of EGCs in primary cultures of enteric nervous system increased neuronal death both under basal conditions and in the presence of oxidative stress (dopamine, hydrogen peroxide). Second, direct or indirect coculture experiments of EGC lines with primary cultures of enteric nervous system or neuroblastoma cell lines (SH‐SY5Y) prevented neurotoxic effects induced by oxidative stress (increased membrane permeability, release of neuronal specific enolase, caspase‐3 immunoreactivity, changes in [Ca2+]i response). Finally, combining pharmacological inhibition and mRNA silencing methods, we demonstrated that neuroprotective effects of EGCs were mediated in part by reduced glutathione but not by oxidized glutathione or by S‐nitrosoglutathione. Our study identified the neuroprotective effects of EGCs via their release of reduced glutathione, extending their critical role in physiological contexts and in enteric neuropathies.—Abdo, H., Derkinderen, P., Gomes, P., Chevalier, J., Aubert, P., Masson, D., Galmiche, J.‐P., Vanden Berghe, P., Neunlist, M., Lardeux, B. Enteric glial cells protect neurons from oxidative stress in part via reduced glutathione. FASEB J. 24, 1082‐1094 (2010). www.fasebj.org

Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system.

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH‐IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH‐IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 μm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP‐IR but not nNOS‐IR enteric neurons. The KCl‐induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 μm), of noradrenaline (1 μm) and of the α2‐adrenoreceptor agonist clonidine (1 μm). Combining pharmacological and calcium imaging studies, we have further shown that L‐type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal‐regulated kinases were required for the increase in the proportion of TH‐IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.