Parkinson’s Disease is a disorder of the midbrain dopaminergic system with characteristic neurodegenerative patterns, recognized for its motor symptoms. The neurodegeneration is most prevalent in the substantia nigra pars compacta, while dopaminergic neurons in neighboring structures are comparatively spared. There are many possible explanations for this disparity, including differences in tolerance to oxidative stress, and vulnerability to α-synuclein aggregates. The substantia nigra is part of the basal ganglia, a network of nuclei in the midbrain and base of the forebrain which are responsible for coordinating voluntary movement. Dopamine has an inhibitory effect in the basal ganglia. It dampens signals to remove noise, so the basal ganglia circuitry is not hyperactive. In the absence of dopamine, the flow of information through the basal ganglia is disrupted. This results in tremor, bradykinesia, and rigidity, known as the classic triad. No cure currently exists and therapies are unable to slow disease progression, so treatments are aimed at symptom management. Degenerative processes in Parkinson’s Disease occur rapidly, early in the disease progression, with about 60% neuronal death in the substantia nigra prior to diagnosis. There is a need for biomarkers or other signs which can be used to clinically to diagnose the disease at an earlier stage. In conclusion this paper provides suggestions for future lines of research.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24786号 / 医博第4978号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 花川 隆, 教授 村井 俊哉, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Les processus compensatoires qui accompagnent les atteintes du système dopaminergique (DA-ergic) nigrostrié illustrent les capacités adaptatives du cerveau adulte. Cette neuroplasticité permet le maintien de la transmission dopaminergique pendant un certain temps de sorte que les symptômes moteurs cardinaux de la Maladie de Parkinson (MP), qui se caractérise par une dégénérescence progressive des neurones DA-ergiques de la substantia nigra (SN), ne se manifestent qu'après une perte neuronale très importante. De ce fait, le diagnostic présymptomatique est une question cruciale pour le développement de traitements neuroprotecteurs. Un autre exemple de neuroplasticité est illustré par la production de nouveaux neurones dans le cerveau adulte (neurogenèse adulte). Cette neurogenèse s’observe principalement dans deux zones: le continuum zone sous-ventriculaire (SVZ)-bulbe olfactif (OB) et le gyrus denté (DG) de l'hippocampe, et se trouve altérée chez les patients parkinsoniens. Ces dernières années, le traitement chirurgical par la stimulation à haute fréquence (SHF) du noyau sous-thalamique (NST) s'est avéré être une option thérapeutique très efficace pour ces patients. Dans ce contexte, mon travail de thèse a été axé sur l’étude de la neuroplasticité dans différents modèles de la maladie de Parkinson et de son traitement avec les objectifs principaux: 1) Développer un modèle de MP présymptomatique; 2) étudier les mécanismes compensatoires impliquant le système nigrostrié; 3) Déterminer les effets de la SHF-NST sur la neurogenèse adulte dans la SVZ-OB et le DG.Dans la première étude, nous avons développé des modèles expérimentaux de la MP à différents stades, basés sur l’administration de MPTP chez la souris. Nous avons montré que le passage du stade avancé présymptomatique au stade symptomatique précoce correspondant au seuil d’atteinte des systèmes DA-ergiques associé à l’apparition des déficits moteurs, se caractérise par : (a) une diminution de DA dans les terminaisons striatales épargnées par la lésion; (b) une augmentation de DA et d’expression de la tyrosine hydroxylase dans les cellules de la SN; (c) une augmentation du renouvellement de la DA dans le striatum et une augmentation moindre dans la SN.La deuxième étude est basée sur un modèle de lésion DA-ergique extensive par injection intranigrale de 6-hydroxydopamine chez le rat, imitant les stades tardifs de la MP. Nous avons étudié séparément les étapes de prolifération et de survie des nouvelles cellules sur des animaux non lésés et des animaux lésés avec ou sans SHF subchronique (8 jours) du NST. Nous avons pu montrer une régulation spécifique des étapes de prolifération et de survie suite à la lésion dopaminergique, et des effets stimulateurs de la SHF du NST sur la survie des cellules néoformées, suggérant un effet neuroprotecteur de ce traitement. / The compensatory processes that accompany a lesion of the nigrostriatal dopaminergic (DA-ergic) system serve to maintain its function and illustrate adult brain neuroplasticity. The typical motor symptoms of Parkinson’s diseases (PD), characterized by progressive degeneration of DA-ergic neurons of substantia nigra (SN), appear only after substantial neuronal loss. Therefore presymptomatic diagnosis is a crucial issue for future neuroprotective therapies. Another good manifestation of neuroplasticity is adult neurogenesis, known to persist in two areas: the subventricular zone (SVZ) – the olfactory bulb (OB) continuum, and the dentate gyrus (DG) of the hippocampus, and to be altered in PD. In recent years, the surgical treatment by high frequency stimulation (HFS) of the subthalamic nucleus (STN) has proven to be an efficient therapeutic option for PD patients. In this context, my PhD work was focused on neuroplasticity under the functional deficiency of the nigrostriatal DA-ergic system (parkinsonism) and its treatment with the following main objectives: 1 - Develop a model of presymptomatic parkinsonism; 2 - study compensatory mechanisms in nigrostriatal system; 3 - Characterize the effects of subchronic STN HFS on adult neurogenesis. In the first part, we have developed models of presymptomatic parkinsonism based on MPTP administration in mice, as defined by sub-threshold DA depletion and degeneration of DA-ergic axons in the striatum followed by a loss of DA-ergic cell bodies in the SN (advanced presymptomatic stage). In the early symptomatic stage, these parameters reach a threshold that is associated with the appearance of motor deficiency. We have shown that the transition from the advanced presymptomatic stage to the early symptomatic stage is characterized by: (a) a decrease of DA content in surviving DA-ergic axons in the striatum; (b) an increase of DA content and TH-expression in surviving neuronal cell bodies in the SN; (c) an increase of DA turnover in the striatum and much less increase in the SN. The last part of my work is based on extensive DA lesion in rats, using intranigral 6-hydroxydopamine injection mimicking late PD stages, to determine a possible effect of STN-HFS on adult neurogenesis. We have completed series of animals with DA lesion either sham implanted or subsequently treated for 8 days by STN-HFS to be compared with unlesioned rats, and studied selective phases of neurogenesis: proliferation and survival. This study demonstrates selective regulation of cell proliferation and survival following DA depletion and provides the first evidence that prolonged STN-HFS might have a neuroprotective action as shown by the selective increase in survival of newly formed cells following this treatment.

A doença de Parkinson (DP) é uma desordem neurodegenerativa caracterizada clinicamente por tremor, rigidez, acinesia (ou bradicinesia) e instabilidade postural. Patofisiologicamente, a DP é classificada como uma sinucleinopatia associada à perda de neurônios dopaminérgicos na substância negra (SN), mas outros neurônios do tronco encefálico podem estar degenerados na DP, contribuindo não só para as alterações motoras, mas também não motoras observadas. Dentre as alterações não motoras, as alterações respiratórias estão presentes tais como obstrução das vias aéreas superiores, pneumonia e ainda a apnéia obstrutiva do sono uma das principais causas de morte na DP. Os mecanismos que levam à degeneração de neurônios envolvidos no controle respiratório ainda não estão bem esclarecidos, mas dados recentes do nosso laboratório mostraram que no modelo de DP induzido pela injeção no caudado-putâmen (CPu) de 6-hidroxidopamina (6-OHDA), um agente neurotóxico seletivo para células catecolaminérgicas, observou-se intensa redução na frequência respiratória e ventilação basais e induzidas pela ativação do quimiorreflexo central por hipercapnia. Além disso, observou-se também intensa redução do número de neurônios bulbares envolvidos no controle neural da respiração, como os neurônios Phox2b+ da região do núcleo retrotrapezóide (RTN), que estão envolvidos com a inspiração e o quimiorreflexo central. Dessa forma, o objetivo do presente trabalho foi investigar se a existência de uma via entre os neurônios da SN e do RTN poderia ser responsável por essa neurodegeneração. Realizamos injeções de traçadores anterógrados e retrógrados na SN e no RTN de ratos para verificar a existência de projeções diretas entre essas regiões, entretanto observamos que não há projeções diretas entre a SN e o RTN, mas há projeções indiretas entre essas duas regiões, utilizando a Substância Cinzenta Periaquedutal (PAG) como região intermediária. Além disso, observamos que no modelo de DP induzido pela injeção bilateral de 6-OHDA no CPu ocorre uma redução no número de varicosidades catecolaminérgicas na PAG e de neurônios que são ativados pelo quimiorreflexo central que se projetam da PAG para o RTN. Nossos experimentos eletrofisiológicos mostraram que a inibição bilateral da PAG pela injeção de muscimol não gera alterações respiratórias basais como ocorre no modelo da DP; entretanto, nesses animais, pudemos também observar, apesar de ser menor do que ocorre com animais submetidos ao modelo da DP, inibição de alterações respiratórias induzidas por hipercapnia. Nossos dados anatômicos mostraram que a comunicação entre os neurônios da SN e do RTN envolve neurônios da PAG e que essa via pode estar reduzida no modelo da DP, o que pode contribuir para a redução de neurônios do RTN; e que a redução neuronal desta via pode alterar as respostas respiratórias frente à ativação do quimiorreflexo central. / Parkinson\'s disease (PD) is a neurodegenerative disorder characterized clinically by tremor, rigidity, akinesia (or bradykinesia) and postural instability. Pathophysiologically, PD is classified as a synucleinopathy predominantly associated with loss of dopaminergic neurons in the substantia nigra (SN), but other brainstem neurons may also be degenerate in PD, contributing not only to the motor but also non-motor alterations observed in this pathology. Among the non-motor changes observed respiratory changes are present and can be characterized as upper airway obstruction, pneumonia and obstructive sleep apnea are one of the main causes of death in PD. The mechanisms that lead to the degeneration of neurons involved in respiratory control are still not well understood but data in the literature have demonstrated the loss of receptors in a region considered to be the respiratory rate generator in postmortem brains of humans. In the model animal DP of 6-hydroxydopamine (6-OHDA), a selective neurotoxic agent for catecholaminergic cells, there was an intense reduction in basal respiratory rate and ventilation, in addition to a intense reduction of neurons involved in neural control of breathing: Phox2b+ neurons in the retrotrapezoid nucleus (RTN) region. Thus, the aim of the present study was to investigate whether the existence of a pathway between SN and RTN neurons could be responsible for this bulbar neurodegeneration. We performed experiments using the injection of anterograde and retrograde tracers in the SN and the RTN to verify the existence of direct projections between these regions in rats. However, our results showed that there are no direct projections between the SN and the RTN, but there are indirect projections between these two regions, using Periaquedutal Gray Substance (PAG) as the intermediate region. In addition, we observed that in the PD model induced by the bilateral injection of 6-OHDA in CPu, a reduction of the projections PAG neurons for RTN and that are activated by the central chemoreflex. Our electrophysiological experiments have shown that in the 6-OHDA PD model there is a reduction of the cardiorespiratory responses induced by the activation of the central chemoreflex, since the bilateral inhibition of the PG of control animals does not alter these cardiorespiratory responses. Therefore, our anatomical results showed that the communication between SN and RTN neurons involves PAG neurons and that this pathway may be reduced in the PD model, which may contribute to the reduction of RTN neurons; and that the neuronal reduction of this pathway may alter respiratory responses to activation of the central chemoreflex.

by Cheng Wai Ming. / Thesis submitted in: October, 1998. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 54-68 (2nd gp.)). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iv / ABSTRACT --- p.v / ABSTRACT (Chinese) --- p.vii / Chapter CHAPTER 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Ionotropic glutamate receptors --- p.1 / Chapter 1.1.1 --- AMP A receptor --- p.3 / Chapter 1.1.1.1 --- Structure of AMP A receptor --- p.3 / Chapter 1.1.1.2 --- Electrophysiological properties of AMPA receptor --- p.4 / Chapter 1.1.1.3 --- Pharmacology of AMPA receptors --- p.6 / Chapter 1.1.1.4 --- Kinetics of AMPA receptors --- p.8 / Chapter 1.1.2 --- NMDA receptor --- p.9 / Chapter 1.1.2.1 --- Structure of NMDA receptor --- p.9 / Chapter 1.1.2.2 --- Electrophysiological properties of NMDA receptor --- p.10 / Chapter 1.1.2.3 --- Pharmacology of NMDA receptor --- p.11 / Chapter 1.1.2.4 --- Kinetics of NMDA receptor --- p.12 / Chapter 1.2. --- The basal ganglia and the SNR --- p.12 / Chapter 1.3 --- Excitatory glutamatergic inputs on SNR --- p.16 / Chapter 1.4 --- Aim of study --- p.17 / Chapter CHAPTER 2 --- Electrophysiological properties of SNR neurones --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.2 --- Methods --- p.19 / Chapter 2.2.1 --- In vitro slice preparation and maintenance --- p.19 / Chapter 2.2.2 --- Whole-cell patch-clamp recording --- p.20 / Chapter 2.2.3 --- Solutions and drugs --- p.21 / Chapter 2.2.4 --- Histological methods --- p.21 / Chapter 2.2.5 --- Data analysis --- p.22 / Chapter 2.3 --- Results --- p.22 / Chapter 2.3.1 --- Passive membrane properties of SNR neurones --- p.22 / Chapter 2.3.2 --- Firing rate and action potential characteristics --- p.23 / Chapter 2.3.3 --- Firing patterns --- p.23 / Chapter 2.3.4 --- Weak hyperpolarization activated inward rectification --- p.24 / Chapter 2.3.5 --- Slow aflerhyperpolarization --- p.25 / Chapter 2.3.6 --- Current-frequency relationship --- p.25 / Chapter 2.3.7 --- Morphology of labelled SNR neurones --- p.25 / Chapter 2.4 --- Discussion and conclusion --- p.26 / Chapter CHAPTER 3 --- AMPA and NMDA induced membrane responses --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Methods --- p.31 / Chapter 3.2.1 --- In vitro slice preparation and maintenance --- p.31 / Chapter 3.2.2 --- Whole-cell patch-clamp recording --- p.31 / Chapter 3.2.3 --- Solutions and drugs --- p.31 / Chapter 3.2.4 --- Drug application --- p.32 / Chapter 3.2.5 --- Immunocytochemistry --- p.32 / Chapter 3.2.6 --- Data analysis --- p.33 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- AMPA induced responses in SNR GABA neurones --- p.33 / Chapter 3.3.1.1 --- AMPA induced membrane depolarization --- p.33 / Chapter 3.3.1.2 --- AMPA induced membrane current --- p.34 / Chapter 3.3.1.3 --- Current-voltage relationship --- p.34 / Chapter 3.3.1.4 --- Effect of NBQX --- p.35 / Chapter 3.3.1.5 --- Effects of JSTX and spermine --- p.35 / Chapter 3.3.2 --- NMDA-induced response in SNR GABA neurones --- p.36 / Chapter 3.3.2.1 --- NMDA induced membrane depolarization --- p.36 / Chapter 3.3.2.2 --- NMDA induced membrane current --- p.36 / Chapter 3.3.2.3 --- APV blocked NMDA-induced current --- p.36 / Chapter 3.3.2.4 --- Effect of glycine on NMDA induced response --- p.37 / Chapter 3.3.2.5 --- Mg2+-sensitivity --- p.37 / Chapter 3.3.2.6 --- Current-voltage relationship --- p.38 / Chapter 3.3.3 --- GluR2 subunit immunostaining --- p.38 / Chapter 3.4 --- Discussion and conclusion --- p.39 / Chapter 3.4.1 --- AMPA receptors in SNR neurones --- p.39 / Chapter 3.4.2 --- NMDA receptors in SNR neurones --- p.41 / Chapter 3.4.3 --- Functional significance --- p.41 / Chapter CHAPTER 4 --- Glutamate-mediated synaptic currents in SNR --- p.43 / Chapter 4.1 --- Introduction --- p.43 / Chapter 4.2 --- Methods --- p.44 / Chapter 4.2.1 --- In vitro slice preparation and maintenance --- p.44 / Chapter 4.2.2 --- Electrophysiological recordings --- p.44 / Chapter 4.2.3 --- Electrical stimulation --- p.45 / Chapter 4.2.4 --- Solutions and drugs --- p.45 / Chapter 4.2.5 --- Data analysis --- p.46 / Chapter 4.3 --- Results --- p.46 / Chapter 4.3.1 --- Characteristics of spontaneous EPSCs --- p.46 / Chapter 4.3.1.1 --- General characteristics --- p.46 / Chapter 4.3.1.2 --- Kinetics --- p.47 / Chapter 4.3.1.3 --- Pharmacology --- p.47 / Chapter 4.3.2 --- Characteristics of evoked EPSCs --- p.48 / Chapter 4.3.2.1 --- General characteristics --- p.48 / Chapter 4.3.2.2 --- Pharmacological characterization --- p.49 / Chapter 4.3.2.3 --- Effects of bicuculline --- p.50 / Chapter 4.4 --- Discussion and conclusion --- p.50 / Chapter 4.4.1 --- Excitatory transmission onto SNR neurones --- p.50 / Chapter 4.4.2 --- Source of excitatory drive --- p.51 / Chapter 4.4.3 --- Interaction with GABA inputs --- p.52 / Chapter 4.4.4 --- Functional significance --- p.52 / REFERENCES --- p.54

INTRODUÇÃO: A presença e extensão da perda neuronal no encéfalo dos idosos ainda é controversa. A substância negra (SN) é uma região localizada no mesencéfalo e caracterizada macroscopicamente pela coloração escura, devido à presença neuromelanina. O envelhecimento da SN ainda é um mistério, mas existem grandes quantidades de estudos relativos às modificações sofridas por essa região com o passar dos anos. Alguns estudos detectaram perda neuronal na SN durante o envelhecimento, enquanto outros afirmam que não há alteração significativa durante esse processo. OBJETIVO: Caracterizar as alterações que ocorrem na SN durante o envelhecimento humano, em indivíduos sem sintomas da doença de Parkinson, do ponto de vista citoarquitetônico em 3D. CASUÍSTICA E METODOLOGIA:O presente projeto faz parte do Projeto Envelhecimento Cerebral (PEC) do Grupo de Estudos em Envelhecimento Cerebral (GEEC) da Faculdade de Medicina da Universidade de São Paulo (FMUSP). Foram utilizados 18 casos, classificados em quatro faixas etárias. Quinze casos foram analisados estereológicamente e recontruídos tridimensionalmente. Para estes, foram determinados número de neurônios, volume da SN e densidade neuronal e submetidos à testes estatísticos não paramétricos. Três casos foram imunocorados com quatro diferentes anticorpos: anti tirosina-hidroxilase (TH), anti proteína tau hiperfosforilada, anti proteína alfa-sinucleína e anti calbindina D28k (Calb) e analisados qualitativamente. RESULTADOS: As lâminas imunocoradas com anticorpo anti -sinucleína e anti-tau, foram negativas. As lâminas imunocoradas com anticorpo anti- TH e anti-Calb D28k foram positivas. Dos quinze casos analisados estereologicamente, Para o número de neurônios, encontrou-se: mediana de 504.575 células, valor mínimo de 348.662 células e valor máximo de 672.172 células. Para o volume, encontrou-se mediana de 190,8mm3; valor mínimo de 134,1 mm3 e valor máximo de 267,3mm3. Para a densidade total média, encontrou-se a mediana de 2.517,4 cel/mm3, valor mínimo de 1.603,1 cel/mm3 e valor máximo de 5.015,7 cel/mm3. Realizou-se o teste de correlação de Spearman para as três variáveis neuronais, correlacionando hemisfério direito e esquerdo, encontrou-se uma correlação moderada para número de neurônios e volume, e correlação forte para densidade. Com o mesmo teste, realizou-se análise das variáveis neuronais em função da idade, e não houve alteração estatisticamente significativa. Apenas uma tendência à diminuição no volume total da SN em relação à idade. Também verificou-se a relação entre as variáveis neuronais, por gênero em função da idade e não houve alteração estatisticamente significativa. Nas reconstruções tridimensionais, verificou-se uma grande variabilidade interpessoal entre as 15 SN reconstruídas. DISCUSSÃO: Diversos estudos estereológicos foram feitos, mas poucos deles com análise estereológica sem viés e a maior parte deles demonstra diminuição no número de neurônios, discordando dos achados atuais. A reconstrução tridimensional também não descreve alterações decorrentes do envelhecimento, mas aparentemente modificações interpessoais. CONCLUSÃO: As características da substância negra se mantém ao longo dos anos, porém, ainda devem ser muito mais estudadas / INTRODUTION: Some aspects about maco and micro braischanges are suffering modifications along the years. The presence and extension of neuronal lost in elderly still a big and polemic issue. The SUBSTANTIA NIGRA (SN) is located at the midbrain and characterized by the dark color due to the pigmented neurons, which contain neuromelanin. The SNs aging is still a mystery, nevertheless, there are great researches related to these changes suffered in this region by the years. Some of the researches noticed that neuronal lost in SN during the aging, while others claim that there are no significant modifications through this process.OBJECTIVE: Describing SN changes during the human aging in individuals with no Parkinsons disease symptoms, from the 3D cytoarchitectonic point of view. METHODS: This project is part of the Brain Aging Project of Brain Aging Study Group, in University of São Paulo Medical School. Eighteen cases had been analyzed and classified into four ages groups. Fifteen cases had been analyzed with stereological methos and 3D reconstructed. During the stereological analysis, were determinate the neuronal number, SN volume, neuronal density and submitted to nonparametric statistics test. Three cases had been immunostained with 4 different antibodies anti tyrosine-hydroxylase (TH), anti hyperphosphorylated tau (anti-tau), anti alfa-synuclein (alpha-syn) e anti calbindin D28k (Calb) and qualitatively analyzed. RESULTS: The immunostained slices with the anti alpha-syn and anti-tau antibodies were negative. The immunostained slices with anti TH and anti calb D28K antibodies were positive. From the 15 cases estereologically analyzed, 53,3% were female and 46,7% male. Had been analyzed neuron numbers, SN volume and neuronal density. For the neural numbers were found median of 504.575 cells, minimum value of 348.662 cells and maximum value of 672.172 cells. For the volume were found median of 190,8mm³; minimum value of 134,1mm³ and maximum value of 267,3mm³. For the total density, was found median of de 2.517,4 cel/mm³, minimum value of 1603,1 cel/mm³ , maximum value of 5015,7 cel/mm³. The Spearmans correlation test had been done to the three neuronal variable correlating the right and left hemispheres and was a moderated correlation between neuron numbers and neuron volume and strong correlation to density. Using the same test, the rate neuron by the age analysis had been concluded and there was no significant change.There was noticed just one trend to the reduction in the SN total volume related to aging. It was also checked the relation between the neurons variety by gender considering aging and there was no significant changes. In the 3D reconstruction, was possible to notice a great interpersonal variability between the 15 SN rebuilt. DISCUSSION: Many stereological researches had been done, however, a few of them used the unbiased stereological analysis and most of them show a reduction in the neuron number, disagreeing from the current conclusions. The 3D reconstruction also dont describe changes as aging result, but apparently interpersonal changes. CONCLUSION: The Substantia Nigra maintain the same characteristics along the years, however, it must be much more explored

Η νόσος του Πάρκινσον χαρακτηρίζεται από την προοδευτική εκφύλιση της μελαινοραβδωτής ντοπαμινεργικής οδού που οδηγεί σε κινητικές διαταραχές. Θεωρείται πολυπαραγοντική νόσος, η αιτιολογία της οποίας παραμένει άγνωστη. Με δεδομένο ότι η διαθέσιμη φαρμακευτική αγωγή της νόσου στηρίζεται στη συμπτωματολογία της και έχει σοβαρές παρενέργειες, η νευροπροστασία από τα πρώϊμα στάδια της νόσου αποτελεί τομέα έντονης έρευνας. Τα τελευταία χρόνια, ένα ευρύ φάσμα παραγόντων ερευνήθηκε ως προς το νευροπροστατευτικό τους ρόλο σε νευροτοξικά μοντέλα οξείας ντοπαμινεργικής εκφύλισης. Ο μεταλλαγμένος μυς “weaver” αποτελεί ένα μοναδικό γενετικό μοντέλο μελαινοραβδωτής νευροεκφύλισης, η οποία λαμβάνει χώρα ενδογενώς και προοδευτικά, αρχίζοντας μετά την 7η μετεμβρυϊκή ημέρα (Ρ7) και προσεγγίζοντας το 50% την 21η μετεμβρυϊκή ημέρα (Ρ21). Στην παρούσα μελέτη, προκειμένου να διερευνηθούν νευροπροστατευτικοί μηχανισμοί κατά τα πρώτα στάδια της νευροεκφυλιστικής διαδικασίας στο μυ “weaver”, και να επιτευχθεί μία πλειοτροπική θεραπευτική δράση, χορηγήθηκαν τρεις φαρμακευτικοί νευροπροστατευτικοί παράγοντες με διαφορετικούς μηχανισμούς δράσης καθώς και ένα σχήμα συνδυασμού τους. Συγκεκριμένα, χορηγήθηκαν, μεμονωμένα και σε συνδυασμό, στους μυς “weaver” N-ακετυλοκυστεΐνη (ΝAC) (αντιοξειδωτική δράση), ασπιρίνη (αντιφλεγμονώδης δράση) και 17β οιστραδιόλη [Ε2] (αντιοξειδωτική, αντιαποπτωτική, νευροτροφική δράση) σε καθημερινή βάση από την Ρ1 μέχρι την Ρ21. Το νευροπροστατευτικό αποτέλεσμα αξιολογήθηκε με ανοσοϊστοχημικό προσδιορισμό των ντοπαμινεργικών νευρώνων της συμπαγούς μοίρας της μέλαινας ουσίας (SNpc) των μυών στους οποίους χορηγήθηκαν τα παραπάνω φάρμακα. Η χορήγηση των NAC και ασπιρίνης δεν επηρέασε την επιβίωση των ντοπαμινεργικών νευρώνων (DA) των weaver μυών. Αντίθετα η χορήγηση της 17β οιστραδιόλης οδήγησε σε σημαντική επιβίωση των DA νευρώνων της SNpc, της τάξης του 48%, στους weaver μυς που έλαβαν την αγωγή, συγκριτικά με τους weaver μυς που έλαβαν φυσιολογικό ορό. Επιπλέον η χορήγηση του συνδυασμού των τριών φαρμάκων (cocktail) προώθησε σε ακόμα μεγαλύτερο βαθμό την επιβίωση των DA νευρώνων της SNpc, σε ποσοστό 86%. Οι weaver μύες που έλαβαν το cocktail εμφάνισαν 26% περισσότερους DA νευρώνες σε σύγκριση με τους weaver μυς που έλαβαν μεμονωμένα 17β οιστραδιόλη προτείνοντας πιθανή συνεργιστική δράση μεταξύ 17β οιστραδιόλης και NAC. Η διερεύνηση του μηχανισμού της νευροεκφύλισης στην SNpc και της παρεχόμενης νευροπροστασίας από τη χορήγηση της 17β οιστραδιόλης και του cocktail πραγματοποιήθηκε σε δύο επίπεδα. Αρχικά με τον προσδιορισμό μιας σειράς δεικτών οξειδωτικού στρες όπως η υπεροξείδωση λιπιδίων και δείκτες της θειολικής κατάστασης του κυττάρου (GSH, GSSG, CSH NPSSC, PSH, PSSP, NPSH, NSPSSR). Ο προσδιορισμός της υπεροξείδωση λιπιδίων πραγματοποιήθηκε στο μεσεγκέφαλο και το ραβδωτό σώμα των φυσιολογικών και weaver μυών που έλαβαν φυσιολογικό ορό (saline +/+ και saline wv/wv), 17 β οιστραδιόλη (17β +/+ και 17β wv/wv) cocktail (cocktail +/+ και cocktail wv/wv). Τα επίπεδα της υπεροξείδωσης λιπιδίων, στο μεσεγκέφαλο, αυξήθηκαν περίπου κατά 98% στους saline wv/wv μυς συγκριτικά με τους saline +/+ δείχνοντας παρουσία έντονου οξειδωτικού στρες στην παθολογική κατάσταση των weaver μυών. Ήταν ενδιαφέρον όμως το γεγονός ότι η λιπιδική υπεροξείδωση ανεστάλη σε ποσοστό 27% στους 17β wv/wv ενώ επανήλθε στα φυσιολογικά επίπεδα στους cocktail wv/wv μύες. Από τους υπόλοιπους δείκτες που εξετάστηκαν μόνο το NPSSC έδειξε διαφορές μεταξύ saline +/+ και saline wv/wv, ενώ οι GSSG, PSSP και PSH ακολούθησαν παρόμοια αύξηση στους cocktail +/+ και cocktail wv/w. Οι παρατηρήσεις αυτές δείχνουν ότι οι συγκεκριμένοι δείκτες από μόνοι τους δεν μπορούν να δώσουν σαφή εικόνα της οξειδωτικής κατάστασης, καθώς αποτελούν ταχέως μεταβαλλόμενα συστατικά αντιοξειδωτικών κύκλων. Στη συνέχεια διερευνήθηκε η έκφραση των γονιδίων Lasp1, Supt14h, Nr4a2 (nurr1), Dlg4 και του γονιδίου του μεταφορέα της σεροτονίνης (SERT), τα οποία φαίνονται να εμπλέκονται στα μονοπάτια της νευροεκφύλισης, στη μεσεγκεφαλική περιοχή και στο ραβδωτό σώμα των weaver μυών. Δεν παρατηρήθηκαν διαφορές στα επίπεδα έκφρασής τους με χρήση της τεχνικής RT-PCR σε καμία από τις υπό εξέταση περιοχές. Τα αποτελέσματα της παρούσας εργασίας οδηγούν στο συμπέρασμα ότι η 17β-οιστραδιόλη παρείχε σημαντική νευροπροστασία στους ντοπαμινεργικούς νευρώνες, για πρώτη φορά, ενός μοντέλου in vivo, ενδογενούς, προοδευτικής μελαινοραβδωτής νευροεκφύλισης, του μοντέλου weaver. Στο μηχανισμό της νευροπροστατευτικής δράσης της Ε2 φαίνεται να παίζει σημαντικό ρόλο η αντιοξειδωτική της δράση αφού η χορήγησή της αναστέλλει τη λιπιδική υπεροξείδωση. Επιπλέον η νευροπροστατευτική δράση της Ε2 ενδυναμώθηκε σημαντικά κατά τη συγχορήγηση του NAC, προτείνοντας την ύπαρξη συνέργειας μεταξύ της Ε2 και της GSH, για πρώτη φορά σε ένα in vivo μοντέλο νευροεκφύλισης. Η ενίσχυση του νευροπροστατευτικού αποτελέσματος από το cocktail δίνει ένα πρόσθετο επιχείρημα στην υπόθεση του αντιοξειδωτικού τρόπου δράσης της Ε2 αφού παράλληλα το cocktail επαναφέρει την υπεροξείδωση των λιπιδίων στα φυσιολογικά επίπεδα. Οι παρατηρήσεις αυτές προτείνουν την Ε2 ως μια μελλοντική υποψήφια φαρμακευτική αγωγή για νευροεκφυλιστικές καταστάσεις, όπως είναι η νόσος του Πάρκινσον, για τα θηλυκά βέβαια άτομα. Eπιπλέον προτείνουν ότι ο συνδυασμός της Ε2 και του NAC μπορεί να οδηγήσει σε εφαρμογή μικρότερων και κατά συνέπεια λιγότερο επιβαρυντικών, από άποψη παρενεργειών, δόσεων που θα οδηγεί σε ίδιο ή και μεγαλύτερο νευροπροστατευτικό αποτέλεσμα με τη μεμονωμένη χορήγηση της 17β-οιστραδιόλης. / Parkinson’s disease (PD) is characterized by the progressive degeneration of the nigrostriatal dopaminergic innervation that leads to motor disturbances. It is considered to be a multifactor disease, the etiology of which still remains unknown. Since currently available treatments are only symptomatic, having severe side-effects, neuroprotection from the early stages of the disease has been given much attention as a promising approach to PD management. Indeed, a broad range of agents has been investigated for their neuroprotective role in neurotoxical models of acute dopaminergic degeneration. “Weaver” mutant mouse represents a unique genetic model, in which the nigrostriatal neurodegeneration occurs endogenously and progressively, starting after postnatal day 7 (P7) and reaching 50% at P21. In the present study, aiming to identify neuroprotective mechanisms in the early progression of the “weaver” degenerative process and to achieve a potentially pleiotropic therapeutic action, we applied three pharmaceutical agents with different mechanisms of action, as well as a scheme combining them. Specifically, “weaver” mice were treated, individually and in combination, with N-acetylcysteine (NAC) (antioxidant), aspirine (anti-inflammatory) and 17b-estradiol [E2] (antioxidant, antiapoptotic, neurotrophic) daily, from P1 to P21. The neuroprotective effect was evaluated by immunohistochemical detection of dopaminergic (DA) neurons in the substantia nigra (SNpc) of treated animals. The administration of ΝΑC and aspirine did not influence the survival of (DA) neurons of weaver mice. On the contrary, the administration of 17b estradiol led to significant survival of DA neurons of SNpc, approximately 48%, in weaver mice that received E2, comparatively with weaver mice that received saline. Moreover the administration of the combination of the three drugs (cocktail) promoted the survival of DA neurons of SNpc, approximately 86% to a higher degree. Weaver mice that received cocktail had 26% more DA neurons compared to weaver mice that received individually 17b estradiol, proposing a possible synergistic action between 17b estradiol and NAC. The investigation of mechanism of neurodegeneration in SNpc and provided neuroprotection by 17b estradiol and cocktail, was realised in two levels. Initiall, by determination of oxidative stress markers, like lipid peroxidation and markers of cellular thiol redox (GSH, GSSG, CSH NPSSC, PSH, PSSP, NPSH, NSPSSR). The determination of lipid peroxidation was realised in the midbrain and striatum of normal and weaver mice that received saline (saline +/+ and saline wv/wv), 17 b estradiol (17b +/+ and 17b wv/wv) cocktail (cocktail +/+ and cocktail wv/wv). Lipid peroxidation levels in the midbrain were increased about 98% in saline wv/wv mice comparatively with the saline +/+, showing the presence of intense oxidative stress in the weaver mutant mouse. It was interesting, however, the fact that lipid peroxidation was inhibited approximately 27% in 17b wv/wv mice, while it was reverted at the normal levels in cocktail wv/wv mice. Regarding to the other oxidative markers that were examined, only NPSSC showed differences between saline +/+ and saline wv/wv, while the GSSG, PSSP and PSH followed similar increasement in both cocktail +/+ and cocktail wv/w animals. This observation indicates that these markers alone cannot give a clear figure of oxidative situation, as they constitute rapidly altered components of antioxidant cycles. Afterwards, we investigated the expression of genes Lasp1, Supt14h, Nr4a2 (nurr1), Dlg4 and serotonin transporter’s gene (SERT), which appear to be involved in neurodegeneration pathways, in the midbrain ant striatum of normal and weaver mice. There were not observed differences in their expression levels (using the RT-PCR technique) in both regions investigated. The results of the present study, lead to the conclusion that 17b-estradiol provided important neuroprotection in the DA neurons, for the first time, in a model of in vivo, endogenous, progressive dopaminergic degeneration, the weaver model. The mechanism of E2’s neuroprotective effect appears to be antioxidant as the administration of E2 suspends lipid peroxidation. Moreover the E2’s neuroprotective effect was strengthened significantly by the co-treatment of NAC, proposing the existence of synergy between E2 and GSH, for the first time in an in vivo model of neurodegeneration. The reinforced cocktail’s result gives an additional argument in the hypothesis of antioxidant mechanism of E2’s action, as cocktail, at the same time, restores lipid peroxidation in normal levels. These observations propose E2 as a future candidate pharmaceutic treatment for neurodegenerative situations, like PD, of course for female individuals. Moreover they propose that the combined treatment of E2 and NAC, can lead to the application of lower and, in consequence, less aggravating doses, concerning the side effects, that will lead to same or even higher neuroprotective result with the individual administration of 17b-estradiol

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Evidências crescentes mostram que os distúrbios de sono associados à doença de Parkinson (DP) são mais relacionados à doença per se, do que apenas fenômenos secundários. Dados apresentados pela literatura sugerem a hipótese de que o sistema dopaminérgico nigroestriatal esteja envolvido na regulação de padrões de sono. Demonstrou-se no presente trabalho que uma lesão de 50% dos neurônios dopaminérgicos residentes na substância negra pars compacta (SNpc) foi capaz de gerar um prejuízo em diversos parâmetros de sono em ratos. Essa redução neuronal provocou uma diminuição importante na porcentagem de sono paradoxal durante os três primeiros dias de registro de sono. Observou-se também uma forte correlação (r=0.91) entre o número de neurônios e a porcentagem de sono paradoxal. A partir disso, propomos que os neurônios dopaminérgicos presentes na SNpc possuem um papel fundamental para a regulação dos padrões de sono, particularmente na promoção de sono paradoxal. Em outro experimento, apresentamos evidências de que a proteína tirosina hidroxilase (TH) encontra-se com sua expressão reduzida no sistema dopaminérgico nigroestriatal após um período de 24 h de privação de sono paradoxal (PSP) em camundongos. De acordo com esses resultados, sugerese que a redução da expressão da TH, produzida pela (PSP), possa explicar em parte a existência da supersensibilidade dopaminérgica de receptores D2. As implicações dessas alterações podem reverberar diretamente sobre anormalidades motoras e de sono encontradas em pacientes portadores da DP. xiii Achados eletrofisiológicos demonstraram que o bloqueio dos receptores D2 (por haloperidol) produziu uma redução de sono paradoxal durante o período de rebote, realizado após 96 h de PSP. Essa redução foi acompanhada por um incremento de sono de ondas lentas, o que possivelmente tenha ocorrido em decorrência de um observado aumento de eficiência de sono. Os resultados também indicaram que a administração de piribedil não pôde gerar um aumento adicional de sono paradoxal. Sugerimos a existência de uma ação particular da neurotransmissão dopaminérgica recaindo sobre a ativação dos receptores D2. As evidências demonstradas no presente trabalho e na literatura permitem sugerir que os neurônios dopaminérgicos presentes na SNpc e na aérea tegmental ventral podem ser considerados essenciais para a regulação de sono, em particular no disparo e manutenção do sono paradoxal, respectivamente. Propõe-se que o paradigma que envolve a dopamina como sendo responsável apenas pela vigília, não é totalmente acurado. A teoria proposta nessa tese alega que esse neurotransmissor pode apresentar uma importante participação em ambos os estados: vigília e sono, e que cada estado deva ser gerado por intermédio de diferentes graus de modulação dopaminérgica. A conclusão delineada a partir desses achados é que a dopamina apresenta implicações significantes na regulação de sono, e essa condição particular deve ser considerada em relação ao tratamento de pacientes com a DP. / Dopamine (DA) is critically involved in regulating neural processes responsible for complex movements and emotions. Alterations in central dopaminergic neurotransmission have been implicated in important neurological and psychiatric disorders such as Parkinson’s disease (PD) and schizophrenia. In addition, DA has recently been recognized as instrumental in the regulation of sleep-wake states. Herein, we present evidence that tyrosine hydroxylase (TH) is down-regulated in the nigrostriatal pathway after 24 h of sleep deprivation (SD) in mice. To identify the involvement of DA in SD and sleep rebound (R) we administered reserpine (1 mg/kg) associated to a-methyl-p-tyrosine (aMT) (250 mg/kg) to produce DA depletion, and rotenone (10 mg/kg) to increase striatal DA turnover. Behavioral tests (catalepsy, grasping and open-field) were conducted to evaluate muscular rigidity and motor alterations inflicted by the drugs immediately after SD and R. Western blot and immunohistochemistry demonstrated that SD alone produced important down-regulation on TH protein expression within the substantia nigra (SN), without affecting the number of dopaminergic neurons. Pharmacological depletion of DA or increase of its turnover affected the entire nigrostriatal pathway. We propose that downregulation of TH expression produced by SD greatly explains the existence of supersensitivity of dopaminergic D2 receptors, especially along the nigrostriatal pathway, and suggest a novel role of DA in the mediation of sleep-wake states as a consequence of the modulation of TH protein expression along that pathway. The implications of these alterations may directly reverberate in motor and sleep abnormalities found in patients with PD. / TEDE / BV UNIFESP: Teses e dissertações