Indiana University-Purdue University Indianapolis (IUPUI) / The serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.

Meperidine has been shown to have potent binding affinity for serotonin transporters (SERT) (Ki = 41 nM) and be an inhibitor of serotonin reuptake. Based upon these pharmacological results meperidine has been identified as a lead compound for the development of a novel class of serotonin-selective reuptake inhibitors (SSRIs). A variety of potent analogues of meperidine have been synthesized and evaluated in vitro as potential ligands for the serotonin transporter. Substitutions have been made on the aryl ring, the ester moiety and the piperidine nitrogen of meperidine. Potent analogues of the aryl substituted series that included 4-iodophenyl, 2-naphthyl, 3,4-dichlorophenyl and 4-biphenyl meperidine derivatives were synthesized and chosen for further optimization of the benzyl ester analogues. Benzyl ester analogues included 4-nitro, 4-methoxyl and 3,4-dichloro benzyl analogues and exhibited high potency for serotonin transporters and high selectivity over the dopamine transporter (DAT) and the norepinephrine transporter (NET). Also the N-demethylated analogues improve the binding affinity and selectivity for serotonin transporter. The analogue 4- (carboxymethoxybenzyl)-4-(4-iodophenyl) piperidine (69f), was found the most potent (Ki=0.6 nM) and selective ligand for serotonin transporter (DAT/SERT >4500; NET/SERT >4500) for the series and has been advanced to in vivo evaluation.

Alzheimer´s disease (AD) is an age-related, irreversible and progressive neurodegenerative pathology that deteriorates cognitive function including learning and memory. AD is characterised neuropathologically by the presence of neuritic plaques (Aβ), neurofibrillary tangles (NFTs), synaptic loss and neuronal death. AD affects specific brain regions involved in mnestic function such as the neocortex and the hippocampus. The dorsal (DR) and the median raphe (MR) nuclei give rise to serotonergic (5-HT) projections that innervate multiple brain regions including the cortex and the hippocampus, playing an important role in learning and memory processes. For a long time the degeneration of cholinergic (ACh) system was considered as the main neurochemical changes in AD brains, however, more recent studies highlight the involvement of other neurotransmitter systems including 5-HT. This thesis entitled “Altered serotonergic neurotransmission as a main player in the pathophysiology of Alzheimer’s disease: structural and ultrastructural studies in a triple transgenic mouse model of the disease” demonstrates that there exist specific alterations in the serotonergic projections of the hippocampus during the progression of AD using the triple transgenic (3xTg-AD) mouse model of the disease, which closely resemble human AD. Mr. Harun N. Noristani is submitting this thesis to the University of Manchester for the degree of PhD in the Faculty of Life Science. The results obtained in this thesis show for the first time a biphasic increase in serotonergic fibre sprouting in the 3xTg-AD mouse model of AD that occurs in parallel with evident intraneuronal/extracellular Aβ deposition in the hippocampus (Chapter 3). In addition, serotonergic fibre sprouting correlated with reduced perforated synapses in the hippocampus, suggesting a structural remodeling process to maintain hippocampal connectivity (Chapter 4). Increased 5-HT neurotransmission (via high dietary intake of tryptophan, 5-HT precursor) reduced intraneuronal Aβ accumulation in the hippocampus, suggesting a direct role of 5-HT neurotransmission in modifying AD neuropathology (Chapter 5). Given the protective role of increased 5-HT neurotransmission, treatment with 5-HT enhancing drugs may be beneficial in reducing the underlying pathology as well as improving the behavioural and cognitive abnormalities associated with AD. Nevertheless, the role of specific 5-HT receptors responsible for such neuro-protective effect of 5-HT in AD awaits further research.

In an effort to identify novel binding sites for cocaine and its analogs, we carried out binding studies with the high-affinity and selective ligand [125I]RTI-121 in rat frontal cortical tissue. Very low densities of binding sites were found. Saturation analysis revealed that the binding was to both high- and low-affinity sites. Pharmacological competition studies were carried out with inhibitors of the dopamine, norepinephrine, and serotonin transporters. The various transporter inhibitors inhibited the. binding of 15 pM [125I]RTI-121 in a biphasic fashion following a two-site binding model. The resultant data were complex and did not suggest a simple association with any single transporter. Correlational analysis supported the following hypothesis: [125I] RTI-121 binds to known transporters and not to novel sites; these include dopamine, norepinephrine, and serotonin transporters. Immunoprecipitation of transporters photoaffinity labeled with [125]RTI-82 and subsequent analysis of SDS-page gels revealed the presence of authentic dopamine transporters in these samples; displacement of the photoaffinity label occurred with a typical dopamine transporter pharmacology. These data are compatible with the binding properties of RTI- 121 and the presence of several known transporters in the tissue studied.

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The 5-HTT gene (SLC6A4) is regulated by a common polymorphism in the promoter region (5-HTTLPR), which has functional consequences. Two major alleles have been observed and shown to have differential transcriptional activity with the long (L) allele having greater gene expression than the short (S) allele. 5-HTTLPR appears to modulate depression, anxiety and personality traits such as neuroticism. Additionally, a significant influence of 5-HTTLPR genotype on amygdala reactivity in response to fearful stimuli has been reported. Moreover, 5-HTTLPR seems to impact on the role of stressful life events (SLEs) in the development of depression. An elevated risk of depression and suicidal behaviors has been found in carriers of at least one low expressing S allele who had experienced SLEs, suggesting a gene x environment interaction. However, a recent meta-analysis showed that several findings failed to replicate this finding. Since genetic polymorphisms of the dopaminergic and serotonergic neurotransmission interact at the molecular, analyses with another polymorphism of the dopaminergic system, the dopamine D4 receptor (DRD4) was included to consider these likely gene-gene interactions (epistasis). The aim of this series of studies was to investigate the role 5-HTTLPR and SLEs on the endocrine stress response in different age samples. While newborns have been examined by a heel prick, stress responses were provoked in children (8-12 yrs) and younger adults (19-31 yrs) and older adults (54-68 yrs.) with the Trier Social Stress Test (TSST). The Life History Calendar (LHC) and Life Events Questionnaire (LEQ) were used to acquire data on SLEs. While in newborns the S/S genotype showed a significantly higher acute endocrine stress response than L/L or S/L genotypes, no significant difference between genotype groups was found in children. In the younger adult sample, the genotype impacted on cortisol stress responsiveness was reversed. Adults carrying the more active L allele of the 5-HTTLPR polymorphism showed a significantly larger cortisol response to the TSST than individuals carrying at least one of the lower expressing S allele. In older adults, no significant difference between genotype groups was found. However, results point in the same direction with showing highest cortisol response in individuals with L/L genotype. These data suggest that the association between 5-HTTLPR and endocrine stress reactivity seems to alter across lifespan, more specific the effects of genotype turns around. In addition, a significant interaction effect of 5-HTTLPR and SLEs has been found in the sample of younger adults, i.e. that early SLE as well as a severe number SLEs across the entire lifespan seem to modulate the interaction between HPA axis activity and 5-HTTLPR genotype. Additionally, a DRD4 by 5-HTTLPR interaction emerged which point to independent and joint effects of these polymorphisms on stress responsivity with regard to the concept of genegene interaction.

Resting frontal electroencephalographic (EEG) asymmetry has been hypothesized to be a biological marker of clinical depression but may reflect an endophenotype specific to women. Frontal EEG asymmetry was assessed in individuals (22% male) with (n = 12) and without (n = 21) a DSM-IV diagnosis of lifetime Major Depressive Disorder (MDD) or Dysthmic Disorder on 4 occasions within a two-week period. Depressed women exhibited greater relative right frontal activity at rest than never-depressed women across occasions. In contrast, depressed men displayed greater relative left frontal activity than never-depressed men. The same participants engaged in a Passive Viewing Face task while undergoing functional magnetic resonance imaging (fMRI). The present study did not replicate previous findings which show a hyperactive hemodynamic response in the amygdalae among depressed individuals. Mixed linear models indicated a lifetime depression by biological sex by amygdala activation interaction. For never-depressed control participants, frontal asymmetry is unrelated to the level of emotion-related amygdalae activation, but for lifetime depression spectrum participants, in both men and women, relatively greater amygdalae activation to emotional faces is associated with less left frontal activity as compared to those with less amygdalae activation to emotional faces. Also, when activation to emotionally expressive faces was closer to the levels of activation observed in the neutral face condition, the predicted pattern of association between frontal EEG asymmetry and depression based on the above findings was disrupted in men, but preserved in women. When levels of activation to emotion faces was considerably lower than that to neutral faces, the pattern was generally preserved for men, but not for women. Preliminary tests were also conducted in an attempt to replicate previous reports that document a positive correlation between the risk allele of the serotonin transporter gene and amygdalae activation. The present study failed to replicate this pattern, perhaps on account of the relatively small sample size available when non-Caucasian participants were excluded from the analysis.

Synthetic cathinones represent threatening and high abuse-potential designer drugs. These are analogs of cathinone (the b-keto analog of amphetamine (AMPH)) a naturally occurring stimulant found in the plant Catha Edulis. Methcathinone (MCAT) was the first synthetic analog of cathinone to be identified in 1987 by Glennon and co-workers and it exerted its action primarily through the dopamine transporter (DAT). Most central stimulants exert their action via monomaine transporters by causing either the release (e.g. cathinone analogs such as MCAT) or by preventing the reuptake (e.g. cocaine) of the neurotransmitter dopamine (DA) thus increasing the extracellular synaptosomal concentration of this neurotransmitter. In 2010, a new class of designer cathinone-like drugs called ‘bath salts’, initially a combination of methylenedioxypyrovalerone (MDPV), methylone (methylenedioxymethcathione, MDMC) and mephedrone (MEPH), soared to popularity. It caused extremely detrimental side effects; it was exceedingly popular for its recreational use and posed a threat to public health. At the time, their mechanisms of action were unknown. Our group identified that MDPV produced actions distinct from other cathinone analogs (i.e., it was identified as the first cathinone-like compound to act as a reuptake inhibitor at the dopamine transporter (DAT)). These findings suggested that not all cathinone-like compounds act uniformly and this insinuated that unique structural features on the cathinone scaffold might contribute to different effects at the transporter level. The overall goal of this project was to study the mechanisms of action of synthetic cathinones (including ‘bath salts’) at the monoamine transporters. We investigated the contribution of each of various structural features on the cathinone scaffold (i.e, the terminal amine, a and b positions, and the phenyl ring). We also constructed homology models of the human dopamine and serotonin transporters (hDAT and hSERT respectively) to help explain differences in selectivity between the neurochemical and behavioral aspects of DAT and SERT. Overall we found that structural features contributed to similar or distinctive mechanisms of action and also contributed to selectivity at monoamine transporters. Our studies provide information that can be useful to drug and health regulatory agencies to help prevent, treat, or curb the future abuse of such drugs.

In the last few years, there has been continued concern about synthetic drug abuse in both the United States and worldwide. Small adjustments in drug compound structure often allow synthetic drug makers to manufacture a legal product that can produce the same highs as illegal counterparts. Unfortunately, this is happening faster than the government can outlaw the drug compounds, and a wide variety of synthetics are now appearing on the street. This study evaluated the effects on the human serotonin transporter of six different 4-para substituted methcathinone compounds. Using a Xenopus oocyte model, the efficacy of each MCAT analogue at hSERT was calculated by applying the Hill equation to the oocyte data. This study suggests that volume, size, and steric bulk of the compound may generally influence efficacy at hSERT in a direct manner, but that other factors, like lipophilicity, may also play an important role in potency at the transporter.