Rozprawy doktorskie na temat „NEUROPROTECTANTS”

Neurodegenerative diseases are a class of conditions that lead to progressive atrophy of different parts of the central nervous system (CNS). These diseases lead to devastating clinical outcomes to patients and give rise to an enormous socio-economical burden on society.1 One commonality among some of the most well-known neurodegenerative disorders, e.g. Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), is neuroinflammation.2-4 Neuroinflammation stems from interactions of the innate immune system with toxins and insults to the central nervous system. In the case of irremovable or chronic insults and toxins, this leads to chronic damaging inflammation that hastens neuronal degeneration and exacerbates disease pathology.5,6 Recently, inflammasomes of the innate immune system have been indicated in playing essential roles in the observed inflammatory responses. The most studied inflammasome is the nod-like receptor pyrin containing 3 (NLRP3) inflammasome.7–9 Recently our research group has successfully developed sulfonamide-based small molecule inhibitors of the NLRP3 inflammasome, such as JC-21 and JC-171, as potential therapeutics for AD and MS. Our studies established that JC-21 is a selective inhibitor of the NLRP3 inflammasome.10,11 Structural modifications led to the development of JC-171 with improved pharmacokinetic properties. More importantly, our studies demonstrated the in vivo activity of JC-171 to effectively ameliorate the experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.12 Our data also strongly suggested that inhibitors based on this chemical scaffold may directly target the NLRP3 inflammasome.10–12 In this dissertation, we conducted biophysical, biochemical, and modeling studies to further elucidate the mechanistic information of these compounds as inhibitors of the NLRP3 inflammasome. In order to conduct further mechanistic studies, the NLRP3 protein was produced via transfection of HEK 293 cells with a modified plasmid of full-length human NLRP3 protein.13 Furthermore, LC-MS studies were conducted to confirm the blood-brain barrier penetration (BBB) of JC-171. Our studies established that JC-171 directly binds to the NLRP3 protein. The results also suggested that JC-171 may bind to the NACHT domain of NLRP3 while in a site that is distinct from the ATP binding site. This notion is supported by the fact that our compounds do not interfere with the ATPase activity of NLRP3. Docking studies of JC-171 to the homology model of the NACHT domain of NLRP3 also supported this assertion by showing the interaction of JC-171 with residues that are not overlapping with the ATP binding pocket. BBB penetration studies in combination with LC-MS analysis confirmed that JC-171 shows better BBB penetration when compared to MCC950. Collectively, our results strongly support that our compounds function as NLRP3 inflammasome inhibitors by directly binding to the NLRP3 protein, a novel and distinct mechanism of action when compared to the known inhibitors that target the NLRP3 inflammasome pathway. These results strongly encourage further development of such inhibitors as potential therapeutics for neurodegenerative diseases.

The discovery of amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) mutations associated with hereditary Alzheimer’s disease (AD) was a fundamental step in the development of rodent models to investigate its underlying pathology. This project examined the effects of pre-treatment with photobiomodulation (PBM) or saffron on learning and memory of the APP/PS1 double transgenic mouse using the Intellicage system which allows fully automated continuous testing of behaviour in a home cage environment. Protocols covering exploration, place and place reversal learning, and drinking behaviours were used to characterize longitudinal behaviours. Transgenic mice were successful in learning and re-learning tasks and showed inconsistent effects from both saffron and PBM treatment which may have been influenced by group housing of transgenic and wild-type mice. In contrast when brains were examined by histological analysis at the conclusion of behavioural experiments, it was evident that both PBM and saffron treatment mitigated Aß deposition in the aging APP/PS1 mice. This correlated with reduced mitochondrial dysfunction and saffron pre-treatment also reduced oxidative damage. The mitigation of these three major pathological hallmarks of Alzheimer’s disease – amyloid-ß plaque pathology, oxidative stress and mitochondrial dysfunction, by PBM and saffron treatment reinforces their potential as viable neuroprotective treatments for chronic neurodegenerative conditions. This work emphasises the importance of behavioural testing of animal models in the assessment of treatments and that dietary saffron and PBM are effective, safe, easily administered and minimally invasive interventions to treat chronic neurodegenerative disease.

Progesterone has been shown to be neuroprotective in a number of central nervous system injury models, including cerebral ischaemia. There is still a lack of understanding behind progesterone’s neuropotective properties, and the purpose of this project is to clarify some of these issues. Osmotic mini-pump infusion was hypothesised to more effective in delivering progesterone to the target organ of the brain, when compared to a bolus intraperitoneal injection. Progesterone pharmacokinetic profiles were compared between different dosing regimes. Intraperitoneal progesterone injection had a short half-life in both plasma and brain, while osmotic mini-pumps delivered higher concentrations of progesterone in plasma and particularly in brain, over a longer period, which supports the hypothesis. It was hypothesised that progesterone will reduce NO production and cell death in in vitro. Progesterone reduced nitric oxide production after challenging microglia with LPS, which supports our hypothesis and the nuclear progesterone receptor was found not to have a major role in nitric oxide attenuation. Neither of the microglial cell lines, BV-2 and HAPI cells produced elevations in NO formation in ischaemic conditions. The in vitro oxygen and glucose deprived model of ischaemia, reduced viability in both microglial and neuronal cells. Also, high pharmacological concentrations of progesterone exacerbated ischaemic injury, which does not support the hypothesis of progesterone in reducing cell death. Progesterone administration, via osmotic mini-pump infusion, was hypothesised to have a better outcome compared to vehicle treatment. After the onset of experimental stroke, progesterone delivery via osmotic mini-pump with loading dose was found to be beneficial in terms of neurological deficit score in adult male mice, which supports the hypothesis. Also, we hypothesise that co-morbidity can affect the efficacy of progesterone treatment in outcomes. Aged animals have an increased sensitivity to experimentally induce stroke and did not display, in the outcomes measured, any benefit from progesterone treatment. NOD/ShiLtJ mice had severe symptoms, resulting in high mortality after surgery and are not recommended as a model of diabetes for experimental stroke. Hypertensive BPH/2 mice are a potential hypertensive model and had better functional outcomes after treatment with intraperitoneally administered progesterone, compared to non-treated hypertensive animals in our small preliminary study. This supports our hypothesis that co-morbidity can affect the efficacy of progesterone treatment in outcomes. The gold-standard for assessing intervention effects across studies within and between subgroups is to use meta-analysis based on individual animal data. We hypothesise meta-analysis would reveal progesterone to reduce lesion volume, but also discover other effects in different subgroups of animals. Progesterone significantly reduced lesion volume, it also appeared to increase the incidence of death following experimental stroke. Furthermore, this negative effect appears to be particularly apparent in young ovariectomised female animals. These findings support the hypothesis that progesterone reduces lesion volume and progesterone having other effects in different subgroups. This investigation has clarified some issues and expanded our understanding on the neuroprotective properties of progesterone. However, these findings indicate further investigation is still required before progesterone can be considered for use in clinical trials as a neuroprotectant in stroke.

Neurological disorders are the biggest concern globally and ageing contributes in worsening the disease scenarios. In AD or AD like diseases, there is abnormal accumulation of extracellular amyloid beta produced due to abnormal processing of the transmembrane amyloid precursor protein, by β and γ-secretases. It spreads in the cortical and limbic regions of the brain leading to neuronal toxicity, impairment in memory and neurological functions. Aβ deposition in the CNS is common in aging HIV patients. Neurotoxic protein Tat, results in increased Aβ in combination with drugs of abuse cocaine. We examined the role of Withaferin A, against Aβ induced neurotoxicity. Our in-vitro dose optimization study demonstrates that lower concentrations (0.5–2 μM) of WA significantly reduce the Aβ40, without inducing cytotoxicity in the APP plasmid transfected SH-SY5Y cells (SHAPP). We demonstrate that Aβ secretion is increased in the presence of Tat (50 ng/ml) and coc (0.1 μM), WA reduces the Tat and coc induced increase in Aβ40. Additionally, we studied the role of WA against NF-kB mediated neuroinflammation, and observed that WA inhibits the expression of NFkB2 and RELA transcription factors, which play a major role in the expression of inflammatory chemokines. Further, to address the issue of minimal drug bioavailability in the CNS, we developed the WA loaded liposomal nanoformulation (WA-LNF) and characterized its size (499+/-50nm), toxicity and drug binding efficacy (28%). Our in-vitro 3D BBB transmigration of WA-LNF demonstrated ~40% transmigration efficiency. Furthermore, it was imperative for us to understand the mechanism of action of WA, therefore we studied the molecular mechanism of interaction of WA with Aβ protein by in-silico molecular dynamics simulations. We demonstrated that WA binds to the middle region of Aβ protein and the amino acid motif involved were FAEDVGS highlighting the mid-region Aβ capture by WA. 3 Hydrogen bonds were formed between WA and the amino acids, ASN17, GLY15 and SER16. This study reports WA as a potent neuroprotectant against amyloid induced neurotoxicity. Our study may have an immense therapeutic potential to target Aβ in the CNS, in the ageing patients and/or PLWH and/or ageing drug abusers.

In the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS), protein misfolding and aggregation are involved in motor neuron death. Heat shock proteins (Hsp), which help refold misfolded proteins or target them to the proteasome for degradation, have been studied as a therapeutic target in ALS, with some success. At the same time, it has been shown that mitochondrial dysfunction is one of the earliest pathogenic events leading to motor neuron death in ALS. The mitochondria have their own Hsps, for protein folding in the matrix, an important example of which is Hsp60. Given the finding that increased expression of another mitochondrial Hsp, Hsp22, preserved motor function, reduced motor neuron death and increased resistance to oxidative stress in a Drosophila model of aging, it is possible that the upregulation of mitochondrial Hsp60 might have similar neuroprotective effects in ALS. The current study examined Hsp60 upregulation in a primary dissociated spinal cord culture model of familial ALS (fALS) due to mutation in the SOD1 gene. Increasing Hsp60 expression failed to improve motor neuron viability, to ameliorate SOD1 protein misfolding/aggregation, and to prevent mitochondrial rounding in motor neurons. Therefore, the upregulation of mitochondrial Hsp60 would not be an effective therapy for ALS.Meanwhile, dominantly-inherited mutations in the gene encoding for Hsp60 have been linked to hereditary spastic paraplegia SPG13 (HSPG13), a disease in which the axons of motor neurons degenerate. Disease mechanisms are not known; however, given the importance of mitochondria to axonal integrity, and the mitochondrial localization of Hsp60, mitochondrial dysfunction seems a likely candidate. Hsp60V72I, a mutation found in HSPG13 patients, was expressed in motor neurons in primary dissociated spinal cord culture to model potential mitochondrial abnormalities. Hsp60V72I expression did not reduce motor neuron viability, alter mitochondrial morphology, or impair mitochondria on functional measures, including membrane potential (ΔΨ), resistance to oxidative stress, or axonal transport. It is possible that Hsp60V72I causes no, or a weak, mitochondrial phenotype. However, the expression of a severe, ATPase-deficient mutant, Hsp60D423A, also failed to induce mitochondrial dysfunction in this culture model. Thus, these phenotypes might not manifest because of culture conditions or because they are masked by the high level of endogenous Hsp60.
Lors de la maladie neurodégénérative de la sclérose latérale amyotrophique (SLA), le mauvais repliement et l'agrégation des protéines ont une incidence sur la mort des motoneurones. Les protéines du choc thermique (Hsp), lesquelles aident les protéines au mauvais repliement à se replier de nouveau ou encore ciblent ces dernières au niveau du protéasome pour leur dégradation, ont été étudiées comme source de traitement possible pour la SLA, non sans réussite. Parallèlement, il a été démontré que le dysfonctionnement mitochondrial est un des événements pathogènes menant à la mort des motoneurones, les anomalies mitochondriales apparaissant avant qu'une agrégation ou un mauvais repliement des protéines significatif ne soit observé. Les mitochondries ont leurs propres Hsp pour les protéines se repliant dans la matrice, les Hsp60 étant l'exemple par excellence. Si on considère que l'expression intensifiée d'autres mitochondries comme les Hsp22 peut préserver la fonction motrice, empêcher la mort de motoneurones et augmenter la résistance au stress oxydatif dans un modèle vieillissant de Drosophila, il semble que la régulation positive des Hsp60 mitochondriales pourrait possiblement protéger les neurones contre la SLA. La présente étude avait pour but d'examiner la régulation positive des Hsp60 dans un modèle de moelle épinière dissociée atteinte de SLA familiale causée par une mutation du gène SOD1. L'intensification de l'expression des Hsp60 n'a pas contribué à préserver la viabilité des motoneurones, ni à rectifier le mauvais repliement ou l'agrégation des protéines SOD1, ni à prévenir l'arrondissement mitochondrial dans les motoneurones. Par conséquent, la régulation positive des Hsp60 mitochondriales n'est pas recommandée pour le traitement de la SLA.Entretemps, les mutations de l'encodage du gène pour les Hsp60 transmises selon le mode dominant ont été associées à la paraplégie spasmodique héréditaire SPG13 (HSPG13), une maladie au cours de laquelle la dégénérescence axonique cause la mort des motoneurones. On ne connait aucun autre mécanisme de mort cellulaire plus précis. Toutefois, étant donné la présence des anomalies mitochondriales dans grand nombre de maladies neurodégénératives, l'importance des mitochondries pour l'intégrité des axones et la localisation mitochondriale des Hsp60, le dysfonctionnement mitochondrial pourrait très bien constituer un tel mécanisme. Les Hsp60V72I, une mutation observée chez les patients atteints de HSPG13, ont pu être exprimées dans les motoneurones provenant de cultures de moelle épinière dissociée afin de servir de modèle pour de potentielles anomalies mitochondriales. L'expression des Hsp60V72I n'a pas mené à une baisse de viabilité chez les motoneurones, ni à une modification de la morphologie mitochondriale, ni à une détérioration des mesures fonctionnelles des mitochondries, incluant le potentiel de membrane (ΔΨ), la résistance au stress oxydatif et le transport axonal. Il se peut que les Hsp60V72I ne causent qu'un faible phénotype mitochondrial, ou encore qu'elles n'en causent aucun du tout. Cependant, l'expression d'un mutant à grave déficience en ATP-ase, soit les Hsp60D423A, n'est pas non plus parvenue à causer un dysfonctionnement mitochondrial dans ce modèle de culture. On ne sait toujours pas si l'absence de phénotype est révélatrice du rôle des Hsp60 dans les maladies touchant les motoneurones, ou si les résultats négatifs sont un artefact du modèle de culture, lequel était caractérisé par un niveau élevé d'expression endogène des Hsp60 de phénotype sauvage qui pourrait potentiellement masquer les effets de l'expression du mutant exogène Hsp60. Les études à venir devraient se pencher sur les effets mitochondriaux de l'expression des Hsp60V72I dans un contexte où le phénotype sauvage Hsp60 endogène est réduit, en utilisant par exemple les Hsp60 shRNA particulières aux souris.

博士
臺北醫學大學
藥學研究所
95
Neuronal injury occurs via various mechanisms, including the initial ischemic insult which in turn initiates a secondary injury cascade of metabolic and biochemical changes. These changes include production of oxygen free radicals with lipid peroxidation, release of excitatory amino acids such as glutamate, failure of energy metabolism, release of inflammatory mediators, disruption of calcium homeostasis, and potentially other unidentified mechanisms. Natural products have been used as medicinal agents for many years. Through a large number of chemical and pharmacological research works, numerous bioactive compounds have been found from Chinese medicinal plants and some of them have been used clinically. The present study was divided into several parts includes Chinese herbal medicine, the extracts of Taiwan folk medicine plant or Taiwan botany, and their active components. The overall goal in this study is to find out the potential natural resources of polyphenols from traditional Chinese Medicine, folk medical plants, and if they can protect neurotoxicity or excitotoxicity in primary cultured cortical neuron. Our experimental design is that polyphenols, specifically found the flavonoids and the medical plant extracts, and to examine their protective effects on the cortical neurons after exposure to different neurotoxic chemicals. In our results, we found four potential candidates with neuroprotective abilities. Baicalein prevents neurotoxicity induced by both glutamate and glucose deprivation via decreasing intracellular calcium. Tangeretin can against glutamate and glucose deprivation. Tangeretin may protect central neurons by increasing the calcium via a synapse activity-dependent manner and might serve as a GABA mimic modulator to protect neurons against overexcitation-related brain injury and neurodegeneration. In addition, in the crude extract of pre-germinated brown rice (PGBR) with high levels of the nutrient GABA and exert a benefit for the prevention on the neurodegenative diseases. The ethanol extract of PGBR exerted the remarkable neuroprotective against glutamate and arachidonic acid-induced neurotoxicity. The ethanol extract of dried flowers Osmanthus fragrans (OFE) contained a high amount of total flavonoid and polyphenol. The neuroprotective activity of OFE was investigated under different insults (glutamate, arachidonic acid, and 6-hydroxydopamine). This is the first demonstration of the neuroprotective, free radical scavenging and anti-oxidative effects of O. fragrans.

碩士
國防醫學院
海底醫學研究所
92
Methamphetamine (METH) is a addictive drug that can cause neurotoxicity in the brain. Nevertheless, the molecular and cellular mechanisms involved in this neurotoxic process remain to be clarified. In this thesis, we design studies to investigate the neurotoxic effects of METH on both dopamonergic (PC 12 cell line) and non-dopaminergic (primary cortical culture) neurons using in vitro culture technique. Our results have shown that METH caused cellular apoptosis both in PC12 cell and primary cortical culture. Herein, we show that METH-induced apoptosis is associated with reactive oxygen species (ROS) accumulation detected at 8th hour and decreases in mitochondrial membrane potential measured at 12 hour, and induce apoptosis after METH exposure for 24 hours. In primary cortical culture, METH can elicit activation of microglia at lower dose, while higher doses of METH lead to damage of microglia. Pretreatment with α-tocopherol (vitE) is found to reduce METH-induced ROS production and neuronal apoptosis in PC12 cells, and only at a higher dose of vitE can restore mitochondrial membrane potential. In primary cortical culture, vitE can reduce METH-induced neurotoxicity but can not inhibit the METH-induced activation of microglia. Pretreatment with LiCl reduces METH-induced ROS production and neuronal apoptosis in PC12 cells, but can not restore mitochondrial membrane potential at the same dose range. In primary cortical culture, LiCl neither reduce the METH neurotoxicity nor inhibit the activation of microglia after METH challenge. Pretreatment with docosahexaenoic acid (DHA) is found to reduce METH-induced ROS production and neuronal apoptosis in PC12 cells, and can restore mitochondrial membrane potential at a higher dose. In addition, DHA can inhibit the activation of microglia in primary cortical culture. In summary, our studies show that apoptotic process plays an important role in neuronal injury in both PC12 cell line and primary cortical culture after METH challenge. Despite both vitE and DHA are found to protect PC12 cell and primary cortical culture, it appears that different mechanisms are involved. LiCl is noted to protect PC12 cell, but can not protect primary cortical culture against METH-induced neurotoxicity.

Among several neurodegenerative disorders, Parkinson’s is one of the utmost widespread diseases, occur due to the degeneration of the dopaminergic neurons in the region of midbrain which is identified by motor signs like tremor & bradykinesia; non-motor features (like impaired vision, depression, sleeping disorders). Currently, there is no treatment available that can cure PD. However, there is an imbalance between signaling pathway in the basal ganglia circuit. The direct pathway is downregulated and mediated via dopamine D1-like receptors. In contrast, the indirect pathway is upregulated and this is mediated via dopamine D2-like receptors. Using dopamine D2-like receptors antagonists, PD symptoms can be ameliorated. Increased histaminergic signaling has been observed in PD. Herein, we want to analyze the interaction between approved antihistamine drugs with dopamine D2-like receptors. We curated a list of 58 approved antihistamine drugs to analyze their affinity with dopamine D2-like receptors as an inhibitor. Literature indicates a substantial correlation between the antihistamines and dopamine D2-like receptors. Thus, targeting these drugs might regulate the disrupted indirect pathway in the basal ganglia circuit. Through molecular docking analysis, we identified that bilastine, a highly potent and selective inhibitor of histamine H1 receptor shows high affinity with dopamine D2-like receptors and might inhibit the aberrant signaling cascade of indirect pathway and upregulated histaminergic signaling simultaneously. Further studies are required to determine the action.

Les dommages au nerf optique (neuropathie optique) peuvent entraîner la perte permanente de la vision ou la cécité causée par la mort des cellules ganglionnaires de la rétine (CGR). Nous avons identifié qu’une surproduction de l'anion superoxyde constitue un événement moléculaire critique précédant la mort cellulaire induite par des lésions. Récemment, Suarez-Moreira et al (JACS 131:15078, 2009) ont démontré que la vitamine B12 peut capter l’anion superoxyde aussi efficacement que l’enzyme superoxyde dismutase. La carence en vitamine B12 peut conduire à une neuropathie optique causée par des mécanismes inconnus. Nous avons étudié la relation entre la captation de superoxyde par la cyanocobalamine (forme de vitamine B12 la plus abondante) et ses propriétés neuroprotectrices dans les cellules neuronales. La cyanocobalamine aux concentrations de 10 μM et 100 μM a réduit le taux de production de superoxyde respectivement par 34% et 79% dans les essais sans-cellule. Dans les cellules RGC-5 traités avec la ménadione, les concentrations de cyanocobalamine supérieures à 10 nM ont diminué l’anion superoxyde à des valeurs similaires à celles traitées par PEG-SOD. La cyanocobalamine aux concentrations de 100 μM et 1 μM a réduit la mort des cellules RGC-5 exposées à la ménadione par 20% et 32%, respectivement. Chez les rats avec section du nerf optique unilatérale, une dose intravitréenne de 667 μM de cyanocobalamine a réduit le nombre de CGRs exposées au superoxyde. Cette dose a également augmenté le taux de survie des CGRs comparativement aux rats injectés avec la solution témoin. Ces données suggèrent que la vitamine B12 peut être un neuroprotecteur important, et sa carence nutritionnelle pourrait causer la mort de CGRs. La vitamine B12 pourrait aussi potentiellement être utilisée comme une thérapie pour ralentir la progression de la mort CGR chez les patients avec les neuropathies optiques caractérisés par une surproduction de superoxyde.
Damage to the optic nerve (optic neuropathy) can result in permanent loss of vision or blindness through retinal ganglion cell (RGC) death. Our prior work identified a burst of superoxide anion as a critical molecular event in RGCs prior to injury-induced cell death. Recently, Suarez-Moreira et al (JACS 131:15078, 2009) demonstrated that vitamin B12 scavenges superoxide as effectively as superoxide dismutase. Vitamin B12 deficiency can lead to optic neuropathy through unknown mechanisms. We investigated the relationship between superoxide scavenging by cyanocobalamin, the most abundant vitamin B12¬¬ vitamer, and its neuroprotective properties in neuronal cells. Cyanocobalamin at concentrations of 10 μM and 100 μM reduced the rate of superoxide generation by 34% and 79% in cell free assays, respectively. In menadione-treated RGC-5 cells, cyanocobalamin concentrations above 10 nM scavenged superoxide anion similar to those treated with pegylated-SOD. Cyanocobalamin at concentrations of 100 μM and 1 mM reduced RGC-5 cell death from menadione by 20% and 32%, respectively. In rats with unilateral optic nerve transection, a single intravitreal dose of 667 μM cyanocobalamin significantly reduced the number of RGCs with superoxide. This dose also increased RGC survival rate compared to rats injected with saline control. These data suggest that vitamin B¬¬12 may be an important neuroprotectant, which could cause death of RGCs when depleted in nutritional deficiency. Vitamin B12 could also potentially be used as a therapy to slow progression of RGC death in patients with optic neuropathies characterized by overproduction of superoxide.