Thibaud Lebouvier

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.

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.

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.

Withdrawing amantadine in dyskinetic patients with Parkinson disease The AMANDYSK trial

Objective: The AMANDYSK trial was designed to assess long-term efficacy of chronic treatment with amantadine in patients with Parkinson disease (PD) and levodopa-induced dyskinesia (LID). Methods: This was a 3-month, multicenter, randomized, double-blind, placebo-controlled, parallel-group, wash-out study conducted in 57 amantadine-treated (≥200 mg/d for ≥6 months) dyskinetic patients with PD. The primary outcome measure was the change from baseline in a Unified Parkinson’s Disease Rating Scale (UPDRS) dyskinesia subscore (items 32 [duration] + 33 [severity]). Secondary outcomes included other LID measurements (“responders” analysis, premature dropout for LID, Abnormal Involuntary Movement Scale). Exploratory outcomes included time with troublesome dyskinesia as measured by diaries, UPDRS Motor Examination (part III) for motor symptoms of PD, and fatigue and apathy scores for nonmotor symptoms. Results: UPDRS items 32 + 33 deteriorated more in patients switched to placebo (“discontinuing” group) (+1.7 ± 2.0 units; 95% confidence interval 0.9, 2.4) as compared with those maintained on amantadine (“continuing” group) (+0.2 ± 1.5 units; 95% confidence interval −0.4, 0.8; p = 0.003). Secondary outcomes confirmed this difference because there were significantly more responders, more dropouts for LID, greater increase in “ON” time with troublesome dyskinesia, and greater worsening of Abnormal Involuntary Movement Scale score in the discontinuing group. There were no between-group differences in the UPDRS Motor Examination, whereas apathy (as measured by caregivers) and fatigue scores tended to worsen more in patients randomized to placebo. Conclusion: Wash-out of amantadine in dyskinetic patients with PD significantly worsened LID. No significant effect was observed on motor parkinsonian symptoms, while exploratory outcomes suggested that amantadine might improve apathy and fatigue in such patients. Classification of evidence: This article provides Class II evidence that in patients with PD, withdrawing amantadine significantly aggravates LID in a median time of 7 days.

Routine colonic biopsies as a new tool to study the enteric nervous system in living patients

Abstract  Better characterization of enteric neuropathies during the course of gastrointestinal diseases could be of great diagnostic and/or therapeutic interest. However, studies using whole mounts of the enteric nervous system (ENS) are restricted to specific diseases requiring surgery and are also limited by the small number of specimens available. Therefore, we here describe a novel method to obtain whole mounts of submucosal plexus in routine colonic biopsies. We show that a single biopsy displays a substantial number of submucosal ganglia and neurons and that it can be reliably used to perform morphometric and neurochemical analysis and Western Blots quantification of neuronal or glial markers. This method of analysis of the human ENS will enable us to gain better insight into the characterization of enteric neuropathies in living patients.

Neurochemical plasticity in the enteric nervous system of a primate animal model of experimental Parkinsonism

Abstract  Emerging evidences suggest that the enteric nervous system (ENS) is affected by the degenerative process in Parkinson’s disease (PD). In addition lesions in the ENS could be associated with gastrointestinal (GI) dysfunctions, in particular constipation, observed in PD. However, the precise alterations of the ENS and especially the changes in the neurochemical phenotype remain largely unknown both in PD and experimental Parkinsonism. The aim of our study was thus to characterize the neurochemical coding of the ENS in the colon of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated monkeys, a well‐characterized model of PD. In the myenteric plexus, there was a significant increase in the number of neurons per ganglia (identified with Hu), especially nitric oxide synthase immunoreactives (IR) neurons in MPTP‐treated monkeys compared to controls. A concomitant 72% decrease in the number of tyrosine hydroxylase‐IR neurons was observed in MPTP‐treated monkeys compared to controls. In contrast no change in the cholinergic or vasoactive intestinal peptide‐IR population was observed. In addition, the density of enteric glial cells was not modified in MPTP‐treated monkeys. Our results demonstrate that MPTP induces major changes in the myenteric plexus and to a lesser extent in the submucosal plexus of monkeys. They further reinforce the observation that lesions of the ENS occur in the course of PD that might be related to the GI dysfunction observed in this pathology.

The Microtubule-Associated Protein Tau is Also Phosphorylated on Tyrosine

Tau protein is the principal component of the neurofibrillary tangles found in Alzheimers disease (AD), where it is hyperphosphorylated on serine and threonine residues. It is hypothesized that this hyperphosphorylation contributes to neurodegeneration through the destabilization of microtubules. There is now evidence that phosphorylation of tau can also occur on tyrosine residues. Human tau has five tyrosines numbered 18, 29, 197, 310, and 394, according to the sequence of the longest CNS isoform. Tyrosines 18, 197, and 394 have been shown to be phosphorylated in the brain of patients with AD whereas tyrosine 394 is the only residue that has been described to date that is phosphorylated in physiological conditions. Src family kinases and spleen tyrosine kinase (Syk) have been shown to phosphorylate tyrosine 18 while c-Abl is capable of phosphorylating tyrosine 394. Recently, a dual specificity kinase termed TTBK1 has been characterized in human brain and shown to be able to phosphorylate residue 197 of tau. Data about the role of tau tyrosine phosphorylation in neuronal physiology are still scarce and preliminary. In contrast, there is mounting evidence suggesting that tau tyrosine phosphorylation is an early event in the pathophysiology of AD and that Fyn and c-Abl are critical in the neurodegenerative process which occurs in tauopathies.

A comparison between colonic submucosa and mucosa to detect Lewy pathology in Parkinson’s disease

Background  Lewy bodies and neurites (LN), the two pathological hallmarks of Parkinson’s disease (PD), are found in the enteric nervous system (ENS). Previously, we have shown that whole mounts of submucosa obtained after microdissection of colonic biopsies can be used for the detection of LN in the submucosal plexus (SMP) of PD patients. Recent reports suggest that Lewy pathology may extend beyond the submucosa to involve the digestive mucosa. The aim of the present research was to determine whether the analysis of the mucosa obtained after microdissection may help improve the sensitivity of colonic biopsies to detect Lewy pathology in the colon of PD patients.

Biopsable neural tissues: toward new biomarkers for Parkinson's disease?

Biomarkers for Parkinsons 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.