Fast Scan Cyclic Voltammetry (FSCV) is an electrochemical technique, based on ramping a voltage through a microelectrode and measuring the resulting redox current to obtain information about an electroactive molecule. FSCV can be used for the detection of dopamine, which is a vital neurotransmitter. Dopamine is central to conditions such as Parkinson’s disease. The purpose of this thesis was to evaluate if a new type of highly biocompatible microelectrode, called tube electrode, can be used to detect dopamine using FSCV and determine how they compare to standard carbon fibre microelectrodes (CFMEs). In order to achieve this, three main tasks were set and fulfilled. First, a station for in-vitro FSCV was set up and CFMEs of varying sizes were used to detect dopamine at different concentrations. Secondly, the same trials were done with tube electrodes. Finally, the results of the tubes were compared to the CFMEs. In total, four CFMEs and four tube electrodes were investigated. The dopamine concentrations ranged from 20 nM to 40 μM. In short, the results indicate that the tubes generally have lower sensitivity than the CFMEs but better linearity between the increasing dopamine concentration and the resulting current. The tubes also had a marginally higher concentration threshold for dopamine detection. The main challenge encountered was a decrease in sensitivity over time. Here, further investigations are necessary to map the causes responsible. In conclusion, the tube electrodes are able to detect dopamine in-vitro, with concentrations relevant for in-vivo sensing, with a performance comparable to CFMEs. However, further studies are necessary before the tube electrodes can be used for dopamine detection in-vivo. / Fast Scan Cyclic Voltammerey (FSCV) är en elektrokemisk teknik, baserad på att ändra en spänning genom en mikroelektrod och sedan mäta den resulterande redox-strömmen för att få information om en elektroaktiv molekyl. FSCV kan användas för detektering av dopamin, som är en livsviktig signalsubstans. Dopamin har en central roll vid tillstånd så som Parkinsons sjukdom. Syftet med detta examensarbete var att utvärdera om en ny typ av mycket biokompatibel mikroelektrod, kallad tubelektrod, kan användas för att detektera dopamin genom FSCV och fastställa hur de jämför sig med vanliga kolfibermikroelektroder (KFME). För att uppnå detta sattes och uppfylldes tre huvuduppgifter. Först upprättades en station för in-vitro FSCV där KFME med varierande storlek användes för att detektera olika koncentrationer dopamin. Sedan gjordes samma försök med tubelektroder. Slutligen jämfördes resultaten med de från KFME. Totalt testades fyra KFME och fyra tubelektroder. Dopaminkoncentrationerna sträckte sig från 20 nM till 40 μM. Kort sagt indikerar resultaten att tuberna generellt hade lägre känslighet än KFME men bättre linjäritet mellan den ökande dopaminkoncentrationen och den resulterande strömmen. Tuberna hade även något högre koncentrationströskel för detektionen av dopamin. Den största utmaningen som påträffades var en minskning i känslighet över tid. Här krävs vidare undersökningar för att helt förstå de bakomliggande anledningarna. Slutsatsen är att tuberna kan detektera dopamin in-vitro, med koncentrationer som är relevanta för mätningar in-vivo, och med en prestation jämförbar med den för KFME. Dock krävs mer studier innan tubelektroderna kan användas för att detektera dopamin in-vivo.

Parkinson's disease (PD) is the second most common neurodegenerative disease, caused by the loss of dopaminergic neurons (DN) in the substantia nigra. It is characterized by significant inflammation occurring in both the central nervous system and peripheral areas. Specifically, PINK1 variants have been detected to cause an autosomal-recessive form of the disease. PINK1 plays a crucial role in developing PD and the inflammation process. Therefore, we developed a PINK1 knock-out (KO) model in Thp1 cells using the CRISPR/CAS9 technique to assess the modulation of macrophage activities in the peripheral immune system. Our PINK1 KO models exhibited a decreased Mitochondrial Complex I activity, confirming the disruption of the PINK1 function. Moreover, PINK1 KO clones displayed changes in the expression levels of Tau protein. Furthermore, PINK1-KO macrophages showed increased expression levels of the cytokines IL-6, IL-1β, and IL-23, confirming their pro-inflammatory predisposition. Finally, immunostaining results revealed a reduction in the average of lipids droplets per cell, similar to the reduced expression levels observed in SCD5 in our PINK1-KO. Altogether, these data support the role of PINK1 in mitochondrial dysfunction, activation of the peripheral immune system, and lipid dysfunction. Our study suggests that SCD5 could be a potential target for novel therapies. These findings could aid in the diagnosis and open the path to unveiling key pathogenic mechanisms in PBMCs/macrophages that could potentially be used as a disease biomarker in the future. / La maladie de Parkinson (MP) est la deuxième maladie neurodégénérative la plus courante et est causée par la perte de neurones dopaminergiques (ND) dans la substance noire. Elle se caractérise par une inflammation importante, survenant à la fois dans le système nerveux central et dans les zones périphériques. Plusieurs éléments soutiennent l'implication de mécanismes immunitaires dans l'étiologie de la maladie. Plus précisément, des variants de PINK1 ont été détectés comme provoquant une forme autosomique récessive de la maladie. PINK1 joue un rôle crucial dans le développement de la maladie de Parkinson et dans le processus d'inflammation. Par conséquent, nous avons développé un modèle PINK1 knock-out (KO) dans les cellules Thp1 en utilisant la technique CRISPR/CAS9 pour évaluer fonctionnellement la modulation des activités des macrophages dans le système immunitaire périphérique. Nos modèles PINK1 KO présentent une diminution de l'activité du complexe mitochondrial I, confirmant la perturbation de la fonction PINK1. De plus, les clones PINK1 KO présentent des changements dans les niveaux d’expression de la protéine Tau. Les macrophages PINK1 KO ont montré une augmentation des niveaux d’expression des cytokines IL-6, IL-1β et IL-23 confirmant leur prédisposition pro-inflammatoire. Enfin, nos résultats d'immunomarquage ont révélé une réduction de la moyenne des gouttelettes lipidiques par cellule, similaire aux niveaux d’expression réduits observés dans SCD5 dans notre PINK1 KO. Ensemble, ces données soutiennent le rôle de PINK1 dans le dysfonctionnement mitochondrial, l'activation du système immunitaire périphérique et le dysfonctionnement lipidique. Notre étude a montré que SCD5 pourrait être une cible potentielle pour de nouvelles thérapies. Les résultats de nos recherches pourraient faciliter le diagnostic et ouvrir la voie à la révélation de mécanismes pathogéniques clés dans les PBMC/macrophages pouvant être potentiellement utilisés comme biomarqueurs de la maladie à l’avenir.

Parkinson’s disease (PD) is a common neurodegenerative movement disorder, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of aggregated alpha synuclein (aSyn). The disease often presents with early prodromal non-motor symptoms and later motor symptoms. Diagnosing PD based purely on motor symptoms is often too late for successful intervention, as a significant neuronal loss has already occurred. Furthermore, the lower prevalence of PD in females is not well understood, highlighting the need for a better understanding of the interaction between sex and aSyn, the crucial protein for PD pathogenesis. Here, we conducted a comprehensive phenotyping study in 1- to 5-month-old mice over- expressing human aSyn gene (SNCA) in a bacterial artificial chromosome (BAC-SNCA). We demonstrate a SNCA gene-dose-dependent increase of human aSyn and phosphorylated aSyn, as well as a decrease in tyrosine hy- droxylase expression in BAC-SNCA mice, with more pronounced effects in male mice. Phosphorylated aSyn was already found in the dorsal motor nucleus of the vagus nerve of 2-month-old mice. This was time-wise associated with significant gait altrations in BAC-SNCA mice as early as 1 and 3 months of age using CatWalk gait analysis. Furthermore, anxiety-related behavioral tests revealed an increase in anxiety levels in male BAC-SNCA mice. Finally, 5-month-old male BAC-SNCA mice exhibited a SNCA gene-dose-dependent elevation in energy expen- diture in automated home-cage monitoring. For the first time, these findings describe early-onset, sex- and gene- dose-dependent, aSyn-mediated disturbances in BAC-SNCA mice, providing a model for sex-differences, early- onset neuropathology, and prodromal symptoms of PD.

Parkinson’s Disease (PD) is the second most common neurodegenerative disease (NDD) affecting approximately 1 - 2% of the population older than 65 and it is characterized by both motor and non-motor symptoms such as rest tremors, stooping posture, and rigidity. The neuromolecular basis of PD is quite complex and that is why there is a need for in vitro systems that can be utilized for studies of PD and NDDs in general. Human-derived cell lines are a good candidate for in vitro systems since they are easy to manipulate and are a less costly alternative to post-mortem human tissue sections or animal models. In this study, I optimize the Lund human mesencephalic (LUHMES) cell line differentiation protocol by determining that the optimal seeding density of cells is 37 500 cells/ml and that the differentiation media can contain quadruple the recommended concentration of tetracycline hydrochloride. Additionally, I use the differentiated LUHMES cells to conduct an exploratory study by treating the cells with cerebrospinal fluid (CSF) from PD patients and CSF from healthy individuals to investigate the neuromodulatory effects of the CSF on the neuronal cell culture. Cell viability assay showed neurotoxicity 24 hours post-treatment for the control CSF and 48 hours post-treatment for both control and PD CSF. Immunohistochemistry showed differential expression of proteins of interest that reflect hallmarks of neurodegenerative diseases. Further studies are needed to reach conclusive results.

Morbus Parkinson (PD) ist eine Bewegungsstörung die durch intrazelluläre Einschlüsse, sogenannte Lewy Bodies, charakterisiert ist. Das synaptische Protein alpha-Synuclein (alpha-Syn) und Polyubiquitin sind Hauptkomponenten von Lewy Bodies. Das Ziel dieser Arbeit war es erstens herauszufinden ob alpha-Syn die proteolytische Aktivität des Proteasoms beeinflusst und zweitens die Auswirkungen der Proteasominhibition auf die Löslichkeit und Lokalisation von alpha-Syn zu prüfen. alpha-Syn inhibierte in vitro reversibel die 20S Proteasomenaktivität während in vivo alpha-Syn Überexpression weder in Neuroblastoma- (SH-SY5Y und SK-N-BE), HEK293 Zellen, noch in alpha-Syn transgenen Mäusen die Aktivität beeinflusste. Umgekehrt wurde der Einfluss pharmakologischer Proteasominhibition (mit MG132 und Epoxomicin) auf die Löslichkeit von alpha-Syn in SH-SY5Y Zellen untersucht. Diese Behandlung führte nicht zur Aggregation von alpha-Syn, wie sie in PD beobachtet wird, jedoch zu einer Verschiebung von mehr löslichem zu mehr membrangebundenem alpha-Syn in Zellen mit endogenem (mock) und mit WT alpha-Syn transfizierten Zellen. Diese Behandlung führte auch zur Aussparung von alpha-Syn und Polyubiquitin im Zellkern und zu cytoplasmatischen alpha-Syn-Einschlüssen ohne Polyubiquitin in einem geringen Anteil der Zellen. Die Kombination aus Proteasominhibition und Serum-Depletion, welches in den Zellen zu oxidativem Stress führen kann, verursachte die Bildung von höhermolekularem alpha-Syn im Western Blot, vergleichbar mit jenem, was in Lewy Bodies gefunden wird. Diese Daten zeigen, dass hohe Konzentrationen an alpha-Syn in vitro die Proteasomfunktion beeinflussen kann, dass dies in unseren Modellsystemen aber nicht in vivo geschieht. Außerdem führt Proteasominhibition zu strukturellen Veränderungen von alpha-Syn. Jedoch muss mehr als eine krankhafte Bedingung (z.B. oxidativer Stress und Proteasominhibition) vorhanden sein, um pathologische Veränderungen zu induzieren. / Parkinson’s disease (PD) is a movement disorder characterized by the appearance of inclusions known as Lewy bodies. The synaptic protein alpha-synuclein (alpha-syn) and polyubiquitin are major components of Lewy bodies. The objective of this study was, firstly, to evaluate whether alpha-syn inhibits proteolytic function of the proteasome and, secondly, to determine the effects of proteasome inhibition on alpha-syn solubility and localization. alpha-Syn inhibited 20S proteasome activity reversibly in vitro, while alpha-syn overexpression did not affect activity in neuroblastoma (SH-SY5Y and SK-N-BE) or HEK293 cells nor in alpha-syn transgenic mice in vivo. A reciprocal approach was used to analyze the effects of pharmacological proteasome inhibition (with MG132 and epoxomicin) on alpha-syn solubility in SH-SY5Y cells. This treatment did not lead to alpha-syn aggregation, as seen in PD. However, it induced a shift from more soluble alpha-syn toward more membrane bound alpha-syn in endogenous (mock) and WT alpha-syn transfected cells. This treatment also led to the clearing of nuclei of alpha-syn and ubiquitin, as well as to cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of the cells. The combination of proteasome inhibition with serum deprivation, which is known to produce oxidative dysfunction, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. So far these data suggest that, although not observed in our in vivo models, high concentrations of alpha-syn can interfere with proteasome function under certain conditions, while proteasome inhibition leads to structural changes in alpha-syn that may precede neurodegeneration. However, more than one condition (e.g. oxidative stress and proteasome inhibition) needs to be met to induce pathological changes.

Thesis (MScMedSC)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental (such as exposure to pesticides and neurotoxins) and genetic factors. A number of PD-causing genes have been found including SNCA, LRRK2, EIF4G1 and VPS35 (for autosomal dominant forms of PD) and parkin, PINK1, DJ-1 and ATP13A2 (for autosomal recessive forms of PD – arPD). Mutations in the parkin gene are the predominant cause of arPD. Parkin plays a role in the ubiquitin-proteasomal system which degrades damaged and unwanted proteins in the cell and it is also thought to be involved in maintaining healthy mitochondria. Numerous studies have implicated mitochondrial function in the pathogenesis of PD. Therefore the aim of the present study was to investigate the role of mitochondrial dysfunction in PD patients with parkin-null mutations. Four South African PD patients, each harbouring two parkin-null mutations, were recruited for this study. A muscle biopsy was performed for analysis of mitochondrial morphology using histology and transmission electron microscopy (TEM). Skin biopsies were taken, from which fibroblasts were cultured. These fibroblasts were used in i) mitochondrial morphological assessments using TEM, ii) mitochondrial network analysis, iii) functional studies via ROS measurement and iv) analysis of the proteome using a LTQ Orbitrap Velos mass spectrometer. In addition, RNA was isolated from peripheral blood samples for gene expression studies using the RT² Profiler PCR Array (SABiosciences, USA) and the RT² PCR Primer Assay (SABiosciences, USA). Heterozygous family members (carriers) and wild-type controls were also recruited for this study. Results from the histological and TEM analysis from the muscle biopsy observed subtle mitochondrial changes including the presence of type II fibres, atrophic fibres, the presence of lipids, and wrinkling of the sarcolemmal membrane. Enlarged mitochondria were also observed in one patient. TEM analysis on the patient’s fibroblasts observed an increase in the number of electron dense vacuoles, speculated to be autolysosomes. The mitochondrial network in two of the patients’ fibroblasts showed fragmented and dot-like networks which are indicative of damaged mitochondria. An increase in mitochondrial ROS levels was observed in three of the four patients. Expression studies found down-regulation of 14 genes from four of the five mitochondrial complexes and a total of 688 proteins were found only in the control and not in the patient fibroblasts. Some of these proteins are known to be part of the ‘mitochondrial dysfunction’ pathway. Taken together, these results indicate that the absence of parkin results in a number of mitochondrial alterations. Based on these findings, a model of PD was proposed: It is speculated that when parkin is absent, electron transport chain complex genes are down-regulated. This results in impaired oxidative phosphorylation, causing an increase in the production of mitochondrial ROS and subsequent oxidative stress. Mitochondria are then damaged; resulting in the fragmentation of the mitochondrial network. The impaired mitochondria are thus tagged for degradation, causing the recruitment of autolysosomes which engulf the mitochondria via mitophagy. Ultimately, as the compensatory mechanisms fail, this triggers the consequential cascade of cellular apoptotic events. This study has elucidated the effect of parkin on the mitochondria, and can act as a ‘stepping stone’ towards future development of therapeutic strategies and/or biochemical markers that will benefit not only patients with PD but also other neurodegenerative disorders. / AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is ‘n neurodegeneratiewe bewegings-afwyking gedefineer deur die verlies van dopaminergiese neurone in die substantia nigra van die midde brein. Alhoewel die spesifieke oorsprong van die afwyking nog nie ten volle begryp is nie, word bydraes van beide omgewings faktore (bv. blootstelling aan plaagdoders en neurotoksienes) asook genetiese faktore gespekuleer. Vanuit ‘n genetiese aspek is ‘n aantal gene al geassosieer met PS. Hierdie gene sluit in SNCA, LRRK2, EIF4G1 en VPS35 (vir outosomale dominante vorms van PS) en parkin, PINK1, DJ-1, en ATP13A2 (vir outosomale resessiewe vorms van PS - orPS). Mutasies in die parkin geen is aangedui as die hoof oorsaak van orPS. Parkin speel ‘n rol in die ubiquitine-proteasomale sisteem wat beskadige en ongewensde proteïne binne in die sel verwyder en is verdink om by te dra tot die instandhouding van gesonde mitokondria. Mitokondriese wanfunksionering is ook deur talle studies gewys as ‘n bydraende faktor in die patologie van PS. Die doel van die studie is om ondersoek in te stel tot die spesifieke rol wat mitokondriese wanfunsionering speel in PS pasiënte met parkin-nul mutasies. Vier Suid-Afrikaanse PS-pasiënte, elk met twee parkin-nul mutasies, is gebruik vir die studie. Deur middel van spierbiopsies is monsters verkry vir mitokondriese morfologiese analises met behulp van histologiese en elektron-oordrag mikroskopie tegnieke (TEM). Vel biopsies is ook geneem en fibroblaste is gekweek vir die gebruik in: i) mitokondriese morfologiese assesering; ii) mitokondriese netwerk analiese; iii) funksionele studies waar vlakke van reaktiewe suurstof spesies (ROS) gemeet is; iv) proteoom analiese met behup van ‘n LTQ Orbitrap Velos massa spektrometer. RNA is ook geisoleer vanaf perifere bloedmonsters vir die gebruik in geen-uitdrukkings studies met behulp van ‘n RT² Profiler PCR Array en ‘n RT² Primer Assay. Selle vanaf famielie lede wat heterosigotiese draers is van die mutasie, asook normale (geen parkin mutasie) selle is gebruik as kontroles in die studie. TEM resultate vanaf die spier monsters het subtiele mitokondriese veranderinge getoon. Hierdie sluit in die teenwoordigheid van tipe II vesels, atrofiese vesels, teenwoordigheid van lipiedes, assook waarnemings van rimpeling van die sarcolemmal membraan. Vergrote mitokondrias is ook in een van die pasiënte opgelet. TEM resultate vanaf die fibroblaste het toename in die aantal elektron-digte vakuole vertoon, moontlik geidentifiseer as autolisosome. Gefragmenteerde en onderbreekte mitokondria netwerke is gelet tydens netwerk analiese van die fibroblaste, ‘n indikasie van beskadigde mitokondria. ‘n Toename in mitokondriese ROS vlakke is gevind in drie van die vier pasiënte. Af-regulering van 14 gene, geassosieerd met vier uit die vyf mitokondria komplekse, is verneem tydens die geen-uitdrukkings studie. Saam met dit is ‘n totaal van 688 proteïene geidentifiseer wat slegs teenwoordig is in die kontrole monsters en nie in die pasiënt monsters nie. Hierdie proteïene is almal uitgedruk en betrokke in die mitokondriese wanfunsionerings-weë. Hierdie resultate dui dat die afwesigheid van parkin mitokondriese afwykings tot gevolg het wat kan lei tot die afsterwing van selle. Dit dra ook by tot die vorming van ‘n beter-verstaande siekte-model vir PS: Mutasies in parkin (wat lei tot die afwesigheid van parkin) kan dus moontlik lei tot die af-regulasie van gene geassosieerd met die elektron-vervoer ketting komplekse in die mitokondria. Dit lei tot gebrekkige oksidatiewe fosforilering en veroorsaak ‘n toename in die vorming van ROS, wat dan ‘n toename in oksidatiewe stres binne in die sel tot gevolg het. Uiteindelik lei dit dus tot die beskadiging van die mitokondria wat gepaard gaan met fragmentering van die mitokondriese netwerk. Beskadigde mitokondrias word geetiketeer vir afbraking. Hierdie etiketering aktiveer omringende autophagosome wat die beskadigde mitokondrias dan verwyder deur middel van ‘n verswelgende proses genaamd mitophagy. Dit veroorsaak die aktivering van ‘n aantal gekorreleerde sellulêre prosesse wat lei tot apoptose (afsterwing van die sel). Hierdie studie dra by tot die verklaring van die spesifieke effek wat parkin mutasies het op die funksionering van die mitokondria. Resultate hier lê ook die grondslag vir toekomstige studies met die doel tot die ontwikkeling van terapeutiese strategeë en biochemiese merkers wat kan bydrae tot die genesing van beide pasiënte met PS, asook pasiënte met ander neurodegeneratiewe afwykings.

帕金森氏症是現今常見的神經退化性疾病，其特徵主要表現在運動功能上的缺失包含靜止時振顫、步態不穩及肢體僵硬等特徵。而基因研究指出當PTEN-induced putative kinase 1 (PINK1)及Parkin基因發生突變可能會導致粒腺體功能失常，進而導致帕金森氏症之神經退化性疾病，另外PINK1基因突變的果蠅亦會產生嗅覺辦識功能障礙。在環境因素方面，一種廣效使用的除草劑百草枯(paraquat)被認為與誘發帕金森氏症有關。根據中醫生理學概念，消化系統功能紊亂，不但影響腸道功能亦會影響其他系統的運作,包含中樞神經系統。因此本實驗採用中草藥紫蘇初萃取物作為實驗藥物，紫蘇(Perilla frutescens)在中醫臨床的主要功能用於治療因飲食不節而導致的腸胃氣滯，進而減緩其他表症的不適感，另外紫蘇另一項功能在於能解食魚蟹之毒。因此我們評估紫蘇可能對於野生型及突變型果蠅因百草枯引發之毒性具有保護作用。實驗結果顯示，紫蘇可以有效的降低因曝露在百草枯(24-72小時)下的致死率，不論在野生型Oregon-R品系及PINK1B9或Parkin25的突變型(年齡：3-7天, 10-14天)。然而紫蘇對於果蠅的運動爬行功能只有在施予百草枯後的野生型Oregon-R 品系(年齡：3-7天)有些微改善，但是在嗅覺辨識功能被百草枯破壞的PINK1B9 突變果蠅，則能經由紫蘇而減少嗅覺辨識的損害。綜合以上實驗結果得知紫蘇對於百草枯所造成的毒性具有保護效果，期望未來可作為治療帕金森氏症的潛在藥物。 / Parkinson’s disease (PD) is the most common neurodegenerative disease characterized by motor deficits including resting tremor, akinesia, and rigidity. The olfactory disturbance appears to be an earlier symptom prior to the onset of motor dysfunction in PD. Genetic research has shown that mutations of the PTEN-induced putative kinase 1 (PINK1) and Parkin genes could lead to mitochondrial dysfunction and neuronal degeneration in PD. Moreover, the environmental neurotoxin paraquat (PQ), a widely used pesticide, is known to induce PD-like symptoms. According to Traditional Chinese medicine physiology theory, that the central nervous system and gut interact bidirectionally in functional gastrointestinal disorders. Perilla frutescens, a traditional herbal medicine, is mainly prescribed for the treatment of gastrointestinal discomfort after eating and symptom of qi stagnation. In addition, perilla has an important role in detoxification of seafood intake. Therefore, this study evaluated the protective effects of perilla on paraquat-induced toxicity in Drosophila PINK1 and parkin mutants and wild type flies. Result showed that perilla can effectively alleviate lethality of drosophila, including PINK1 and parkin mutant flies, exposed to paraquat for 24-72 hours. However, their motor dysfunctions induced by paraquat were little ameliorated by perilla. Importantly, the olfactory dysfunction, particularly olfactory discrimination, elicited by paraquat in PINK1 mutant fly was improved by perilla. Taken together, our findings provide the protective potential of perilla for treatment of PD syndromes.

Due to the blood-brain barrier, delivery of many drugs to the brain has required intracranial surgery which is prone to complication. Here we show that Dopamine Neuron Stimulating Peptide 11 (DNSP-11), following non-invasive intranasal administration, protects dopaminergic neurons from a lesion model of Parkinson’s disease in the rat. A significant and dose-dependent increase in an index of dopamine turnover (the ratio of DOPAC to dopamine) was observed in the striatum of normal young adult Fischer 344 rats by whole-tissue neurochemistry compared to vehicle administered controls. Among animals challenged with a moderate, unilateral 6-hydroxy-dopamine (6-OHDA) lesion of the substantia nigra, those treated repeatedly with intranasally administered DNSP-11 exhibited greater numbers of tyrosine hydroxylase (TH) positive dopaminergic neuronal cell bodies in the substantia nigra and greater TH+ fiber density in the striatum when compared to animals treated intranasally with vehicle only or a scrambled version of the DNSP-11 sequence. Lesioned animals that received intranasal DNSP-11 treatment did not exhibit abnormal, apomorphine-induced rotation behavior, contrasted with animals that received only vehicle or scrambled peptide that did exhibit significantly greater rotation behavior. In addition, the endogenous expression of DNSP-11 from the pro-region of GDNF was investigated by immunohistochemistry with a custom, polyclonal antibody. Signal from the DNSP-11 antibody was found to be differentially localized from the mature GDNF protein both spatially and temporally. While DNSP-11-like immunoreactivity extensively colocalizes with GDNF immunoreactivity at post-natal day 10, the day of maximal GDNF expression, DNSP-11-like signal was found to be present in the 3 month old rat brain with signal in the substantia nigra, ventral thalamic nucleus, dentate gyrus of the hippocampus, with the strongest signal observed in the locus ceruleus where GDNF is not expressed. Results from immunoprecipitation of brain homogenate were not consistent with the synthetic, amidated 11 amino-acid rat DNSP-11 sequence. However, binding patterns in the literature of NPY, the only homologous sequence present in the CNS, do not recapitulate the immunoreactive patterns observed for the DNSP-11 signal. This study provides evidence for a potential easy-to-administer intranasal therapeutic using the DNSP-11 peptide for protection from a 6-OHDA lesion rat model of Parkinson’s disease.

Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-motor symptoms, of which cognitive dysfunction is an example, also frequently occur. Current therapy provides symptomatic relief mainly by augmentation of dopaminergic signalling (levodopa, dopamine agonists, MAO and COMT enzyme inhibitors), but disease progression is not adequately addressed. New therapies that can prevent further neurodegeneration in addition to providing symptomatic relief are therefore urgently required. Adenosine has an important function as neuromodulator in the central nervous system. The adenosine A2A receptor in particular plays an essential role in the regulation of movement. This, coupled to the fact that it is uniquely distributed in the basal ganglia, contributes to its attractiveness as non-dopaminergic target in the treatment of movement disorders, such as Parkinson’s disease. The efficacy of adenosine receptor antagonists has been illustrated in animal models of Parkinson’s disease and several adenosine receptor antagonists have also reached clinical trials. The neuroprotective properties of adenosine A2A receptor antagonists are further attributed to their ability to modulate neuro-inflammation and decrease the release of the excitatory neurotransmitter glutamate, which is implicated in neurotoxicity. While adenosine A1 receptor antagonism has a synergistic effect on the motor effects of adenosine A2A receptor antagonism, it has the additional benefit of improving cognitive dysfunction, a cardinal non-motor symptom of Parkinson’s disease. Dual antagonism of adenosine A1 and A2A receptors therefore offers the potential of providing symptomatic relief as well as the neuroprotection so desperately needed in the clinical environment. Amino substituted heterocyclic scaffolds, such as those containing the 2-aminopyrimidine motif, have been shown to exhibit good efficacy as dual adenosine receptor antagonists. Since the structure activity relationships of 2-aminopyrimidines have not been comprehensively explored, it is in this regard that this study aimed to make a contribution. Results - Fourteen 2-aminopyrimidines were synthesised successfully over three steps, (although in low yields) and characterised by nuclear magnetic resonance and infrared spectroscopy, mass spectrometry, by determination of melting points and high performance liquid chromatography. Structure modifications explored included variation of the aromatic substituent on position 4, as well as variations in the substituents of the phenyl ring, present on position 6 of the pyrimidine ring. Radioligand binding assays were performed to determine the affinities of the synthesised compounds for the adenosine A1 and A2A receptor subtypes. Several high dual affinity derivatives were identified during this study; the compound with the highest affinity was 4-(5- methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) with Ki values of 0.5 nM and 2.3 nM for the adenosine A2A and adenosine A1 receptors, respectively. A few general structure activity relationships were derived, which included: The effect of the aromatic substituent (position 4) on A2A affinity could be summarised (in order of declining affinity) as follows: 5-methylthiophene > phenyl > furan > pyridine > p-fluorophenyl > benzofuran. On the other hand, the effect of this substituent on A1 receptor affinity could be summarised (in order of declining affinity) as follows: phenyl > 5-methylthiophene > pfluorophenyl > benzofuran > pyridine. The affinities as exhibited by the methylthiophene derivatives 39f, 39h – 39j, further showed that while piperidine substitution (39f) resulted in optimal A2A and A1 affinity, pyrrolidine substitution (39j) was less favourable. Substitution at the 4ʹ position of the phenyl ring, as well as thiazole substitution, generally resulted in poor adenosine A1 and A2A receptor affinity. However, 4-[2-amino-6-(5-methylfuran-2-yl)pyrimidin- 4-yl]-N-(1,3-benzothiazol-2-yl)benzamide (39l) surprisingly demonstrated good affinity and selectivity for the adenosine A1 receptor. The results obtained during radioligand binding assays were rationalised by QSAR and molecular modelling (Discovery Studio 3.1, Accelrys) studies. The inverse relationship seen between log Ki (as indicator of affinity) and polar surface area, illustrated the importance of this physico-chemical property in the design of 2-aminopyrimidine A2A antagonists. The results from the docking study further showed that the orientation adopted by derivatives in the binding cavity (and particular hydrogen bonding to Asn 253 and Glu 169) is of importance. Results from the MTT cell viability assay indicated that none of the high affinity derivatives had a significant effect on cell viability at 1 μM, a concentration much higher than their Ki values. However, incorporation of the furan, benzofuran and p-fluorophenyl groups as aromatic substituent and a pyrrolidine as amine substituent, presented liabilities. Lastly, the haloperidol induced catalepsy assay (in rats) was used to give a preliminary indication of adenosine receptor antagonism or agonism. Compound 39f failed to reverse catalepsy under standard conditions, but showed some reversal after an increased time period. Indications therefore exist that 39f is an adenosine receptor antagonist that suffers from bioavailability issues. Compound (39c), 4-phenyl-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine which also demonstrated promising affinity in the radioligand binding assays however showed a statistically significant reduction in catalepsy, indicating adenosine A2A receptor antagonism, and in vivo efficacy. Highly potent, dual affinity aminopyrimidine derivatives with acceptable toxicity profiles were identified in this study, with compound 39c demonstrating in vivo activity. The aim of designing and synthesising a promising dual adenosine A1/A2A receptor antagonist is therefore realised, with compound 39c as the most favourable example. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

The diagnosis of Parkinson’s disease (PD) follows the UK Brain Bank Criteria, which demands bradykinesia and one additional symptom, i.e. rigidity, resting tremor or postural instability. The latter is not a useful sign for the early diagnosis of PD, because it does not appear before Hoehn and Yahr stage 3. Early symptoms of PD which precede the onset of motor symptoms are hyposmia, REM sleep behavioral disorder, constipation, and depression. In addition, an increasing number of patients whose PD is related to a genetic defect are being described. Thus, genetic testing may eventually develop into a tool to identify at-risk patients. The clinical diagnosis of PD can be supported by levodopa or apomorphine tests. Imaging studies such as cranial CT or MRI are helpful to distinguish idiopathic PD from atypical or secondary PD. SPECT and PET methods are valuable to distinguish PD tremor from essential tremor if this is clinically not possible. Using all of these methods, we may soon be able to make a premotor diagnosis of PD, which will raise the question whether early treatment is possible and ethically and clinically advisable. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.