Magister Pharmaceuticae - MPharm / The cascade of neurotoxic events involved in the pathogenesis of Alzheimer's disease may explain the inefficacy of currently available treatment based on acetylcholinesterase inhibitors (AChEI - donepezil, galantamine, rivastigmine) and N-methyl-D-aspartate (NMDA) antagonists (memantine). These drugs were designed based on the "one-moleculeone- target" paradigm and only address a single target. Conversely, the multi-target drug design strategy increasingly gains recognition. Based on the versatile biological activities of tacrine, trolox and β-carboline derivatives, the attention they have received as lead structures for the design of multifunctional drugs for the treatment of Alzheimer's disease, and the topology of the active site of AChE, we have designed tacrine-trolox and tacrine-tryptoline hybrids with various linker chain lengths. The aim with these hybrids was to provide additive or synergistic therapeutic effects that might help overcome the limitation of current anti Alzheimer's disease drugs. All synthesized compounds were designed from lead structures (tacrine, tryptoline and trolox) to obtain cholinesterase (ChE) multisite binders and multifunctional AD agents. The study was rationalized by docking all structures in the active site of TcAChE using Molecular Operating Environment (MOE) software before proceeding with the synthesis. ChE inhibition was assessed in a UV enzyme inhibition assay using Ellman's method. Antioxidant activities were assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH.) absorbance assay. The hybrids containing the trolox moiety (compounds 8a-e) showed moderate to high AChE inhibitory activity in the nano to micro molar range (IC₅₀: 17.37 - 2200 nM), BuChE inhibition was observed in the same range (IC₅₀: 3.16 – 128.82 nM), and free radical scavenging activities in micro molar range (IC50: 11.48 – 49.23 µM). These are comparable or slightly higher than their reference compounds donepezil (AChE IC₅₀ = 220 nM), tacrine (BuChE IC₅₀: 14.12 nM), and trolox (DPPH IC₅₀: 17.57 µM). The hybrids with longer linker chain lengths, 6 and 8 carbons (8d and 8e), showed better ChE inhibitory activity than the shorter ones, 2, 3, and 4 carbons (8a-c respectively). This correlates well with literature. Free radical scavenging activities, however, seems not to be significantly affected by varying linker chain lengths. The hybrid compound (14) containing the tryptoline moiety linked with a 7 carbon spacer displayed the best AChE and BuChE inhibitory activity (IC₅₀ = 17.37 and 3.16 nM) but poor free radical scavenging activity. Novel anti-Alzheimer's disease drugs with multi-target neuroprotective activities were thus obtained and hybrid molecules that exhibit good ChE inhibition (8d, 8e and 14) and anti-oxidant (8d and 8e) activity were identified as suitable candidates for further investigation. / National Research Foundation (NRF)

Alzheimer's disease is a progressive neurodegenerative disorder, in which there is a marked decline in neurotransmitters, especially those of the cholinergic pathways. One of the approaches to the symptomatic treatment of Alzheimer's disease is the inhibition of the breakdown of the neurotransmitter acetylcholine, using an acetylcholinesterase inhibitor. One such drug tested, is the organophosphate, metrifonate. Any drug used for the treatment of neurodegenerative disorders should preferably not induce further neurological damage. Thus, in the present study, we investigated whether or not metrifonate is neuroprotective. The in vivo and in vitro effect of this drug on free radicals generation shows that metrifonate increases the level ofthese reactive species. Lipid peroxidation induced using quinolinic acid (QA) and iron (II) and show that metrifonate increased the peroxidative damage induced by using quinolinic acid. Metrifonate is also able to induce lipid peroxidation both in vivo and in vitro. This was reduced in vitro in the presence of melatonin. Using iron (II), in vi/ro, there was no significant difference in the level of lipid peroxidation in the presence of this drug. An investigation of the activity of the mitochondrial electron transport chain and complex I of the electron transport chain in the presence of metrifonate revealed that metrifonate reduces the activity of the electron transport chain at the level of complex I. The activity of the mitochondrial electron transport chain was restored in the presence of melatonin. Pineal organ culture showed that metrifonate does not increase melatonin production. Histological and apoptosis studies show that tissue necrosis and apoptosis respectively, occur in the presence of this agent, which is reduced in the presence of melatonin. Metal binding studies were performed USing ultraviolet spectroscopy, and electrochemical analysis to examine the interaction of metrifonate with iron (II) and iron (III). No shift in the peak was observed in the ultraviolet spectrum when iron (ll) was added to metrifonate. Electrochemical studies show that there may be a very weak or no ligand formed between the metal and drug. This study shows that while drugs such as metrifonate may be beneficial in restoring cognitive function in Alzheimer's disease, it could also have the potential to enhance neurodegeneration, thus worsening the condition, in the long term. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

The potent analgesic property of acetylsalicylic acid and acetaminophen makes these the most commonly used analgesics in the world. Easy accessibility and cost effectiveness of these agents are attractive to patients seeking pain relief. However, the abuse of nonnarcotic analgesics such as acetaminophen and acetylsalicylic acid by alcoholics and patients seeking to relieve dysphoric moods is well documented. These agents therefore impact on the brain neurotransmitter levels and therefore all processes involved in the synthesis and metabolism of neurotransmitters may be affected. The use of non-narcotic analgesics has been reported to reduce the incidence of neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). The mode of action by which acetylsalicylic acid and acetaminophen elicit neuroprotection is however unclear as many mechanisms of action have been inconclusively postulated. The first part of this study aims to elucidate the various mechanisms by which acetylsalicylic acid and acetaminophen affect the enzymes responsible for the catabolism of tryptophan, which is a precursor for the mood elevating neurotransmitter serotonin, as well as to investigate whether these agents alter the interplay between serotonin and pineal indole metabolism. The second part of this study focuses on the neuroprotective properties of acetylsalicylic acid and acetaminophen utilizing the neurotoxic metabolite of the kynurenine pathway, quinolinic acid and the potent Parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). The ability of acetylsalicylic acid and acetaminophen to alter TRP metabolism was determined by investigating the effects of these agents on the primary enzymes of the kynurenine pathway i.e. tryptophan 2, 3-dioxygenase and indoleamine 2,3-dioxygenase as well as to investigate whether these agents would have any effects on 3-hydroxyanthranilic acid oxygenase. 3-Hydroxyanthranilic acid oxygenase is the enzyme responsible for the synthesis of quinolinic acid. Acetylsalicylic acid and acetaminophen alter tryptophan metabolism by inhibiting tryptophan 2, 3-dioxygenase and indoleamine 2,3-dioxygenase thus increasing the availability of tryptophan for the production of serotonin. Acetylsalicylic acid and acetaminophen also inhibit 3-hydroxyanthranilic acid oxygenase thus implying that these agents could reduce quinolinic acid production. Acetaminophen administration in rats induces a rise in serotonin and norepinephrine in the forebrain. Acetylsalicylic acid curtails the acetaminophen-induced rise in brain norepinephrine levels as well as enhances serotonin metabolism, indicating that analgesic preparations containing both agents would be advantageous, as this would prevent acetaminophen-induced mood elevation. The results from the pineal indole metabolism study show that acetylsalicylic acid enhances pineal metabolism of serotonin whereas acetaminophen induces an increase in melatonin levels in the pineal gland. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders such as AD and PD. The second part of the study aims to elucidate and characterize the mechanism by which acetylsalicylic acid and acetaminophen afford neuroprotection. The hippocampus is an important region of the brain responsible for memory. Agents such as quinolinic acid that are known to induce stress in this area have detrimental effects and could lead to various types of dementia. The striatum is also a vulnerable region to oxidative stress and hence (MPP+), which is toxic for this particular region of the brain, was also used as a neurotoxin. The results show that ASA and acetaminophen alone and in combination, are potent superoxide anion scavengers. In addition, the results imply that these agents offer protection against oxidative stress and lipid peroxidation induced by several neurotoxins in rat brain particularly, the hippocampus and striatum. Histological studies, using Nissl staining and Acid fuchsin, show that acetylsalicylic acid and acetaminophen are able to protect hippocampal neurons against quinolinic acidinduced necrotic cell death. Immunohistochemical investigations show that QA induces apoptotic cell death in the hippocampus, which is inhibited by ASA and acetaminophen. In addition, ASA and acetaminophen inhibited MPP+ induced apoptotic cell death in the rat striatum. The study also sought to elucidate possible mechanisms by which ASA and acetaminophen exert neuroprotective effects in the presence of MPP+ as these agents are shown to prevent the MPP+-induced reduction in dopamine levels. The results show that acetylsalicylic acid and acetaminophen inhibit the action of this neurotoxin on the mitochondrial electron transport chain, a common source of free radicals in the cell. In addition, these agents were shown to block the neurotoxic effects of MPP+ on the enzymatic defence system of the brain i.e. superoxide dismutase, glutathione peroxidase and catalase. The reduction in glutathione levels induced by MPP+ is significantly inhibited by acetylsalicylic acid and acetaminophen. The results imply that these agents are capable of not only scavenging free radicals but also enhance the cell defence mechanism against toxicity in the presence of MPP+. These agents also block the MPP+-induced inhibition of dopamine uptake into the cell. This would therefore reduce auto-oxidation of dopamine thus implying another mechanism by which these agents exert a neuroprotective role in MPP+-induced neurotoxicity. The discovery of neuroprotective properties of acetylsalicylic acid and acetaminophen is important considering the high usage of these agents and the increased incidence in neurological disorders. The findings of this thesis point to the need for clinical studies to be conducted as the results show acetylsalicylic acid and acetaminophen to have a definite role to play as antioxidants. This study therefore provides novel information regarding the neuroprotective effects of these agents and favours the use of these agents in the treatment of neurodegenerative disorders, such as AD and PD, in which oxidative stress is implicated.

Understanding pathways of neurological disorders requires extensive research on both functional and structural characteristics of the brain. This dissertation introduced two interrelated research endeavors, describing (1) a novel integrated approach for constructing functional connectivity networks (FCNs) of brain using non-invasive scalp EEG recordings; and (2) a decision aid for estimating intracranial volume (ICV). The approach in (1) was developed to study the alterations of networks in patients with pediatric epilepsy. Results demonstrated the existence of statistically significant (p

Background: The rise in the total number of seniors will lead to a considerable increase in the prevalence of persons with dementia (PWD), the number of senior drivers and the amount of drivers with dementia. Understanding how this life event is experienced by PWD and their caregivers is paramount to policy development and planning. Methods: Descriptive qualitative study using secondary data. There were 25 participants over the age of 65, whose monthly phone call conversations were analyzed using analytic induction, to find links and create a theoretically based hypothesis regarding the experience of driving cessation. A standardized questionnaire was used to guide the telephone data collection. Results: PWD may experience emotions of anger when they don’t understand why they are no longer allowed to drive. When PWD understand why they are no longer allowed to drive, they seem to accept and self-regulate their driving cessation. Cognitive tests and physicians instructing PWD they have to cease driving, are not perceived to be helpful in this understanding. Public transportation and alternate means of personal transportation are potentially associated with the acceptance of driving cessation. Conclusions: PWD might not understand the link between cognitive assessments, their memory, and their driving performance. A systems level approach to improved access to transportation and on-road driving tests might make it easier for PWD to understand and accept the lifestyle changes that come with driving cessation.

The amyloid beta peptide (Aβ) plays an important role in Alzheimer’s disease (AD). Increasing evidence suggest that overactivation of extrasynaptic N-methyl-D-aspartate receptors (NMDARs) mediate Aβ-induced excitotoxicity. In serine racemase knockout (SRKO) mice with significantly depleted D-serine levels, Aβ-induced excitotoxicity is attenuated. Using SRKO mice, this thesis attempts to determine the effects of Aβ on synaptic and extrasynaptic NMDAR function, and how D-serine can alter these Aβ- mediated effects. In CA1 pyramidal neurons, Aβ significantly depresses evoked synaptic NMDAR excitatory postsynaptic currents (EPSCs), and this effect is even greater in SRKO mice. The same effect was also observed on isolated evoked extrasynaptic NMDAR currents. During synaptic NMDAR current recordings, Aβ potentiated the holding current in wild type (WT) mice, but not SRKO mice, suggesting an increase in extrasynaptic NMDAR activation in WT, but not in SRKO mice. SRKO mice attenuated Aβ-induced holding current shift and had reduced basal tonic NMDAR activation. These data, along with evidence from previous studies in the literature, suggest that low levels of D-serine can alter NMDAR function in the presence of Aβ. These findings provide insight for future experiments in exploring the importance of D-serine in AD.

This dissertation describes separate but related studies that explore the prospective memory abilities of older adults and individuals with Mild Cognitive Impairment/Early Alzheimer’s disease. Prospective memory (PM) refers to the type of memory utilized to execute planned actions in accordance with a specific event. PM is critical to maintaining functional independence in older adults, as it can refer to such basic acts as remembering to turn off a stove or taking one’s medication. Research suggests PM abilities decline within normal aging and to a greater extent in Mild Cognitive Impairment (MCI) and early Alzheimer’s Disease (AD). Together, the studies assessed and compared the PM abilities across healthy younger and older adults, individuals with MCI, and individuals with early AD while exploring two major theories that seek to explain PM retrieval. The preparatory attentional and memory process theory of PM (PAM) assumes that PM retrieval requires resource-demanding preparatory attentional processes, whereas the Dynamic Multiprocess theory (DMPT) assumes that retrieval can also occur spontaneously (Scullin, McDaniel, & Shelton, 2013; Smith & Bayen, 2006). Study 1 used a novel laboratory PM task in which the focality and the frequency of PM cues were manipulated to compare the PM abilities of cognitively healthy younger and older adults. The results revealed significant differences in the patterns of performance between the younger and older adults based on the focality and frequency of cues which indicated different attentional allocation strategies. Study 2 examined the impact of cognitive impairment on PM abilities by using the same paradigm to compare the performance of cognitively healthy older adults to individuals with MCI and early AD. The results again revealed significant differences in the patterns of performance which indicated that these groups may have used different strategies of attentional allocation depending on the focality and cue frequency. Taken together, the findings in Studies 1 and 2 were mixed with respect to the predictions of the DMPT and PAM. The MCI group, in particular, demonstrated a unique performance profile that suggests the neuropathophysiological changes associated with this diagnosis may lead to the reliance on different PM retrieval processes compared to healthy older adults. Finally, Study 3 explored the use of a more naturalistic and ecologically valid PM task to compare the PM performance of individuals with MCI and early AD to healthy older adults without cognitive impairment. The results showed that, after taking the learning and retrospective memory scores into account, the significant differences between groups in PM accuracy on this task can mostly be accounted for by these factors. Nevertheless, the AD group was found to display significantly lower PM accuracy with event-based cues with a weak association between cue and action compared to the older adult and MCI groups after controlling for these factors. These findings provide valuable theoretical, methodological, and clinical contributions which will be discussed.

Alzheimer's disease (AD) is an incurable disease characterized by a slow, progressive decline in cognitive functions as well as the presence of amyloid-beta (Aβ) plaques and neurofibrillary tangles. Interestingly, two thirds of AD patients are women who have a faster disease progression. Despite this clinical profile, sex differences in AD pathophysiology are largely ignored at the basic and clinical levels. Current therapies provide only mild to moderate improvement in patient symptoms. There is, therefore, an urgent need to expand our understanding of the underlying pathophysiology of AD, and to obtain alternative hypotheses and therapeutics. A recent and promising development involves the sigma-1 receptor (Sig1R), a protein regulated by steroid hormones, which has been implicated in AD. Most interestingly, Sig1R agonists have been shown to ameliorate cognitive deficits in an AD mouse model. Here, we investigated the role of Sig1Rs in an Aβ25-35-infusion mouse model of AD, using behavioural paradigms. Previous studies employing this model have demonstrated Aβ-induced impairments in learning and memory in young male rodents, while no work has been done on females. We examined cognitive function following Aβ25-35 infusion in wild-type and knock-out Sig1R adult male and female mice using the Morris water maze, spontaneous alternation in the Y-maze, and forced alternation in the Y-maze tasks. Overall, the data unexpectedly shows that genotype, Aβ25-35-treatment, and sex had no effect on cognitive functions. These results suggest that additional efforts are required to obtain a working Aβ25-35-infusion model in our Sig1R mice and behavioural tasks. Future experiments will hopefully shed some light on the link between Sig1Rs and AD, which could lead to the development of therapeutics and disease prevention.

Alzheimer’s disease (AD) is characterized by amyloid-β (Aβ) and tau pathologies, insulin resistance, neuro-inflammation and dysregulation of cholesterol homeostasis, all of which play a role in neuro-degeneration. The main aim of this study was to determine possible relationships between insulin signaling, cholesterol biosynthesis and their effects on Aβ, and inflammatory response in vitro. Insulin treatment increased cholesterol synthesis in human Neuroblastoma SH-SY5Y (SHY) and mouse neuroblastoma 2a (N2a) and N2a transfected with human APP (N2a-APP) by up-regulating biosynthesis enzymes including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3methyl-glutaryl-CoA reductase (HMGCR) through sterol regulatory element binding protein-2 (SREBP2) up-regulation. Aβ caused insulin resistance in N2a-APP cells by phosphorylating IRS-1 at Ser612, inhibiting signaling to downstream targets. Aβ1-42-treated SHY exhibited similar IRS-1 phosphorylation at Ser612 and inflammatory response of JNK activation. Aβ1-42 caused down-regulation of neuro-protective/anti-inflammatory DHCR24, and an increase in HMGCR levels indicating dysregulation of cholesterol homeostasis in SHY cells. Insulin resistance, Aβ toxicity, neuro-inflammation and dysregulation of cholesterol homeostasis appear to be intertwined processes in AD that should be studied simultaneously.

This thesis is devoted to Alzheimer's disease, which affects millions of people worldwide and the costs of this disease reaching almost $ 400 billion crowns. The first part is devoted to the definition of the disease, symptoms, symptoms and treatment. The second part focuses on the costs associated with this disease. Costs are identified and quantified by Cost of Illness analysis.