Investigation of ghrelin’s neuroprotection potential in a cellular model of parkinsonism

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Rapid eye movement sleep behavior disorders

Rapid eye movement sleep behavior disorders are characterized by an excessive motor activity of the eyes during the dreams. It is retrospectively described in many PD patients. Indeed, among 29 men diagnosed with RBD, 38% developed PD in the 13 years following the onset of RBD (Schenck et al., 1996). Moreover, a study performed in PD patients and healthy controls with an interval of two years also showed a greater increase in the occurrence of RBD in PD patients compared to controls. Within two years, the amount of PD patients suffering from RBD increased from 25% to 43% whereas in the control group it increased non significantly from 2% to 4% (Sixel-Döring et al., 2016). Although they are not specific of PD and are usually misdiagnosed (Stores, 2007), sleep disorders are currently considered as early non motor symptoms of PD which greatly impact the quality of life of PD patients. A better understanding of these symptoms and their neurobiological causes should improve the patient’s quality of life and alleviate the burden of PD in the daily life.

Gastro-intestinal disorders

Gastro-intestinal disorders, including excess of saliva, constipation and delayed gastric emptying are common in all stages of PD (Pfeiffer, 2011). Indeed, 70-78% of PD patients display an excess of saliva whereas only 6% of control subjects report it (Eadie and Tyrer, 1965; Edwards et al., 1991). As the amount of saliva produced is lower in PD patient, this excess of saliva in the mouth is thought to be the result of swallowing difficulties and could cause lack of saliva control (Bagheri et al., 1999; Edwards et al., 1991). Concerning constipation, a prospective study undertaken in 6790 control men during 24 years showed that subjects with one bowel movement or less per week had a three times higher risk of developing PD in the ten years following the beginning of constipation (Abbott et al., 2001). Delayed gastric emptying, also called gastroparesis, is present in all stages of PD. It affects the quality of life of the patients, their nutrition states and the absorption rate of their medications (Heetun and Quigley, 2012). Indeed, a study showed that an intra-duodenal L-DOPA administration was able to improve motor symptoms in a PD patient with motor fluctuations while oral-administered L-DOPA could not (Kurlan et al., 1988). This could be explained by the fact that, in patients with gastroparesis, L-DOPA stays longer in the stomach and is therefore more susceptible to degradation into dopamine by dopa-decarboxylase present in the stomach. Since dopamine cannot cross the blood-brain-barrier, the amount of available L-DOPA reaching the brain is lower than it would be in a patient with normal transit time.
These non-motor symptoms, their frequency and their severity can be evaluated with a rating scale named Non-Motor-Symptoms questionarry (NMS-Quest, see Annex 2; Chaudhuri and Martinez-Martin, 2008; Romenets et al., 2012). Although these non-motor symptoms are not specific of PD, they are known risk factors for the disease and could be useful in understanding the early stages of PD (Lin et al., 2015, 2014; Ponsen et al., 2004; Postuma et al., 2015; Walter et al., 2015). Moreover, the presence of several of these non-motor symptoms, now recognized in the general population as early signs of PD, might lead the patient to consult a neurologist without delay, therefore allowing the early treatment of the first motor deficits and a longer time for the patient to adjust to his/her new condition.

Braak’s staging hypothesis

Braak and colleagues studied the presence and the localization of Lewy bodies and Lewy neurites in the brain of 41 PD patients, 69 subjects with Lewy body pathology but no clinical sign of PD and 58 age- and gender-matched controls (Braak et al., 2003). Based on the observations undertaken after the staining of the brains for alpha-synuclein inclusions, β-amyloid plaques and neurofibrillary tangles, they documented the pattern of progression of the disease, known as Braak´s staging. Based on the severity of the lesions, referred to as Lewy pathology, and their localization in the brain, these neuropathologists identified six stages in PD progression (Fig. 8). For all stages described, Lewy neurites seem to appear before Lewy bodies. At stage 1, Lewy neurites are observed in the dorsal motor nucleus of the vagus nerve and the intermediate reticular zone. At stage 2, Lewy neurites are more numerous in these initial areas and are also found in some neurons of the caudal raphe nuclei and the reticular formation. Stage 3 is characterized by the aggravation of the lesions described for stages 1 and 2, i.e. more Lewy neurites in the concerned areas of the brain and the appearance of Lewy bodies, as well as the propagation of the lesions to melanin containing neurons of the SN. Several projection neurons in the basal forebrain are also affected and Lewy neurites are observed in the compact portion of the pedunculopontine tegmentum nucleus. At the same stage, Lewy bodies are observed in the tuberomammillary nucleus.

Basal ganglia dysfunction in PD4

PD is the most emblematic disorder affecting the nigro-striatal pathway. Its characteristic dopaminergic neuronal death within the SN leads to modifications of the basal ganglia motor circuits. Indeed, as presented previously, in physiological condition dopamine is essential to balance the activation between the direct and the indirect pathway. This neurotransmitter is able to prevent inadequate movement and to facilitate the adapted sequence of movement. In PD patients, the amount of dopamine produced by the neurons of the SN and released in the striatum is decreased. This dopamine depletion induces an imbalance in the basal ganglia motor circuits. On one hand, dopamine depletion in the striatum induces a lower activation of the D1-type receptors present in the striatum, therefore leading to a lower inhibitory signal to the SNr/ globus pallidus internalis which in turn will send stronger inhibitory signal to the thalamus, thus leading to a weaker signal from the thalamus to the cortex. This sequence of modifications finally induces a decrease in the motor function of the patient in the contralateral side of the body. On the other hand, the dopamine depletion in the striatum leads to a higher activation of the indirect pathway, starting with a stronger inhibitory signal from the striatum to the external part of the globus pallidus which in turn inhibits the subthalamic nuclei to a lesser extend, therefore inducing a more important signal from the subthalamic nuclei to the SNr/ globus pallidus internalis. As it is the case for the modulation of the direct pathway, the dopamine depletion in the striatum leads also to a weaker activation of the thalamus and a decrease in motor function (Fig. 10; Albin et al., 1989; DeLong, 1990). It is estimated that PD characteristic motor symptoms appear only after more than 50% of the dopaminergic of the SN are dead, suggesting that during the pre-motor stages of the disease, the feedback networks present in the basal ganglia circuits allow a compensation of the slight decrease in the dopamine content.

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Table of contents :

GENERAL OVERVIEW
1. History of the discovery of Parkinson’s disease
2. Epidemiological data
CHAPTER 1: PARKINSON’S DISEASE
1. Symptomatology and clinical diagnosis
1.1. Motor symptoms
1.1.1. Bradykinesia
1.1.2. Resting tremor
1.1.3. Rigidity or stiffness of the muscles
1.1.4. Postural instability
1.2. Non-motor symptoms
1.2.1. Olfactory dysfunctions
1.2.2. Mood disorders
1.2.3. Dementia
1.2.4. Rapid eye movement sleep behavior disorders
1.2.5. Gastro-intestinal disorders
1.3. Evolution of the disease and clinical diagnosis
1.3.1. Evolution of the disease
1.3.2. Clinical diagnosis
2. Neuropathological characterization
2.1. Neuropathological hallmarks of Parkinson’s disease
2.1.1. Dopaminergic cell death
2.1.2. Lewy bodies and Lewy neurites
2.1.2.1. In the central nervous system
2.1.2.2. In the enteric nervous system
2.1.3. Braak’s staging hypothesis
2.1.4. Other neuropathological hallmarks
2.2. Functional consequences
2.2.1. Anatomy of basal ganglia
2.2.2. Basal ganglia dysfunction in PD
2.2.3. Potential for neuroimaging investigations
3. Aetiology
3.1. Environmental factors
3.1.1. Environmental risk factors for Parkinson’s disease
3.1.1.1. Von Economo disease
3.1.1.2. MPTP
3.1.1.3. Rotenone and paraquat
3.1.1.4. Brain injury
3.1.2. Environmental factors inversely correlated to Parkinson’s disease
3.2. Genetic factors
3.2.1. Genetic risk factors
4. Currently available therapeutic options
4.2. Pharmacological therapies
4.2.1. The L-DOPA replacement therapy
4.2.2. The use of dopamine agonists
4.2.3. The use of inhibitors of dopamine catabolism
4.3. Surgical therapies
4.3.1. Dopaminergic cell grafts
4.3.2. Deep-brain stimulation
4.4. Alternative therapies
CHAPTER 2: RELEVANCE AND CONTRIBUTION OF EXPERIMENTAL MODELS OF PARKINSON’S DISEASE IN UNDERSTANDING THE UNDERLYING PATHOPHYSIOLOGICAL MECHANISMS.
1. What is an animal model?
1.1. The definition of a model in experimental sciences
1.2. Internal validity criteria
1.3. External validity criteria
1.3.1. Construct validity
1.3.2. Face validity
1.3.3. Predictive validity
1.4. What criteria for modelling Parkinson’s disease?
2. Currently available models of Parkinson’s disease
2.1. In vitro models
2.1.1. Established cell lines
2.1.2. Primary neuronal cells
2.2. In vivo models
2.2.1. Genetic models of Parkinson’s disease
2.2.2. Environmentally-induced experimental analogs of Parkinson’s disease
2.2.1.1. Reserpine
2.2.1.2. 6-hydroxydopamine
2.2.1.3. MPTP
2.2.1.4. Paraquat
2.2.1.5. Rotenone
3. Contribution of PD experimental models to the understanding of underlying molecular mechanisms
3.1. Mitochondrial dysfunction and oxidative stress
3.2. Degradation of misfolded protein: autophagy and ubiquitin proteasome system
3.3. Alpha-synuclein: conformation and aggregation
3.4. Proximal cell-to-cell alpha-synuclein spreading and the “prion-like” hypothesis
CHAPTER 3: THE POTENTIAL OF THE OREXIGENIC PEPTIDE GHRELIN IN PARKINSON’S DISEASE.
1. Ghrelin: a pleiotrophic hormone?
1.1. Origin and biosynthesis
1.2. Ghrelin receptors
1.3. Functions of ghrelin
2. Ghrelin / dopamine interactions in the nigrostriatal pathway and therapeutical potential in PD 
2.1. Diagnostic potential of ghrelin in PD
2.2. Therapeutic potential of ghrelin against PD non-motor symptoms
2.3. Therapeutic potential of ghrelin as a disease-modifying agent in PD
OBJECTIVES OF THE Ph.D. WORK
CHAPTER 4: MATERIAL AND METHODS
1. In vivo experiments
1.1. Animal model of early parkinsonism
1.1.1. Rotenone exposure
1.1.2. Non-invasive intestinal motility test
1.1.3. Spontaneous activity cylinder test
1.1.4. Challenging beam traversal test
1.1.5. Euthanasia and collection of samples
1.2. Transcriptome analysis
1.2.1. RNA extractions
1.2.2. Reverse transcription
1.2.3. Primer design
1.2.4. Quantitative PCR
1.3. Immunohistochemistry
1.4. Ghrelin dosage in the plasma
1.4.1. Blood sampling
1.4.2. Dosage of acyl- and desacyl-ghrelin in plasma samples
1.5. Western blot analyses
1.5.1. Protein extractions for western blot
1.5.2. SDS-PAGE and western blot
2. In vitro experiments
2.1. Primary cultures of mouse mesencephalic neurons
2.1.1. Coating conditions
2.1.2. Isolation of mouse primary mesencephalic cells
2.1.3. In vitro model of dopaminergic degeneration
2.2. Immunocytology
2.3. Automated PI-positive nuclei quantification
2.4. Sampling of the culture medium
3. Statistical analyses
CHAPTER 5: RESULTS
I. Ghrelin: a potential disease-modifying agent in PD? Study in primary mesencephalic cultures
1. Effect of acyl-ghrelin exposure on primary mesencephalic cells
1.1. Long term ghrelin exposure and survival of TH+ cells
1.2. Acyl-ghrelin half-life in the culture medium
2. Investigation of ghrelin’s neuroprotection potential in a cellular model of parkinsonism
2.1. Assessing construct and face validities of the cellular model
2.2. Effect of short and long-term acyl-ghrelin on primary mesencephalic cells exposed to rotenone
2.3. Effect of short-term desacyl-ghrelin in primary mesencephalic cultures exposed to rotenone
II. Investigation of ghrelin’s potential as a biomarker of PD early stages
1. Variation of GHRLOS in samples from PD patients
2. In vivo study in an experimental analog of early parkinsonism
2.1. Physiologic variations of plasma ghrelin in mice
2.1.1. Plasma ghrelin variations in healthy 4-5 months old female mice before and after a meal
2.1.2. Plasma ghrelin variations in healthy 1 year old male mice before and after a meal
2.2. Validation of the mouse model of early parkinsonism after chronic exposure to low doses of the pesticide rotenone
2.2.1. Animals well-being throughout the experimental procedure
2.2.2. Evaluation of non-motor symptoms after 1.5 months of exposure to low doses of the pesticide rotenone
2.2.3. Investigation of the motor behavior after 1.5 months of exposure to low doses of the pesticide rotenone
2.2.3.1. Analysis of the global motor behavior
2.2.3.2. Analysis of the fine motor coordination between mouse fore- and hindlimbs
2.2.4. Post-mortem analyses
2.2.4.1. Histopathological analysis of the substantia nigra
2.2.4.2. Analysis of the intestinal neuronal populations
2.3. Ghrelin variations in a mouse model of early parkinsonism
2.3.1. Plasma ghrelin variations after 1.5 months of rotenone exposure
2.3.2. Ghrelin-related gene expression in the duodenum of rotenone-exposed mice
CHAPTER 6: DISCUSSION
1. Ghrelin as a disease-modifying agent in PD: is the good player acyl- or desacyl-ghrelin?
2. Can ghrelin be used as a biomarker of PD early stages?
3. Implication for patient-based investigations: is the translation « from the bench to the bedside » always possible?
4. Experimental perspectives
CHAPTER 7: CONCLUSION
CHAPTER 8: REFERENCES
CHAPTER 9 : ANNEXES
Annex 1: Document de synthèse en français
Annex 2: NMS-Quest
Annex 3: Hoehn and Yahr scale

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