The developing nervous system depends largely on extracellular cues to shape its complex network of neurons. Classically, neurotrophins are known to be important mediators in this process. More recently, Bone Morphogenetic Proteins (BMPs), belonging to the Transforming Growth Factor beta (TGFβ) superfamily of secreted cytokines, have been shown to exert a wide range of effects, such as cellular growth, differentiation, survival and apoptosis, both in the developing and adult nervous system. They signal via serine/threonine kinase receptor essentially to the Smad pathway, which carries the signal to the nucleus where the transcription of target genes is regulated. This thesis investigates the functions of BMPs in the nervous system, using a set of different models. Firstly, a targeted deletion of GDF10 (BMP3b) in the mouse was established to evaluate the role of this growth/differentiation factor in the hippocampal formation, a brain area known to be involved in memory processing. Other members of the TGFβ superfamily likely compensate for the lack of GDF10, since no detectable alterations in hippocampal function or gene transcription profile have been found. Secondly, a mouse model was set up, with the aim to study impaired BMP-signalling in dopaminergic neurons. The tyrosine hydroxylase (TH) locus was used to drive the expression of dominant negative BMP receptors by means of bicistronic mRNAs. TH is the rate-limiting enzyme in the biosynthesis of catecholamine and the mice described, show a graded decrease of TH-activity resulting in severe to mild dopamine deficiency. The contribution of the dominant negative BMP receptors to the phenotype is however secondary to the apparent TH hypomorphism. The final theme of this thesis is the potentiating effects of BMPs on neurotrophin-induced neurite outgrowth as studied in explanted ganglia from chick embryos and in the rat phaeochromocytoma cell line PC12. A number of pharmacological inhibitors of intracellular signalling kinases were applied to the cultures in order to reveal the contribution of different pathways to the enhanced neurite outgrowth. We made the unexpected finding that inhibition of MEK signalling mimicked the potentiating effects of BMP stimulation in the chick system. The underlying mechanisms for the synergistic effects, however, are still an enigma.

Der Morbus Parkinson ist durch den selektiven Zelltod der dopaminergen Neurone der Substantia nigra pars compacta gekennzeichnet. Hierbei sind die verschiedenen Populationen pigmentierter Neurone innerhalb der SNc unterschiedlich stark betroffen. Die Ursachen für diese unterschiedliche Schädigung sind noch nicht bekannt. Möglicherweise besteht aber ein Zusammenhang mit der Verteilung der extrazellulären Matrix innerhalb der Substantia nigra. Für die Untersuchung wurden immunhistochemische Methoden an Hirnschnittserien von menschlichen Kontrollgehirnen angewandt. Zur Darstellung von Komponenten der extrazellulären Matrix wurden drei verschiedene Antikörper genutzt. Dazu gehörten anti- CRTL-1, welcher das Link- Protein 1 von CSPGs detektiert, ein Aggrecan- Antikörper ( Klon HAG7D4), welcher an das Kern- Protein menschlichen Aggrecans bindet, sowie anti- Proteoglykan- Di-0S (Klon 1B5), der die Reste der Chondroitin- Sulfat- Seitenketten verschiedener Proteoglykane detektiert, die nach Verdau mit Chondroitinase ABC übrigbleiben. Zur räumlichen Orientierung und strukturellen Gliederung der Substantia nigra nach der von Damier et al. ( 1999) beschriebenen Calbindin- Methode, auf deren Grundlage die SNc in eine Calbindin-reiche Matrix und Calbindin- arme Bereiche, die sogenannten Nigrosomen, gegliedert wird, wurden benachbarte Hirnschnitte mit anti- Calbindin D₂₈K behandelt. Es zeigte sich, dass extrazelluläre Matrix in Form von perineuronalen Netzen nur an den nicht pigmentierten Neuronen der SNr und SNl vorkommt, während die pigmentierten Neurone der SNc keine perineuronalen Netze besitzen, aber von einer Vielzahl von ACs kontaktiert werden. Deren Dichte war an großen, stark Melanin- haltigen Neuronen am höchsten, sodass in der dorsalen Schicht der SNc, also in den Nigrosomen 3 und 4, besonders viel extrazelluläre Matrix detektiert werden konnte. Im ventralen Anteil der SNc war entsprechend der unterschiedlichen Zellgrößen, insbesondere in Nigrosom 1, eine heterogene Verteilung der extrazellulären Matrix festzustellen. Zur Untersuchung über mögliche Veränderungen der extrazellulären Matrix im Verlauf des Morbus Parkinson wurden Hirnschnitte menschlicher Gehirne mit diagnostiziertem Morbus Parkinson ebenfalls mit den drei Antikörpern zur Darstellung der extrazellulären Matrix behandelt. Dabei zeigte sich, dass insgesamt die Menge extrazellulärer Matrix verringert scheint. Eine Darstellung der perineuronalen Netze mit anti- Proteoglykan- Di-0S (Klon 1B5) war nicht mehr möglich. Wie bereits in früheren Studien verschiedener Autoren festgestellt, waren die stärksten Auswirkungen der neurodegenerativen Prozesse im ventralen Anteil der SNc, vor allem in Nigrosom 1, auszumachen, während die Neurone der Nigrosomen 3 und 4 im dorsalen Anteil weniger vulnerabel erscheinen. Diese Ergebnisse verstärken die Annahme, dass die extrazelluläre Matrix eine protektive Funktion für bestimmte Neuronengruppen besitzt. Bei der Parkinsonschen Erkrankung wird möglicherweise zuerst dieses Schutzsystem zerstört bevor es zum progressiven Neuronenverlust kommt. Ungeklärt bleibt weiterhin was die Ursachen dafür sind.

A recent model of Parkinson’s disease (PD) suggests that the neuropathological, behavioural and cognitive symptoms progress in stages. There is substantial evidence for a prodromal stage of PD, during which time pre-motor symptoms develop. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a risk factor for developing PD and may be part of the pre-motor stage. In both disorders, neuropathological α-synuclein aggregates are thought to be a direct cause of the resulting symptoms. One model has shown that in rats, proteasome inhibition produced by systemic exposure to environmental toxins results in α-synuclein pathology and motor behaviour dysfunction that mimics the progression of PD in humans. The present study examined the hypothesis that the systemic proteasome inhibition model would produce pre-Parkinsonian RBD-like pathology in rats. It was expected that sleep disturbances would be seen prior to behavioural disturbances in rats treated systemically with PSI (a proteasome inhibitor). Following baseline sleep recording and training on the inclined beam-traverse task, rats were injected with PSI (a proteasome inhibitor) or ethanol (control), 6 times over 2 wk. Sleep recording over 8 wk and behavioural testing over 16 wk provided no evidence of sleep disturbances or motor dysfunction. Post-mortem immunohistochemical analyses of brain tissue provided no evidence of PSI-associated α-synuclein aggregates in the locus coeruleus, subcoeruleus (dorsal part), or substantia nigra (areas involved in RBD and/or PD). These results did not provide support for RBD as a prodromal phase of PD within the systemic proteasome inhibitor-based model and add to a growing body of research reporting inconsistent findings using this model. We suggest that systemic PSI exposure in rats does not produce a viable model of RBD or PD. Whether RBD is an early symptom in the progression of PD remains to be established. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-04-28 12:04:50.613

Purpose Monoamine oxidase (MAO) consists of two isoforms, namely MAO-A and MAO-B. Both these isoforms are involved in the oxidation of dopamine. In Parkinson’s disease (PD) therapy, the inhibition of the oxidation of dopamine by MAO may elevate the levels of dopamine in the brain and prevent the generation of toxic by-products such as hydrogen peroxide. MAO-B inhibitors have found application as monotherapy in PD and it has been shown that MAO-B inhibitors may also be useful as adjuvants to L-dopa in PD therapy. For example, an earlier study has shown that the combination of L-dopa with (R)-deprenyl (a selective MAO-B inhibitor), may lead to a reduction of the dose of L-dopa required for alleviating the motor symptoms in PD patients. However, older MAO inhibitors may possess adverse side effects such as psychotoxicity, liver toxicity and cardiovascular effects. The irreversible mode of inhibition of the older MAO-B inhibitors, such as (R)-deprenyl, may also be considered as less desirable. After the use of irreversible inhibitors, it may require several weeks for the MAO enzyme to recover activity. In contrast, after administration of a reversible inhibitor, enzyme activity is recovered as soon as the inhibitor is cleared form the tissues. The adverse effects and disadvantages of the older MAO-B inhibitors prompted us to undertake the discovery of safer and reversible inhibitors of MAO-B. Such compounds may find application in the treatment of PD. Rationale It was recently discovered that (E)-8-(3-chlorostyryl)caffeine (CSC) is a potent inhibitor of MAO-B, with an IC50 value of 0.128 µM. CSC has a caffeine moiety, which is thought to be essential for MAO-B inhibition. It was also reported that a related series of 8- (phenoxymethyl)caffeine derivatives are potent and reversible inhibitors of MAO-A and –B. The IC50 values of the 8-(phenoxymethyl)caffeines ranged from 0.148–5.78 µM for the inhibition of MAO-B. For the purpose of this study the phenoxymethyl side-chain was replaced with a phenylsulfanyl moiety at C8. The aim of this study was therefore to synthesize a series of 8-[(phenylsulfanyl)methyl]caffeine analogues and to compare their MAO-B inhibition potencies to the previously synthesised 8-(phenoxymethyl)caffeine derivatives. A series of five 8-[(phenylsulfanyl)ethyl]caffeine analogues was also synthesized in order to determine the effect of carbon chain elongation on the potency of MAO inhibition. O C-8 N N O N N Caffeine Cl O N N (E) O N N CSC O N N O O N N 8-(Phenoxymethyl)caffeine O N N O N N S 8-[(Phenylsulfanyl)methyl]caffeine O N N S O N N 8-[(Phenylsulfanyl)ethyl]caffeine Compound R1 R2 1a H H 1b Cl H 1c Br H 1d F H 1e CH3 H 1f OCH3 H 1g OCH2CH3 H 1h H Cl 1i H Br Compound R1 R2 2a H H 2b Cl H 2c Br H 2d H Cl 2e H Br Methods The C8 substituted caffeine analogues were synthesised by reacting 1,3-dimethyl-5,6-diaminouracil with an appropriately substituted 2-(phenylsulfanyl)acetic acid or 3-(phenylsulfanyl)propanoic acid in the presence of a carbodiimide activating reagent, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC). Ring closure of the intermediary amide was effected by reaction with sodium hydroxide. Resulting theophylline analogues were subsequently methylated in the presence of iodomethane to yield the target compounds. The structures of the C8 substituted caffeine analogues were verified by NMR and MS analysis. The purities thereof were subsequently estimated by HPLC analysis. The 8-[(phenylsulfanyl)methyl]caffeine and 8-[(phenylsulfanyl)ethyl]caffeine analogues were evaluated as MAO-A and –B inhibitors. The recombinant human enzymes were used as enzyme sources. The inhibitory potencies of the caffeine derivatives were expressed as IC50 values (the concentration of a drug that is required for 50% inhibition in vitro). The time- dependency of inhibition of MAO-B by the most potent inhibitor was also evaluated in order to determine the reversibility of inhibition of the test compound. A study was also conducted to determine the inhibition mode of the most potent test compound, by constructing a set of Lineweaver Burk plots. Results The results showed that the 8-[(phenylsulfanyl)methyl]caffeine analogues were inhibitors of MAO-A and –B. The most potent inhibitor in the first series (1a–i) of this study were 8-[(3- bromophenylsulfanyl)methyl]caffeine and 8-[(4-bromophenylsulfanyl)methyl]caffeine with IC50 values of 4.90 and 4.05 µM, respectively. When these results were compared to those of the previously studied 8-(phenoxymethyl)caffeine derivatives it was found that, for these compounds, the bromine substituted homologues were also the most potent MAO-B inhibitors. The bromine substituted 8-(phenoxymethyl)caffeine derivatives exhibited IC50 values of 0.148 and 0.189 µM for those homologues containing bromine on the meta and para positions of the phenoxy side chain, respectively. In general, the 8- [(phenylsulfanyl)methyl]caffeine derivatives were found to be less potent MAO-B inhibitors than the 8-(phenoxymethyl)caffeine derivatives. The 8-[(phenylsulfanyl)methyl]caffeine derivatives also did not show as high a degree of selectivity for MAO-B (compared to MAO- A) as did the 8-(phenoxymethyl)caffeines. Similar to the 8-(phenoxymethyl)caffeines, the 8- [(phenylsulfanyl)methyl]caffeines also proved to be weak MAO-A inhibitors. The most potent inhibitor of MAO-A among the test compounds exhibited an IC50 value of 19.4 µM. The most potent MAO-A inhibitor among the previously studied 8-(phenoxymethyl)caffeines was more potent with an IC50 value of 4.59 µM. From these results it may be concluded that the phenoxy side chain is more suited for the design of caffeine derived MAO inhibitors than the phenylsulfanyl side chain. The results for the second series investigated in this study, the 8-[(phenylsulfanyl)ethyl]caffeines (2a–e), revealed the chlorine substituted derivatives to be the most potent MAO-B inhibitors. The meta and para chlorine substituted derivatives exhibited IC50 values of 5.67 and 7.79 µM, respectively, for the inhibition of MAO-B. Interestingly, the meta substituted derivative exhibited no inhibition toward the MAO-A isoenzyme. However, the 8-[(phenylsulfanyl)ethyl]caffeine derivatives were found to be very weak inhibitors of both MAO-A and –B and may be considered as less potent than the 8-[(phenylsulfanyl)methyl]caffeine derivatives. Since one of the aims of this study was to synthesise reversible MAO inhibitors, a time- dependency study was carried out with the best inhibitor (1i). The aim of this study was to determine the reversibility of inhibition by the 8-[(phenylsulfanyl)methyl]caffeine derivatives. From the results, it was concluded that the inhibition of MAO-B by compound 1i is reversible. To determine the mode of inhibition, a set of Lineweaver-Burk plots was constructed and since the plots were linear and intersected on the y-axis, it was concluded that 1i is a competitive inhibitor of MAO-B. Conclusion This study concludes that the phenoxymethyl side-chain is more suited for the design of caffeine derived MAO-B inhibitors than the (phenylsulfanyl)methyl side-chain. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Purpose Monoamine oxidase (MAO) consists of two isoforms, namely MAO-A and MAO-B. Both these isoforms are involved in the oxidation of dopamine. In Parkinson’s disease (PD) therapy, the inhibition of the oxidation of dopamine by MAO may elevate the levels of dopamine in the brain and prevent the generation of toxic by-products such as hydrogen peroxide. MAO-B inhibitors have found application as monotherapy in PD and it has been shown that MAO-B inhibitors may also be useful as adjuvants to L-dopa in PD therapy. For example, an earlier study has shown that the combination of L-dopa with (R)-deprenyl (a selective MAO-B inhibitor), may lead to a reduction of the dose of L-dopa required for alleviating the motor symptoms in PD patients. However, older MAO inhibitors may possess adverse side effects such as psychotoxicity, liver toxicity and cardiovascular effects. The irreversible mode of inhibition of the older MAO-B inhibitors, such as (R)-deprenyl, may also be considered as less desirable. After the use of irreversible inhibitors, it may require several weeks for the MAO enzyme to recover activity. In contrast, after administration of a reversible inhibitor, enzyme activity is recovered as soon as the inhibitor is cleared form the tissues. The adverse effects and disadvantages of the older MAO-B inhibitors prompted us to undertake the discovery of safer and reversible inhibitors of MAO-B. Such compounds may find application in the treatment of PD. Rationale It was recently discovered that (E)-8-(3-chlorostyryl)caffeine (CSC) is a potent inhibitor of MAO-B, with an IC50 value of 0.128 µM. CSC has a caffeine moiety, which is thought to be essential for MAO-B inhibition. It was also reported that a related series of 8- (phenoxymethyl)caffeine derivatives are potent and reversible inhibitors of MAO-A and –B. The IC50 values of the 8-(phenoxymethyl)caffeines ranged from 0.148–5.78 µM for the inhibition of MAO-B. For the purpose of this study the phenoxymethyl side-chain was replaced with a phenylsulfanyl moiety at C8. The aim of this study was therefore to synthesize a series of 8-[(phenylsulfanyl)methyl]caffeine analogues and to compare their MAO-B inhibition potencies to the previously synthesised 8-(phenoxymethyl)caffeine derivatives. A series of five 8-[(phenylsulfanyl)ethyl]caffeine analogues was also synthesized in order to determine the effect of carbon chain elongation on the potency of MAO inhibition. O C-8 N N O N N Caffeine Cl O N N (E) O N N CSC O N N O O N N 8-(Phenoxymethyl)caffeine O N N O N N S 8-[(Phenylsulfanyl)methyl]caffeine O N N S O N N 8-[(Phenylsulfanyl)ethyl]caffeine Compound R1 R2 1a H H 1b Cl H 1c Br H 1d F H 1e CH3 H 1f OCH3 H 1g OCH2CH3 H 1h H Cl 1i H Br Compound R1 R2 2a H H 2b Cl H 2c Br H 2d H Cl 2e H Br Methods The C8 substituted caffeine analogues were synthesised by reacting 1,3-dimethyl-5,6-diaminouracil with an appropriately substituted 2-(phenylsulfanyl)acetic acid or 3-(phenylsulfanyl)propanoic acid in the presence of a carbodiimide activating reagent, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC). Ring closure of the intermediary amide was effected by reaction with sodium hydroxide. Resulting theophylline analogues were subsequently methylated in the presence of iodomethane to yield the target compounds. The structures of the C8 substituted caffeine analogues were verified by NMR and MS analysis. The purities thereof were subsequently estimated by HPLC analysis. The 8-[(phenylsulfanyl)methyl]caffeine and 8-[(phenylsulfanyl)ethyl]caffeine analogues were evaluated as MAO-A and –B inhibitors. The recombinant human enzymes were used as enzyme sources. The inhibitory potencies of the caffeine derivatives were expressed as IC50 values (the concentration of a drug that is required for 50% inhibition in vitro). The time- dependency of inhibition of MAO-B by the most potent inhibitor was also evaluated in order to determine the reversibility of inhibition of the test compound. A study was also conducted to determine the inhibition mode of the most potent test compound, by constructing a set of Lineweaver Burk plots. Results The results showed that the 8-[(phenylsulfanyl)methyl]caffeine analogues were inhibitors of MAO-A and –B. The most potent inhibitor in the first series (1a–i) of this study were 8-[(3- bromophenylsulfanyl)methyl]caffeine and 8-[(4-bromophenylsulfanyl)methyl]caffeine with IC50 values of 4.90 and 4.05 µM, respectively. When these results were compared to those of the previously studied 8-(phenoxymethyl)caffeine derivatives it was found that, for these compounds, the bromine substituted homologues were also the most potent MAO-B inhibitors. The bromine substituted 8-(phenoxymethyl)caffeine derivatives exhibited IC50 values of 0.148 and 0.189 µM for those homologues containing bromine on the meta and para positions of the phenoxy side chain, respectively. In general, the 8- [(phenylsulfanyl)methyl]caffeine derivatives were found to be less potent MAO-B inhibitors than the 8-(phenoxymethyl)caffeine derivatives. The 8-[(phenylsulfanyl)methyl]caffeine derivatives also did not show as high a degree of selectivity for MAO-B (compared to MAO- A) as did the 8-(phenoxymethyl)caffeines. Similar to the 8-(phenoxymethyl)caffeines, the 8- [(phenylsulfanyl)methyl]caffeines also proved to be weak MAO-A inhibitors. The most potent inhibitor of MAO-A among the test compounds exhibited an IC50 value of 19.4 µM. The most potent MAO-A inhibitor among the previously studied 8-(phenoxymethyl)caffeines was more potent with an IC50 value of 4.59 µM. From these results it may be concluded that the phenoxy side chain is more suited for the design of caffeine derived MAO inhibitors than the phenylsulfanyl side chain. The results for the second series investigated in this study, the 8-[(phenylsulfanyl)ethyl]caffeines (2a–e), revealed the chlorine substituted derivatives to be the most potent MAO-B inhibitors. The meta and para chlorine substituted derivatives exhibited IC50 values of 5.67 and 7.79 µM, respectively, for the inhibition of MAO-B. Interestingly, the meta substituted derivative exhibited no inhibition toward the MAO-A isoenzyme. However, the 8-[(phenylsulfanyl)ethyl]caffeine derivatives were found to be very weak inhibitors of both MAO-A and –B and may be considered as less potent than the 8-[(phenylsulfanyl)methyl]caffeine derivatives. Since one of the aims of this study was to synthesise reversible MAO inhibitors, a time- dependency study was carried out with the best inhibitor (1i). The aim of this study was to determine the reversibility of inhibition by the 8-[(phenylsulfanyl)methyl]caffeine derivatives. From the results, it was concluded that the inhibition of MAO-B by compound 1i is reversible. To determine the mode of inhibition, a set of Lineweaver-Burk plots was constructed and since the plots were linear and intersected on the y-axis, it was concluded that 1i is a competitive inhibitor of MAO-B. Conclusion This study concludes that the phenoxymethyl side-chain is more suited for the design of caffeine derived MAO-B inhibitors than the (phenylsulfanyl)methyl side-chain. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Purpose: Monoamine oxidase (MAO) plays a key role in the treatment of Parkinson‟s disease (PD), since it is the major enzyme responsible for the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO-B may conserve dopamine in the brain and provide symptomatic relief. The MAO-B inhibitors that are currently used for the treatment of PD, are associated with a variety of adverse effects (psychotoxic and cardiovascular effects) along with additional disadvantages such as irreversible inhibition of the enzyme. Irreversible inhibition may be considered a disadvantage, since following treatment with irreversible inhibitors, the rate by which the enzyme activity is recovered may be variable and may require several weeks. In contrast, following the administration of reversible inhibitors, enzyme activity is recovered when the inhibitor is cleared from the tissues. There exists therefore, a need to develop new reversible inhibitors of MAO-B which are considered to be safer than irreversible MAO-B inhibitors. Rationale: Recently discovered reversible MAO-B inhibitors include safinamide and (E)-8-(3-chlorostyryl)caffeine (CSC). Safinamide has a benzyloxy side chain, which is thought to be important for inhibition of MAO-B. CSC, on the other hand, consists of a caffeine moiety with a styryl substituent at C-8, which is also a critical feature for its inhibitory activity. In a previous study, the caffeine ring and the benzyloxy side chain were combined to produce a series of 8-benzyloxycaffeine analogues which proved to be potent new MAO-B inhibitors. In this study, caffeine was substituted with the phenoxymethyl functional group at C-8, instead of the benzyloxy moiety. The aim of this study was therefore to compare the MAO-B inhibition potencies of selected 8-(phenoxymethyl)caffeine analogues with the previously studied 8-benzyloxycaffeine analogues. In the current study, 8-(phenoxymethyl)caffeine (1) and nine 8-(phenoxymethyl)caffeine analogues (2-10) were synthesized and evaluated as inhibitors of recombinant human MAOA and –B. These analogues only differed in substitution on C3 and C4 of the phenoxymethyl phenyl ring. The substituents that were selected were halogens (Cl, F, and Br), the methyl group, the methoxy group and the trifluoromethyl group. These substituents are similar to those selected in a previous study where 8-benzyloxycaffeine analogues were evaluated as MAO inhibitors. This study therefore explores the effect that a variety of substituents on C3 and C4 of the phenoxymethyl phenyl ring will have on the MAO-A and –B inhibition potencies of 8-(phenoxymethyl)caffeine. Based on the results, additional 8-(phenoxymethyl)caffeine analogues with improved MAO-A and –B inhibition potencies will be proposed for investigation in future studies. Methods: The target, 8-(phenoxymethyl)caffeine, analogues were synthesized by reacting 1,3- dimethyl-5,6-diaminouracil with the appropriately substituted phenoxyacetic acid in the presence of a carbodiimide coupling agent. Ring closure was catalyzed in basic conditions and methylation of the resulting theophyline intermediates at C-7 was carried out with iodomethane. The structures and purities of all the target compounds were verified by NMR, MS and HPLC analysis. All of the 8-(phenoxymethyl)caffeine analogues were subsequently evaluated as MAO-A and –B inhibitors using the recombinant human enzymes. The inhibition potencies of the analogues were expressed as the IC50 values (concentration of the inhibitor that produces 50% inhibition). In addition, the time-dependency of inhibition of both MAO-A and –B was evaluated for two inhibitors in order to determine if these inhibitors interact reversibly or irreversibly with the MAO isozymes. A Hansch-type quantitative structure-activity relationship (QSAR) study was carried out in order to quantify the effect that different substituents on the phenyl ring of the 8-(phenoxymethyl)caffeine analogues have on MAO-B inhibition activity. Results: The results showed that among the test compounds, several analogues potently inhibited human MAO-B. The most potent inhibitor was 8-(3-bromophenoxymethyl)caffeine with an IC50 value of 0.148 μM toward human MAO-B. There were also inhibitors which displayed inhibition activities towards human MAO-A with IC50 values ranging from 4.59 μM to 34.0 μM. Compared to the 8-benzyloxycaffeine analogues, that were in general non-selective inhibitors, the 8-(phenoxymethyl)caffeine analogues, evaluated here, were selective for MAO-B. For example, 8-(3-bromophenoxymethyl)caffeine was found to be 141 fold more selective as an inhibitor of MAO-B than of MAO-A. Also, compared to the 8-benzyloxycaffeine analogues, the 8-(phenoxymethyl)caffeine analogues were slightly less potent MAO-B inhibitors. For example, 8-benzyloxycaffeine is reported to have an IC50 value of 1.77 μM for the inhibition of human MAO-B while 8-(phenoxymethyl)caffeine was found to have an IC50 value of 5.78 μM for the inhibition of human MAO-B. This study also shows that two selected analogues bind reversibly to MAO-A and –B, respectively, and that the mode of MAO-B inhibition is competitive for one representative compound. Qualitative inspection of the results revealed interesting structure-activity relationships. For the 8-(phenoxymethyl)caffeine analogues, bearing both the C3 and C4 substituents on the phenyl ring, the MAO-B activity significantly increases with halogen substitution. Furthermore, increased MAO-B inhibition was observed with increased electronegativity of the halogen substituent. To quantify these apparent relationships, a Hansch-type QSAR study was carried out. The results showed that the logarithm of the IC50 values (logIC50) correlated with Hansch lipophilicity (π) and the Swain-Lupton electronic (F) constants of the substituents at C-3 of the phenoxymethyl ring. The correlation exhibited an R2 value of 0.87 and a statistical F value of 13.6. From these results it may be concluded that electron-withdrawing substituents at C3 with a high degree of lipophilicity enhance MAO-B inhibition potency. These results are similar to those previously obtained for the series of 8-benzyloxycaffeine analogues. For this series, the MAO-B inhibition potencies correlated with the Hansch lipophilicity (π) and Hammett electronic (σ) constants of the substituents at C-3 of the benzyloxy ring. Similarly to the 8-(phenoxymethyl)caffeine analogues, electron-withdrawing substituents with a high degree of lipophilicity also enhance the MAO-B inhibition potencies of 8-benzyloxycaffeine analogues. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011

Dopamine (DA) neurons of the substantia nigra pars compacta (SNc) have a key role in regulation of voluntary movement control. Their death is a hallmark of Parkinson’s disease, characterised by inhibited motor control, including muscle rigidity and tremor. Excitatory input to SNc-DA neurons is primarily from the subthalamic nucleus, and in PD these afferents display a higher frequency firing, as well as increased burst firing, which could cause increased excitatory activity in SNc-DA neurons. NMDA receptors (NMDARs) bind the excitatory neurotransmitter glutamate, and are essential for learning and memory. In SNc-DA neurons, NMDARs have a putative triheteromeric subunit arrangement of GluN1 plus GluN2B and/or GluN2D. Wild type (WT) mice, and those lacking the gene for GluN2D (Grin2D-null), were used to explore its role in various aspects of DA neuronal function and dysfunction using patch-clamp electrophysiology, viability assaying, and immunofluorescence. Pharmacological intervention using subunit-specific inhibitors ifenprodil and DQP-1105 on elicited NMDAR-EPSCs suggested a developmental shift from primarily GluN2B to GluN2B/D. Activity dependent regulation was assessed by high frequency burst stimulation of glutamatergic afferents: in comparison to controls, significant downregulation of NMDARs was observed in SNc-DA neurons, though no differences were observed based on genotype. This regulatory function may be a neuroprotective or homeostatic response. Ambient extracellular glutamate elicits tonic NMDAR activity in SNc-DA neurons, which may be important for maintaining basal levels of excitability: the role of GluN2D was assessed by recording the deflection in baseline current caused by application of competitive NMDAR antagonist D-AP5. There was a significantly larger NMDAR-mediated current in WT vs Grin2D-null mice, indicating that GluN2D has a role in binding ambient glutamate. Dysfunction of glutamate uptake could be a secondary pathophysiological occurrence in the SNc, leading to increased ambient glutamate: the effect of this was explored by application of the competitive glutamate transporter blocker TBOA. Here, the NMDAR-mediated portion of this current was significantly higher in WT mice in comparison to Grin2D-null. Interestingly, dose-response data obtained from bath application of NMDA showed significantly larger currents in Grin2D-null animals vs WT, but only at the top of the response curve (~1-10 mM), which may indicate a capability for larger conductance in Grin2D-null animals at high NMDAR saturation due to replacement of GluN2D with GluN2B. GluN2D may therefore be neuroprotective, by attenuating peak current flow in response to very high agonist concentrations. Lastly, GluN2D has been found to decrease NMDAR open probability under hypoxic conditions, potentially conferring resistance to hypoxia / ischemia related excitotoxicity. Therefore, low (15% O2 / 80% N2 / 5% CO2) vs high (95% O2 / 5% CO2) oxygen conditions were used along with immunofluorescent propidium iodide cell death assaying and immunofluorescent labeling for DA neurons in order to compare levels of DA neuronal death in the SNc based on oxygen status and genotype. Whilst there was a significant submaximal effect based on O2 status, genotype did not confer a practical resistance under these conditions. In summary, NMDARs have diverse roles in SNc-DA neurons which may both serve to maintain normal function and protect the cell against potentially pathological conditions.

Numa primeira etapa, estudamos as conexões da VTA para o complexo VTA-substância negra (SN) utilizando a leucoaglutinina do Phaseolus vulgaris (PHA-L). Estas conexões são substanciais, topograficamente organizadas, com destaque para o pólo caudal da VTA que inerva bilateralmente toda a extensão deste complexo. Numa segunda etapa, estudamos as projeções da VTA para o hipotálamo. A VTA se projeta principalmente para a área pré-óptica lateral e área hipotalâmica lateral, a região subfornical posterior e o núcleo dorsomedial. Foram vistas poucas aposições entre varicosidades PHA-L+ e neurônios imunorreativos para orexina ou para hormônio concentrador de melanina. Por fim, estudamos a colocalização do substrato do receptor de insulina (IRS-1), IRS-1 fosforilado e fosfatidilinositol-3 quinase (PI3K) com tirosina hidroxilase (TH) ou com a subunidade B da toxina colérica (CTb) injetada no estriado. A maioria dos neurônios TH+ da VTA-SN expressa IRS-1; injeções de CTb no estriado resultaram em células duplamente marcadas para CTb/IRS-1, CTb/PI3K e CTb/IRS-1 fosforilado. / In a first step, we studied the connections of the VTA to the complex VTA-substantia nigra (SN) using the Phaseolus vulgaris leucoagglutinin (PHA-L). These connections are substantial, topographically organized, especially the caudal pole of the VTA, which innervates bilaterally throughout the length of this complex. In a second step, we studied the projections of the VTA to the hypothalamus. The VTA projected mainly to the lateral preoptic area, lateral hypothalamic area, posterior subfornical region and dorsomedial nucleus. Were observed few appositions between PHA-L+ varicosities and neurons immunoreactive for orexin or melanin-concentrating hormone. Finally, we studied the co-localization of the insulin receptor substrate-1 (IRS-1), IRS-1-phosphorylated and phosphatidylinositol-3 kinase (PI3K) with tyrosine hydroxylase (TH) or cholera toxin B subunit (CTb) injected into the striatum. Most TH+ neurons of the VTA-SN expressed IRS-1; CTb injections in the striatum resulted in cells double-labeled for CTb/IRS-1, CTb/PI3K and CTb/IRS-1 phosphorylated.

Le récepteur aux cannabinoïdes de type 1 (CB1) est un récepteur couplé aux protéines G, abondamment exprimé dans le cerveau et régulant plusieurs processus physiologiques. Cependant, les mécanismes cellulaires par lesquels les CB1 régulent ces processus n’ont été que peu analysés. Bien que les CB1 localisés dans les membranes plasmiques sont connus pour induire la transduction de signal; une partie de ces récepteurs sont aussi fonctionnels au niveau des mitochondries (mtCB1), où leur stimulation réduit la respiration mitochondriale. L’objectif de cette thèse fut d’évaluer l’impact de l’activation des récepteurs mtCB1 du cerveau sur les effets connus des cannabinoïdes. Afin de distinguer la fonction des mtCB1 de celle des autres populations de récepteurs, nous avons développé des outils basés sur la signalisation induite par les mtCB1. Dans les mitochondries isolées de cerveau, l’activation des protéines Gαi/o, dépendante des mtCB1 diminue l’activité de l’adénylyl cyclase soluble (sAC). L'inhibition locale de l’activité de sAC prévient l’amnésie, la catalepsie et partiellement l’hypolocomotion induite par les cannabinoïdes. De plus, nous avons généré une protéine fonctionnelle mutante CB1 (DN22-CB1) dépourvue des 22 premiers acides aminés des CB1 ainsi que de sa localisation mitochondriale. Contrairement aux CB1, l'activation des DN22-CB1 n’affecte pas l'activité mitochondriale. Enfin, l’expression des DN22-CB1 dans l’hippocampe bloque à la fois la diminution de la transmission synaptique et l’amnésie induites par les cannabinoïdes. Ces travaux démontrent l’implication des mtCB1 dans certains effets des cannabinoïdes et le rôle clé des processus bioénergétiques contrôlant les fonctions cérébrales. / Type-1 cannabinoid receptor CB1 is a G protein-coupled receptor (GPCR), widely expressed in the brain, which regulates numerous physiological processes. However, the cellular mechanisms of CB1-mediated control of these functions are poorly understood. Although CB1 are known to signal at the plasma membrane, a portion of these receptors are also present in mitochondria (mtCB1), where mtCB1 activation decreases mitochondrial activity. The goal of this thesis was to dissect the impact of brain mtCB1 signaling in known behavioral effects induced by cannabinoids. To distinguish the functions of mtCB1 from other receptor pools, we developed tools based on the characterization of the intra-mitochondrial molecular cascade induced by mtCB1 receptors. In isolated brain mitochondria, we found that intra-mitochondrial decrease of soluble-adenylyl cyclase (sAC) activity links mtCB1- dependent activation of Gαi/o proteins to decrease cellular respiration. Local brain inhibition of sAC activity blocks cannabinoid-induced amnesia, catalepsy and contributes to the hypolocomotor effect of cannabinoids. In addition, we generated a functional mutant CB1 protein (DN22-CB1) lacking the first 22 amino acid of CB1 and its mitochondrial localization. Differently from CB1, activation of DN22-CB1 does not affect mitochondrial activity. Hippocampal in vivo expression of DN22-CB1 abolished both cannabinoid-induced impairment of synaptic transmission and amnesia in mice. Together, these studies couple mitochondrial activity to behavioral performances. The involvement of mtCB1 in the effects of cannabinoids on memory and motor control highlights the key role of bioenergetic processes as regulators of brain functions.

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