Neuroprotection in neurodegenerative processes associated with parkinsonism and aging. Correlation between dopaminergic neuronal death and glial activation.

Résumé

Different shreds of evidence have been point out that the neuroinflammatory processes have a key role in the initial state and progression of Parkinson's disease. These mechanisms are mainly regulated by glial cells from which microglial cells and astrocytes stand out. These cells are the main components involve when neuroinflammation processes are triggered after a dopaminergic insult. In this line, therapeutic strategies based on anti-inflammatory drugs are studied in detail in order to design new ones more effective that reduce or avoid the side effects produced by the current treatments commonly used in Parkinson's disease. With this in mind, the work presented in this thesis examines and explores the involvement of neuroinflammatory processes in the dopaminergic neurodegeneration produced by the induction of Parkinsonism by the administration of MPTP in both young and old mice. First, Chapter 1 familiarizes the reader with the pathology and pathogenesis of Parkinson's disease and draws the attention to the main problem to be addressed in this work and how it has been approached. After this brief introduction, Chapter 2 presents a thorough overview of the role of neuroinflammation in Parkinson's disease. This report unfolds the timeline of the main contributions, from experimental, genetic and epidemiological studies, that involve inflammatory processes as crucial in the development of the disease. Finally, it is exposed the main challenges that the research focus on therapeutic strategies to treat PD patients has to overcome. As an alternative, it is suggested the different advantages that the "drug repositioning" offers to avoid possible side effects, since the compounds have already passed the safety studies in Phase I, and to accelerate the identification of new pharmacological targets. The first experimental part of this thesis, Chapter 3, starts with an investigation about the effect of systemic inflammation in the neurodegeneration and glial activation in Parkinsonian mice. The hypothesis of this work is inspired by the literature that suggests that both brain and peripherally inflammation could play a key role in the progression of this disorder. In the study, a model of ulcerative-colitis was carefully combined with an experimental model of Parkinsonism. The post-mortem studies showed a very significant decrease of dopaminergic neurons in the SNpc as well as a significant decrease of dopaminergic fibers in the striatum of the MPTP+DSS-treated group compared with the control animals. In addition, there was a significant exacerbation of microglial and astroglial activation in the MPTP+DSS animals compared to the untreated group. Overall, the data indicated that a specific gastrointestinal injury, which induces a systemic inflammatory response, is able to exacerbate cell death mechanisms of the remaining dopaminergic neurons and then, contributes to the persistent progression of the disease. These results open new lines of research about the role of exclusive colonic inflammation and the progression of nigrostriatal dopaminergic degeneration. The design of therapeutic strategies focus on "Drug repositioning", the re-use of anti-inflammatory and anti-oxidant drugs are a great bet to slow down the progression of neurodegenerative disorders. In Chapter 4, it was evaluated the possible neuroprotective effect of the combination of two different common drugs: (i) the N-acetylcysteine (NAC), a glutathione precursor and JNK inhibitor with anti-oxidant actions, and (ii) HA-1077, a ROCKinase inhibitor and microglia polarizer. Along with this, it was studied the effect of the combination of NAC and HA-1077 on the neurodegeneration and glial response in aged 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Interestingly, the post-mortem studies showed that NAC+HA-1077-based treatment produced a significant increase in the degeneration of the dopaminergic striatal terminals. This event was accompanied by an increase of the glial activation. Overall, the unexpected toxic effects, found after the combined administration of NAC and HA-1077 in old-Parkinsonian mice, highlight the importance of taking into account that the combination of some drugs in elderly Parkinsonian patients (most of them used for other different age-related alterations) can have side effects that may result in the exacerbation of the neurodegenerative process. Chapter 5 covers a similar line of research as the previous chapter. Thus, according to the neuroprotective effects obtained by the administration of NAC, this study aimed to analyse if there was a synergistic positive effect of NAC along voluntary physical activity (PA) on dopaminergic neurodegeneration and glial response in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism model after sub-chronic intoxication. The results from post-mortem studies in the substantia nigra pars compacta (SNpc) showed a significant decrease in the number of TH+ cells in all MPTP groups. TH+ expression in the striatum was significantly decreased in all MPTP groups. Thus, the combined treatment does not overprotect dopaminergic neurons against a subchronic intoxication of MPTP. Regarding glial response, the results obtained from microglial analysis do not show a significant increase in the number of Iba-1+ cell in MPTP+NAC and MPTP+PA+NAC. These results are reinforced by those obtained from the analysis of astroglial activation, in which a decrease in the expression of GFAP+ cells are observed in MPTP+NAC and MPTP+PA+NAC compared with MPTP groups both in the nigrostriatal pathway. Our results show a potential positive effect, only due to NAC treatment, on the neuroinflammatory response after subchronic MPTP intoxication. However, we believe that physical activity, used for therapeutic purposes, has a beneficial long-term effect. In this line, these results open the door to design longer studies to demonstrate its promising effect as a neuroprotective strategy. The final experimental part of this thesis is presented in Chapter 6, which delves into the changes, over time, of the events related to the dopaminergic degeneration, the astroglial response and the expression of the MAPKinases. Mitogen-activated protein kinases (MAPKs) has been pointed out as one of the main metabolic pathways involved in the regulation of inflammation and, as a consequence, it has been related to different neurodegenerative diseases. The results showed that both processes related to neurodegeneration and astroglial response started to increase significantly in comparison to untreated animals from 24h. Moreover, no significant differences were found in the expression phospho-ERK while the levels of phospho-p38 increased from 4h in the nigrostriatal pathway and, specifically, they became significant at 48h only in the striatum. The importance of these data lies in the description of the primary events triggered in old mice after the MPTP intoxication. In conclusion, these results open the door to deeper studies to evaluate the different metabolic pathways both upstream and downstream together with their comparison between different intoxication regimens (acute and chronic). In this project, treatments are designed based on the use of anti-inflammatories and/or antioxidants to see its effect on dopaminergic neuronal death and on the activation of neuroinflammatory processes in parkinsonian mice (young and old). The importance of these studies lies in the possibility of elucidating the underlying mechanisms of neurodegenerative processes to improve the quality of life of patients and to provide knowledge for the search for solutions that slow down the development and progression of Parkinson's disease. Thus, this dissertation provides different observations that highlight the importance of the involvement of neuroinflammatory processes, mediated by glial cells, in the development and exacerbation of neurodegenerative processes in Parkinson's disease.