Tuberous sclerosis complex (TSC) is a multisystem genetic disease that is caused by a germ line mutation in the genes TSC1 and TSC2. Patients with the disease tend to suffer from benign tumors of the brain, heart, kidneys, skin, and other organs that contain giant cells. Although mTORC1 inhibitors (rapamycin and rapalogs) are often used to treat TSC because of their efficacy in promoting tumor shrinkage, clinical studies in the past have shown that when treatment is taken away, the tumor size returns to its original state. The objective of this study was to identify compounds that selectively inhibit proliferation of TSC2-deficient cells. A high-throughput screen of about 4000 compounds was performed using a lysosomal inhibitor (chloroquine [CQ], 5 μM) and a “repurposing” library of compounds. Through some yet to be determined mechanism, the combination of ritanserin (a selective serotonin reuptake inhibitor [SSRI]) and chloroquine was found to synergize to selectively inhibit the cell viability of DJK MEFs and TSC2-/-KO cells (TFFs) starting at 48 hours after treatment. The effects of this combination treatment were confirmed in a second cell line (TFFs) exhibiting similar reduction in proliferation. Interestingly, treatment with CQ (5 μM) and ritanserin (20 μM) showed synergistic action (combination index [CI] = 0.6) against TSC2-deficient cells. This combination treatment induced apoptosis (41%) in TSC2-deficient cells but not in TSC2-expressing cells. These results suggest a novel treatment approach in tuberous sclerosis complex and provide an incentive for further investigation of the mechanisms contributing to the vulnerability of TSC2-deficient cells. Moreover, the use of an already Food and Drug Administration (FDA)-approved compound can lead to a more rapid pharmacologic approach for TSC patients.

Addiction is a complex process in which behavioral sensitization may be an important component. While the behavioral effects of sensitization are well established, the intricate neurobiology of the phenomenon is still largely unknown. Dopamine systems mediate the induction of behavioral sensitization in adult rats, but there is a large amount of evidence showing that other neurotransmitter systems also modulate the induction process. For example, the α1b-adrenergic and 5-HT2A receptor systems are known to modulate the sensitized responding of adult rats, but the roles that these receptor systems play in the induction and expression of behavioral sensitization during the preweanling period has yet to be investigated. Therefore, the purpose of this thesis was to determine whether the serotonergic and adrenergic receptor systems mediate the induction and/or expression of cocaine-induced one-trial behavioral sensitization in preweanling rats. I used a novel approach to address this question, as the receptors of interest were “protected” from the alkylating effects of EEDQ (an irreversible nonselective receptor antagonist) by prior treatment with selective antagonist drugs. More specifically, rats were given ritanserin (a serotonergic receptor antagonist), prazosin (an adrenergic receptor antagonist), or a combination of the two drugs prior to an injection of EEDQ. To study the induction of behavioral sensitization, this series of injections was administered on PD 18 (24 h before the pretreatment injection of cocaine). To study the expression of behavioral sensitization, the injections were administered on PD 20, which was the day between the drug pretreatment day and the test day. In all experiments, the test day (i.e., the day on which the challenge dose of cocaine was given) was on PD 21. Control experiments were performed for both the induction and expression paradigms in order to determine whether prazosin and ritanserin independently affected sensitization. Results showed that the receptor inactivation caused by EEDQ blocked both the induction and expression of cocaine-induced one-trial behavioral sensitization. Importantly, administering prazosin and ritanserin did not protect the induction of the sensitized locomotor response, which suggests that serotonergic and adrenergic receptors do not mediate cocaine-induced one-trial behavioral sensitization in preweanling rats. This conclusion should be tempered, however, because co-administration of prazosin and ritanserin affected the locomotor activity and sensitized responding of cocaine-treated rats independent of the actions of EEDQ. Considering both past and present results, the most harmonious conclusion is that multiple receptor systems (i.e., dopaminergic, serotonergic, adrenergic, etc.) work in unison to produce the complex phenomenon of behavioral sensitization.

Depressive disorders are among the most frequent psychiatric diseases in the Western world with prevalence between 9% and 18%. Poor compliance and inappropriate antidepressant discontinuation invokes long-term morbidity, and appear linked to hippocampal shrinkage. Despite major advances in pharmacological treatment of the illness over the past 3040 years, currently available agents have distinct shortfalls both in clinical efficacy and in maintenance of response. This implies a greater long-term morbidity with significant impact on the patient, the patient's family as well as economic implications to health care managers and providers. The major reason for this state of affairs is our poor understanding of the neurobiology of depression and hence, of antidepressant (AD) action. AD drugs are thus not addressing the crucial neurobiological target underlying the illness, and new strategies and treatments are urgently needed. In recent years, depression has been associated with disturbances in excitotoxic glutamatergic activity, yet this has not been systematically evaluated. While the role of neurotransmitters such as serotonin, noradrenaline and dopamine has been extensively studied, new evidence suggests a role for the unique neurotransmitter nitric oxide (NO). Nitric oxide (NO), is activated by glutamatergic systems in various limbic and other regions of the brain, and has recently also been implicated in anxiety and affective disorders. Of special interest is the putative role of NO in cellular memory, synaptic plasticity and cell survival, all-important processes in the neuropathology and neurodevelopment of depression. Recent clinical studies have provided evidence of the role of the NO-pathway in depression, while preclinical studies have demonstrated the anxiolytic and antidepressant actions of nitric oxide synthase (NOS)-inhibitors. Moreover, NO interacts with other classical transmitters that have a regulatory role on mood, particularly the monoamines, as well as glutamate and gammaaminobutyric acid (GABA). In the current study the role of the NO-cGMP pathway in AD action was investigated, after chronic imipramine (IMI) and after IMI withdrawal, using a learned helplessness paradigm. Behavioural changes, hippocampal NOS activity and cGMP accumulation was determined together with pharmacological manipulation of the NO-cGMP pathway. Chronic IMI, 15 mg/kg/day intraperitoneal (ip) administration induced a pronounced reduction in swim immobility time in the forced swim test (FST), with no effect on horizontal or vertical locomotor activity. These behavioural changes were accompanied by a significant reduction in NOS enzyme activity and cGMP accumulation. In order to confirm the involvement of the NO-cGMP pathway in the AD action of IMI, chronic (3 weeks) IMI treatment was followed by an acute withdrawal of 7 days. Acute withdrawal, after chronic IMI treatment, resulted in a significant increase in swim immobility time and an increase in NOS enzyme activity and cGMP levels. In fact, NOS activity was raised above that of control, not just higher than the effect of chronic IMI. In order to assess the possible role of the NMDA-NO-cGMP pathway in AD withdrawal, the NMDA receptor antagonist, memantine, and the NOS/guanylyl cyclase (GC) inhibitor, methylene blue (MB), were administered during the 7 day IMI withdrawal period. Memantine (5 mg/kg/d ip), during the 7 day IMI withdrawal period, significantly reversed the increase in immobility time evoked after IMI withdrawal. This was accompanied by a significant reduction in NOS enzyme activity and a tendency to decrease cGMP levels. This data confirms that the antidepressant action of IMI, as well as IMI withdrawal, is associated with actions on the NMDA-GIu-NO-cGMP pathway. Particularly. IMI withdrawal evokes an increase in glutamate activity that is responsible for NOS activation. During the 7 day IMI withdrawal period, MB (15 mg/kg/d ip) also significantly reversed the increased immobility time after IMI withdrawal and was accompanied by a tendency to decrease NOS enzyme activity and cGMP levels in the rat hippocampus, however statistical significance was not reached. Although not emphatic, this data implies a possible role of the NO-cGMP pathway in AD action and AD withdrawal. In order to determine whether the observed IMI withdrawal effects on the NO-cGMP pathway may occur through an initial destabilisation in the serotonergic system, the 5-HT2a/2c receptor antagonist, ritanserin (4 mg/kg/d ip), was administered during the IMI withdrawal period. These studies revealed that antidepressant withdrawal evokes an increase in 5-HT2-mediated activity, and that antidepressant-induced NOS activation after withdrawal has its origin in serotonergic hyperactivity. Clearly, this is supportive of a distinct relationship between the NO and serotonergic system in antidepressant response. On its own, ritanserin was found to increase NOS and cGMP levels, yet during IMI withdrawal this response was lost, suggesting that IMI withdrawal alters the response to a 5-HT2a/2c receptor antagonist, which may have major clinical implications. In conclusion, the AD action of IMI, as well as chronic IMI withdrawal, involves actions on the NO-cGMP pathway. Withdrawal of ADS is associated with a loss of AD efficacy together with an increase in release of NO and cGMP. The NMDA antagonist, memantine, and the NOS/GC inhibitor, MB, reversed these responses therefore suggesting that the NMDA-GIu-NO-cGMP pathway may be a new putative target in understanding the neurobiology of AD action. Finally, NOS activation following withdrawal suggest that inappropriate withdrawal during the treatment of depression may mediate neurodegenerative pathology observed in recurrent depression, possibly by severely increased hippocampal NOS activity which is toxic to neurons. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Depressive disorders are among the most frequent psychiatric diseases in the Western world with prevalence between 9% and 18%. Poor compliance and inappropriate antidepressant discontinuation invokes long-term morbidity, and appear linked to hippocampal shrinkage. Despite major advances in pharmacological treatment of the illness over the past 3040 years, currently available agents have distinct shortfalls both in clinical efficacy and in maintenance of response. This implies a greater long-term morbidity with significant impact on the patient, the patient's family as well as economic implications to health care managers and providers. The major reason for this state of affairs is our poor understanding of the neurobiology of depression and hence, of antidepressant (AD) action. AD drugs are thus not addressing the crucial neurobiological target underlying the illness, and new strategies and treatments are urgently needed. In recent years, depression has been associated with disturbances in excitotoxic glutamatergic activity, yet this has not been systematically evaluated. While the role of neurotransmitters such as serotonin, noradrenaline and dopamine has been extensively studied, new evidence suggests a role for the unique neurotransmitter nitric oxide (NO). Nitric oxide (NO), is activated by glutamatergic systems in various limbic and other regions of the brain, and has recently also been implicated in anxiety and affective disorders. Of special interest is the putative role of NO in cellular memory, synaptic plasticity and cell survival, all-important processes in the neuropathology and neurodevelopment of depression. Recent clinical studies have provided evidence of the role of the NO-pathway in depression, while preclinical studies have demonstrated the anxiolytic and antidepressant actions of nitric oxide synthase (NOS)-inhibitors. Moreover, NO interacts with other classical transmitters that have a regulatory role on mood, particularly the monoamines, as well as glutamate and gammaaminobutyric acid (GABA). In the current study the role of the NO-cGMP pathway in AD action was investigated, after chronic imipramine (IMI) and after IMI withdrawal, using a learned helplessness paradigm. Behavioural changes, hippocampal NOS activity and cGMP accumulation was determined together with pharmacological manipulation of the NO-cGMP pathway. Chronic IMI, 15 mg/kg/day intraperitoneal (ip) administration induced a pronounced reduction in swim immobility time in the forced swim test (FST), with no effect on horizontal or vertical locomotor activity. These behavioural changes were accompanied by a significant reduction in NOS enzyme activity and cGMP accumulation. In order to confirm the involvement of the NO-cGMP pathway in the AD action of IMI, chronic (3 weeks) IMI treatment was followed by an acute withdrawal of 7 days. Acute withdrawal, after chronic IMI treatment, resulted in a significant increase in swim immobility time and an increase in NOS enzyme activity and cGMP levels. In fact, NOS activity was raised above that of control, not just higher than the effect of chronic IMI. In order to assess the possible role of the NMDA-NO-cGMP pathway in AD withdrawal, the NMDA receptor antagonist, memantine, and the NOS/guanylyl cyclase (GC) inhibitor, methylene blue (MB), were administered during the 7 day IMI withdrawal period. Memantine (5 mg/kg/d ip), during the 7 day IMI withdrawal period, significantly reversed the increase in immobility time evoked after IMI withdrawal. This was accompanied by a significant reduction in NOS enzyme activity and a tendency to decrease cGMP levels. This data confirms that the antidepressant action of IMI, as well as IMI withdrawal, is associated with actions on the NMDA-GIu-NO-cGMP pathway. Particularly. IMI withdrawal evokes an increase in glutamate activity that is responsible for NOS activation. During the 7 day IMI withdrawal period, MB (15 mg/kg/d ip) also significantly reversed the increased immobility time after IMI withdrawal and was accompanied by a tendency to decrease NOS enzyme activity and cGMP levels in the rat hippocampus, however statistical significance was not reached. Although not emphatic, this data implies a possible role of the NO-cGMP pathway in AD action and AD withdrawal. In order to determine whether the observed IMI withdrawal effects on the NO-cGMP pathway may occur through an initial destabilisation in the serotonergic system, the 5-HT2a/2c receptor antagonist, ritanserin (4 mg/kg/d ip), was administered during the IMI withdrawal period. These studies revealed that antidepressant withdrawal evokes an increase in 5-HT2-mediated activity, and that antidepressant-induced NOS activation after withdrawal has its origin in serotonergic hyperactivity. Clearly, this is supportive of a distinct relationship between the NO and serotonergic system in antidepressant response. On its own, ritanserin was found to increase NOS and cGMP levels, yet during IMI withdrawal this response was lost, suggesting that IMI withdrawal alters the response to a 5-HT2a/2c receptor antagonist, which may have major clinical implications. In conclusion, the AD action of IMI, as well as chronic IMI withdrawal, involves actions on the NO-cGMP pathway. Withdrawal of ADS is associated with a loss of AD efficacy together with an increase in release of NO and cGMP. The NMDA antagonist, memantine, and the NOS/GC inhibitor, MB, reversed these responses therefore suggesting that the NMDA-GIu-NO-cGMP pathway may be a new putative target in understanding the neurobiology of AD action. Finally, NOS activation following withdrawal suggest that inappropriate withdrawal during the treatment of depression may mediate neurodegenerative pathology observed in recurrent depression, possibly by severely increased hippocampal NOS activity which is toxic to neurons. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.