The number of people living with Alzheimer’s Disease (AD) is projected to increase dramatically over the next few decades, making the search for treatments and tools to measure the progression of AD increasingly urgent. The antisaccade task, a hands- and language-free metric, may provide a functional index of the Dorsolateral Prefrontal Cortex (DLPFC), which is damaged in the later stages of AD. Patients with AD make significantly more antisaccade errors than controls, however, performance in mild AD has remained unexplored. We hypothesized that mild patients will make more errors than controls. Thirty AD patients and 31 age-match controls completed both laptop-based and clinical versions of the antisaccade task. Two thirds of patients with AD made significantly more errors and corrected less of their errors than age-matched controls. Our findings indicate that antisaccade impairments exist in mild AD, suggesting DLPFC pathology may be present earlier than suggested by previous studies.

The purpose of this study was to investigate a) PTTG expression in pituitary adenoma subtypes, b) the correlation between PTTG expression and clinico-pathological variables in patients with Cushing’s disease with ACTH secreting pituitary adenomas, and c) PTTG expression in brain tumour subtypes. PTTG expression was investigated in 89 pituitary and 88 brain tumours, and 54 ACTH adenomas of patients with Cushing’s disease. Our results show that PTTG is expressed in the cytoplasm of pituitary adenoma cells, and at higher levels in GH adenomas, with lower PTTG expression levels being exhibited by PRL adenomas. Significant differences were noted between PTTG expression in medically treated and non-treated GH adenomas. However, PTTG expression in ACTH adenomas did not correlate with clinico-pathological variables, making it an unsuitable prognostic indicator in such tumours. In brain neoplasms, PTTG was expressed at higher levels in malignant tumours, whereas in less aggressive tumours, PTTG expression was considerably lower.

The Notch receptor and its ligands constitute a ubiquitous signaling pathway found throughout all multicellular animal life. In addition to its highly conserved function in development, a growing body of evidence suggests Notch signaling has important roles to play in adult processes, including long-term memory (LTM) formation. Building on previous work showing a specific requirement for the Notch1 receptor in spatial memory in mice, I show here a similar requirement for the Notch ligand Jag1. Mice with mutations to Dll1 (another Notch ligand) and Lfng (a Notch regulatory protein) do not display such phenotypes. I propose a model in which signaling between Notch pathway components found in the adult mouse hippocampus (such as Notch1 and Jag1) is required for LTM encoding, with no requirement for pathway components not expressed in this tissue (such as Dll1 and Lfng).

The chemical synapse is an asymmetric structure consisting of presynaptic and postsynaptic terminals in direct apposition to each other. Synapses function to mediate the transmission of signals between neurons and their targets. The formation of synapses is a tightly regulated process requiring the interaction of many genes and molecular pathways. I am interested in identifying genes and signaling pathways that are required for proper synapse formation. Using the GABAergic neuromuscular junctions of C. elegans as a model system, I have identified fsn-1 (F-box synaptic protein), a gene required for the control of synaptic growth. fsn-1 mutants exhibit a synaptic defect characterized by both synaptic over differentiation and under differentiation. FSN-1 is an F-box protein with a SPRY (SPla and RYanodine receptor) domain that functions cell-autonomously in neurons to regulate synaptic growth. I have shown that it functions in an E3 ubiquitin ligase-like complex with the RING-H2 finger protein RPM-1 (Regulator of presynaptic morphology), SKR-1 and Cullin. The composition of this complex is similar to SCF (Skp1, Cullin, F-box) E3 ubiquitin ligases. We hypothesize that this complex controls synapse formation by down regulating synapse promoting factors through an ubiquitin mediated process. We have identified two receptor tyrosine kinases that genetically interact with fsn-1, the Anaplastic Lymphoma Kinase homolog SCD-2 (Suppressor of Constitutive Dauer) and the C. elegans insulin receptor DAF-2 (abnormal DAuer Formation). Loss of function mutations in scd-2 or daf-2 partially suppress the synaptic differentiation defects of fsn-1 mutants, suggesting that they participate in signaling pathways whose activities are normally inhibited by FSN-1 during synapse formation. Unlike FSN-1 that functions in GABAergic neurons, I found that SCD-2 and DAF-2 have cell non-autonomous functions at GABAergic neuromuscular junctions. SCD-2 is required in the nervous system in the RID interneuron where it could modulate synapse formation through ligands present on the motoneuron cell surface. The DAF-2/insulin pathway functions in postsynaptic muscle cells to regulate FSN-1 dependent presynaptic growth likely through a retrograde or feedback mechanism. I propose a model where FSN-1 regulates synapse formation by attenuating signals that converge upon the presynaptic terminal to stimulate or inhibit synaptic growth.

Neuroplastic changes in motor representations within the primary motor cortex (M1) have been described after peripheral manipulations and implicated in motor learning and adaptation processes. It is unclear whether dental manipulations, which may result in altered oral sensorimotor functions, are associated with analogous changes within face-M1. This project applied intracortical microstimulation (ICMS) and recordings of evoked muscle electromyographic (EMG) activity to test if changes occur in the ICMS-defined motor representations of tongue-protrusion (genioglossus, GG) and jaw-opening (anterior-digastric, AD) muscles within face-M1 and adjacent face primary somatosensory cortex (face-S1) following trimming or extraction of the rat’s right mandibular incisor, or a change in diet consistency. ICMS mapping was carried out in anaesthetised adult male rats. Consistent with previous findings, AD and GG had extensive motor representations showing considerable overlap in naïve and sham control rats. AD and GG motor representations were also found within face-S1. Left and right AD (LAD, RAD) had significantly larger representations with shorter onset latency of ICMS-evoked EMG responses within contralateral face-M1. A change in diet consistency for 2-3 weeks was not associated with significant changes in AD and GG motor representations within face-M1. Compared to control rats, iii incisor trimming out of occlusion for a period of 1 week resulted, 1 day later, in a significantly longer GG onset latency in ipsilateral than in contralateral face-M1; 1 week later, despite a regain of normal occlusion, GG and GG/AD overlapping representations were significantly larger and the centre of gravity (at AP 4.0) was significantly deeper in contralateral than in ipsilateral face-M1. Incisor extraction was associated, 1 week later, with significantly larger RAD and RAD/GG overlapping representations and a lateral shift of LAD and RAD centre of gravity. Extraction also induced significant changes in AD and GG motor representations within the contralateral face-S1. These novel findings indicate that face-M1 can undergo neuroplastic changes in association with intraoral manipulations and also suggest similar neuroplastic capabilities for face-S1 motor outputs. These findings contribute to our understanding of the role of face-M1 and face-S1 in sensorimotor adaptations to an altered oral state and provide the basis for several future studies.

A great deal of our current understanding about the biology of neurodegenerative diseases has come from studying the function of genes linked to inherited forms of these disorders. Work performed in animal models, including vertebrates as well as invertebrates, has been instrumental in deciphering the cellular, physiological and behavioural deficits arising from the expression of disease-causing genes. Using the fruit fly, Drosophila melanogaster, as a model we examined the normal and aberrant function of two genes linked to the onset of neurodegeneration in humans, presenilin and superoxide dismutase. Drosophila is an extremely versatile model and in many ways is ideal for studying the genetic basis of human disease. The high degree of genetic conservation coupled with low genetic redundancy make this model particularly well suited for studying the function of disease causing genes. We demonstrate a novel genetic,physical and physiological interaction between presenilin and calmodulin and describe how this interaction impacts a very early cellular defect associated with Alzheimer?s Disease, intracellular calcium dyshomeostasis. We also describe progressive locomotory deficits in flies expressing mutant alleles of the superoxide dismutase gene, which have been linked to the onset of familial amyotrophic lateral sclerosis. Collectively, our work demonstrates that Drosophila can be used to study the cellular, physiological and behavioural basis of human neurodegenerative diseases and may provide a model to identify novel therapeutic avenues for neurodegenerative diseases.

A challenge in the treatment of central nervous system (CNS) diseases is the transport of drug candidates into the brain. Inositol stereoisomers have show promise as therapeutic agents for CNS disorders. scyllo-Inositol was an effective prophylactic and therapeutic for Alzheimer’s disease (AD) in TgCRND8 mice, a model of AD. This suggests inositol stereoisomers have excellent CNS bioavailability. They enter the brain through inositol transporters, of which there are three: one hydrogen myo-inositol transporter (HMIT) and two sodium myo-inositol transporters (SMIT1, SMIT2). HYPOTHESIS: Given the high CNS bioavailability of inositol stereoisomers, it may be possible to use inositol transporters to shuttle other compounds into the CNS. OBJECTIVES: 1. To confirm the CNS bioavailability of the two main inositol stereoisomers, myo- and scyllo-inositol, in both TgCRND8 and wild-type mice. 2. To examine inositol transporter expression in the brains, as a function of time and disease pathology, in both groups. 3. To evaluate the flexibility of the inositol transporters for transporting compounds by determining the substrate structural features required for active transport. RESULTS: myo-Inositol and scyllo-inositol accumulated in the brain following oral administration. Disease pathology did not alter baseline inositol levels or uptake. Brain subregional transporter expression was unaltered as a function of age or disease pathology. In vitro cell culture experiments found HMIT inactive and therefore not a contender for drug transport. In contrast SMIT1 and SMIT2 were both active and competitive transport assays, revealed distinct criteria for active transport through each system. However, both were stringent in the substitutions to the structure of myo-inositol possible to maintain active transport. CONCLUSION: Active transport through the inositol transporters is very sensitive to changes in the structure of myo-inositol and only conservative changes are possible. Therefore, these transporters would not make effective shuttling systems for drug transport into the brain.

This thesis represents a culmination of work which seeks to examine the prelimbic and anterior cingulate cortex (ACC) during trace fear memory across sleep and wakefulness states. In order to accomplish this task, a technical platform needed to be developed. Accordingly, the first chapter demonstrates that fear behavior can recorded utilizing neck electromyography (EMG). The second chapter examines the role of the ACC in trace fear memory, discovering that many neurons have premotor activity related to freezing behavior. Additionally, auditory-evoked potentials in the ACC demonstrate learning curves which match learning curves of fear. We suggest that the ACC is involved in affective-motor integration. The third chapter examines how genetic enhancement of trace fear learning, with calcium/calmodulin-dependent protein kinase IV (CaMKIV) over-expressed mice, can influence electro-cortical potentials during wakefulness, learning and sleep. We found that CaMKIV potentiates electro-cortical brain waves during learning and sleep. In particular 4-7.5Hz rhythms were potentiated in CaMKIV over-expressed mice during learning, and are likely to be localized to regions of the prelimbic cortex. Taken together the results of this thesis demonstrate that the trace fear memory paradigm engages the ACC and prelimbic regions, as evidenced at the single cell and cortical field potential level, for sensory-affective and premotor functions related to anticipating painful stimulation. CaMKIV appears to be a protein which modulates learning and electro-cortical potentials and may be a potential target for sleep-dependent memory consolidation in the prefrontal cortex.

The molecular mechanisms that regulate survival of embryonic neural precursors are still relatively ill-defined. Here, we have asked whether the p53 family member p63 plays any role during this developmental window, focusing upon the embryonic cerebral cortex. We show that genetic knockdown of p63 either in culture or in the embryonic telencephalon causes apoptosis of cortical precursors and newly-born cortical neurons, and that this can be rescued by expression of ΔNp63, but not TAp63 isoforms. This cortical precursor apoptosis is the consequence of deregulated p53 activity, since both basal precursor apoptosis and that induced by loss of p63 are rescued by coincident genetic silencing of p53. Finally, we demonstrate that the third p53 family member, ΔNp73, does not regulate survival of cortical precursor cells, but that it collaborates with ΔNp63 to ensure the survival of newly-born cortical neurons. Thus, the balance of ΔNp63 versus p53 determines the life versus death of embryonic cortical precursors. To assess if p63 plays a similar role in the adult brain, we examined mice haploinsufficient in p63 (p63+/-); we observed that aging, but not young, mice show deficits in short term memory and vertical exploratory behaviour. These results establish a foundation upon which the role of p63 in aging can be further characterized. Given the importance of p73 in aging and in preventing neurodegeneration, we aimed to create a conditional p73 knock out mouse (floxed p73). This mouse will allow for lineage-specific characterization of p73 function and will circumvent the reduced life span displayed by 4 mice lacking p73 (p73-/-). The work presented in this thesis has advanced our knowledge on the role of the p53 family members (p53, p63, and p73) in regulating survival and death in the nervous system. This knowledge will be important to understand neuropathology and to design appropriate therapeutic interventions.

Inherited photoreceptor degenerations (IPDs) are the most common monogenic cause of blindness in humans. To discover genes that may influence the risk of death in IPDs, microarray studies were used, and ET-2 was identified as the most differentially expressed transcript. ET-2 mRNA was 32-fold (p< 0.004), 70-fold (p< 0.009) and 72-fold (p<0.0009) increased in the Rds+/- , Tg(RHO P347S) and Rd1-/- mouse models of IPD, respectively, and the ET-2 peptide was minimally three-fold upregulated in Rds+/- retinas. The increased ET-2 transcript was detected solely in the photoreceptors (PRs) of Rds+/- and Tg(RHO P347S) retinas, but not in wild-type (wt) retinas by in situ hybridization. To determine the biological role of ET-2 in IPDs, mouse IPD models were crossed to ET-2-null mice. At age 40 and 15 days, ET-2-/-; Tg(RHO P347S) and ET-2-/-;Rd1-/- retinas showed, respectively, a 41% (n=6;p<0.003) and 49% rescue of PR degeneration (n=5;p<0.007). Unexpectedly, however, the PRs in ET-2-/- Rd1-/-retinal explants were not rescued (n=6;p>0.05), suggesting that the rescue observed in vivo might be due to extraocular mechanisms. Additionally, the expression of ET-2 mRNA from an rAAV5-CBA-ET-2 vector in ET-2-/-; Rd1-/- retinas did not restore PR degeneration (n=6;p>0.05). A survey of the extraocular phenotypes of ET-2 null mice showed them to be hypoxic owing to aberrant lung development, with a loss of normal alveolarization of the lung. Erythropoietin (EPO) levels were 11-fold elevated in the serum of ET-2 null mice (n=7;p<0.05) and retinal vascular endothelial growth factor (VEGF) was increased 4-fold (n=4;p<0.05). To examine the role of hypoxia in PR degeneration and to exclude increased EPO levels as the sole factor accounting for the rescue of mutant PRs in ET-2-/-; Tg(RHO P347S) and ET-2-/-;Rd1-/- mice in vivo, the effect of hypoxia on PR death in Rd1-/- retinal explants was examined. Rd1-/- explants cultured in 6% O2 from PN10 to PN17 showed a 32% rescue of PR death (n=5;p<0.05). Although ET-2 may mediate PR death through a direct role in mutant PRs, the PR rescue observed in ET-2-/-; Tg(RHO P347S) and ET-2-/-;Rd1-/-retinas may also result from systemic hypoxia due to poor lung function in ET-2-/- animals.