Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the growing population of elderly people. Although the etiology of the disease is yet to be fully elucidated, pathological hallmarks have been consistently described, including the accumulation of amyloid plaques, dysfunctional ionic homeostasis, synaptic disruption and neurodegeneration. The amyloid hypothesis postulates that aberrant production of amyloid-β (Aβ) proteins, which have a high propensity to aggregate, lies at the center of the pathological mechanism of AD. In particular, soluble oligomeric Aβ structures have been identified as primary toxic species. The interaction of these structures with several cellular targets, including ion channels such as nicotinic acetylcholine receptors (nAChR) and voltage operated Ca²⁺ channels (VOCC), has also been implicated in Aβ toxicity and AD. The aim of this thesis is to investigate how the acute and chronic actions of Aβ in vitro are affected by nicotinic ligands. Acute application of Aβ₁₋₄₂ to fluo-3-loaded PC12 cells potentiated Ca²₊ increases evoked by stimulation of nAChR and VOCC, while chronic application reduced redox potential, disrupted membrane integrity and initiated apoptosis in PC12 cells. In addition to mimicking the toxic responses of PC12 cells, Aβ₁₋₄₂ also reduced neurite outgrowth and synaptogenesis in rat primary cortical neurons. All actions of Aβ were prevented by inhibitors of Aβ₁₋₄₂ oligomerisation, including the hexapeptide KLVFFA. Neuroprotection afforded by (+)-nicotine also occurred via inhibition of Aβ₁₋₄₂ oligomerisation, rather than by a receptor-mediated mechanism. No other pharmacological approaches, including application of two novel ligands selective for α7 nAChR: the partial agonist SSR180711 and antagonist α-conotoxinArIB[V11L,V16D], characterized herein, protected against Aβ₁₋₄₂ toxicity. While inhibiting oligomerisation prevented the actions of Aβ₁₋₄₂, enhanced oligomerisation evoked amplified toxic responses. However, the potentiation of Ca²⁺ signalling diminished following enhanced oligomerisation. This, coupled with a lack of VOCC-involvement in Aβ toxicity and the differential actions of truncated Aβ peptides on toxicity and Ca²⁺ signaling, indicates that the acute disruption of Ca²⁺ signaling by Aβ does not underpin the chronic toxic effects of Aβ.

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) and is a core component of the neuromuscular junction, facilitating fast synaptic transmission leading to muscle contraction. Mutations to the human adult muscle nAChR lead to various forms of congenital myasthenic syndrome (CMS), a disease characterized by progressive fatigable muscle weakness. A central channel pore constriction formed by a ring of five leucine residues (L9’) forms part of the nAChR channel gate. CMS-causing mutations in the L9’ residues lead to a form of CMS that results in longer channel opening times and a delayed signal decay. To understand better how L9’ mutations in the human adult muscle nAChR influence channel function, I used two-electrode voltage clamp electrophysiology to perform a comprehensive mutant screen of all L9’ residues in each subunit of the human adult muscle nAChR. This resulted in a total of 76 unique mutations: 19 L9’ mutations consisting of every possible natural amino acid substitution in each subunit (α, β, ε, δ). The results of this screen show that while the polarity and size of a substituted residue contribute to its effect on channel function, increasing the polarity of the side chain typically has a more potentiating effect on channel function than does a change in size. The subunit in which the mutation is expressed also tailors the effect of a given mutation on channel function, with several δL9’ mutations producing qualitatively different effects than equivalent mutations in other subunits. Because the majority of L9’ mutations resulted in a gain-of-function, I originally postulated that interactions between L9’ and surrounding residues stabilize the resting state with the elimination of such interaction through mutations destabilizing the resting state to promote channel gating. Using a double mutant cycle, I explored interactions between the L9’ and adjacent non-L9’ residues but found that there are only weak or no interactions that contribute to channel function. Instead, my data support the hypothesis that the nAChR operates via a hydrophobic gating mechanism, and that adjacent L9’ residues are driven together by the hydrophobic effect to form a closed pore. L9’ mutations that either increase the polarity or decrease residue size likely reduce the hydrophobic driving forces that stabilizes the resting state, thus leading to an enhancement in channel function.

In an effort to search for a more selective, less toxic neuronal nicotinic acetylcholine receptor analgesic agent in comparison to epibatidine, a series of analogs with hybrid structures of epibatidine and ABT-594 were designed and synthesized. The 1-(pyridyloxymethyl)-7-azabicyclo[2.2.1]heptane ring systems were furnished via an intramolecular cyclization from a trans 1, 4 disubstitituted amino-cyclohexane derivative. The functionalized cyclohexane ring was formed via a [4+2] Diels-Alder cyclization reaction between the acetamidoacrylate and Danishefsky's diene. These 1- (pyridyloxymethyl)-7-azabicyclo[2.2.1]heptane ring systems were then tested in vitro as potential á4â2 nicotinic acetylcholine receptor ligands with high potency and selectivity. In addition, a series of rigid acetylcholine analogs were synthesized from cocaine to study the conformation of acetylcholine, the endogenous neurotransmitter at the nicotinic acetylcholine receptor. A stereoselective reduction of 2-tropinone led to the enantioselective synthesis of the desired acetoxytropane systems. These compounds were also tested in in vivo models for binding affinity and efficacy responses. Anabasamine, an alkaloid isolated from the Central Asian shrub, Anabasis aphylla, was synthesized for the first time. It was targeted due to interesting preliminary biological activity such as exhibiting anticholinesterase activity, anti-inflammatory activity, and facilitated an increase in hepatic alcohol dehydrogenase levels. Only preliminary studies were performed as anabasamine is limited in quantity due to its difficult isolation. A versatile synthetic methodology was developed for the synthesis of anabasamine and related nicotine analogs. This new methodology employed a pyridyl anion addition to valerolactone, for anabasamine, or butyrolactone for the nicotine analog, to afford 5-hydroxy-1-(6-methoxy-pyridin-3-yl)-pentan-1-one or 4-hydroxy-1-(6- methoxy-pyridin-3-yl)-butan-1-one, respectively. A reductive amination provided the piperidine ring moiety and a Suzuki coupling reaction introduced the bipyridyl moiety to anabasamine in five steps and 23% overall yield. In addition, this methodology was applied successfully to the synthesis of nicotine and other related analogs. In particular the synthesis of 6-methoxynicotine, a useful drug intermediate, was generated improving the yield from 16% over five steps to 54% over three steps.

No description available.

Alzheimers disease (AD) is pathologically characterized by amyloid plaques, neurofibrillary tangles, inflammation, and neurodegeneration. According to the amyloid hypothesis of AD, the central mediating event of the disease is the deposition of amyloid. The inflammation hypothesis of AD states that it is the inflammatory response to plaques and tangles, rather than the actual lesions, which causes the disease. Studies described here combine the two approaches into a single model. Four studies are presented using a basic protocol of intrahippocampal lipopolysaccharide (LPS) injection to stimulate inflammation in transgenic mice. The first study looked at alpha7 nicotinic receptors during the glial response to Abeta deposits and LPS. Reactive astrocytes which immunolabeled for alpha7 were co-localized with Congophilic deposits in APP and APP+PS1 mice, and increased after LPS injection. Unfortunately, LPS injection into alpha7 knock out mice revealed the alpha7 labeling to be nonspecific. The second study evaluated the time course of protein and gene expression after LPS injection into nontransgenic mice. This experiment identified both a transient and chronic microglial inflammatory response, with changes in cell morphology. The third study evaluated a similar time course in APP mice. Concurrent with the inflammatory response, transient reductions in Abeta burden were seen, though compact plaque load was unaffected. The fourth and final study used dexamethasone to inhibit LPS-induced inflammation in APP mice. LPS injection reduced Abeta burden, but was completely blocked by dexamethasone co-treatment. Though dexamethasone inhibited LPS-induced CD45 and complement receptor 3 levels (markers of general microglial activation), dexamethasone had no effect on scavenger receptor A or Fc gamma receptor II/III levels. An overall hypothesis regarding LPS mediated reductions in Abeta can be proposed: It is not the presence of the LPS molecule, nor the upregulation of receptors involved in phagocytosis, but rather general glial cell activation that mediates Abeta removal. Thus, a phagocytic cell must not only bind Abeta (by various receptors) but must also be capable of engulfing the material (via general cell activation). Taken together, these studies suggest that some level of inflammation in AD is beneficial and responsible for maintaining a balance between amyloid deposition and removal.

abstract: Patients with Alzheimer's disease (AD) exhibit a significantly higher incidence of unprovoked seizures compared to age-matched non-AD controls, and animal models of AD (i.e., transgenic human amyloid precursor protein, hAPP mice) display neural hyper-excitation and epileptic seizures. Hyperexcitation is particularly important because it contributes to the high incidence of epilepsy in AD patients as well as AD-related synaptic deficits and neurodegeneration. Given that there is significant amyloid-β (Aβ) accumulation and deposition in AD brain, Aβ exposure ultimately may be responsible for neural hyper-excitation in both AD patients and animal models. Emerging evidence indicates that α7 nicotinic acetylcholine receptors (α7-nAChR) are involved in AD pathology, because synaptic impairment and learning and memory deficits in a hAPPα7-/- mouse model are decreased by nAChR α7 subunit gene deletion. Given that Aβ potently modulates α7-nAChR function, that α7-nAChR expression is significantly enhanced in both AD patients and animal models, and that α7-nAChR play an important role in regulating neuronal excitability, it is reasonable that α7-nAChRs may contribute to Aβ-induced neural hyperexcitation. We hypothesize that increased α7-nAChR expression and function as a consequence of Aβ exposure is important in Aβ-induced neural hyperexcitation. In this project, we found that exposure of Aβ aggregates at a nanomolar range induces neuronal hyperexcitation and toxicity via an upregulation of α7-nAChR in cultured hippocampus pyramidal neurons. Aβ up-regulates α7-nAChRs function and expression through a post translational mechanism. α7-nAChR up-regulation occurs prior to Aβ-induced neuronal hyperexcitation and toxicity. Moreover, inhibition of α7-nAChR or deletion of α7-nAChR prevented Aβ induced neuronal hyperexcitation and toxicity, which suggests that α7-nAChRs are required for Aβ induced neuronal hyperexcitation and toxicity. These results reveal a profound role for α7-nAChR in mediating Aβ-induced neuronal hyperexcitation and toxicity and predict that Aβ-induced up-regulation of α7-nAChR could be an early and critical event in AD etiopathogenesis. Drugs targeting α7-nAChR or seizure activity could be viable therapies for AD treatment. / Dissertation/Thesis / Ph.D. Neuroscience 2011

Rationale and objectives: Because of known and imputed roles of dopaminergic and nicotinic cholinergic systems in a variety of neurological and neuropsychiatric disorders, combined neurochemical and behavioral methods assessments were made to study the intermodulatory roles of these neurochemical systems. Methods: Rats were treated daily during postnatal ontogeny with the dopamine D2/D3 agonist, quinpirole (QNP) HCl (1.0 mg/kg/day), for the first 3 weeks from birth. This priming process replicated previous findings of behavioral sensitization, manifested as hyperlocomotion, increased paw treading with jumping, and increased yawning. Results: All effects were partially or totally blocked by acute treatment with nicotine (0.3 mg/kg, i.p.). The effects of nicotine, in turn, were partially or totally blocked by the nicotinic antagonist, mecamylamine (1.0 mg/kg, i.p.). In concert with these behavioral actions, QNP-primed rats displayed greater binding of [3H]cytisine in midbrain and cerebellum and greater [125I]α- bungarotoxin binding in hippocampus and striatum. Conclusions: Accordingly, these selective ligands for α4β2 and α7 nicotinic receptors, respectively, demonstrate that nicotinic receptors are altered by dopamine D2/D3 agonist treatment of rats with primed dopamine receptors. We propose that nicotinic agonists may have a therapeutic benefit in behavioral disorders brought about by central dopaminergic imbalance.

No description available.

Bladder incontinence in patients who suffer from sacral spinal cord injury can wreak havoc on one's quality of life. A 2012 survey suggests that patients who sustain spinal cord injury prioritize the recovery of bladder function over other faculties. With about 12,000 new spinal cord injury cases reported in the United States each year, finding ways to combat the disabilities that result from lower spinal cord dysfunction should be of utmost importance to the scientific research community. Prior to studying the effects of surgical reinnervation on the bladder after long-term decentralization, it was critical to understand the effects that decentralization had on the integrity of both smooth muscle and intramural nerves of the bladder, the function of which could determine the success of surgical reinnervation. Chapter 2 describes in vivo stimulation, ex vivo smooth muscle contractility studies, and immunohistochemical techniques that were used to assess the condition of the functional components of the bladder. Collective results showed that although pelvic plexus-induced stimulation decreased when decentralization included the bilateral transection of the L7 dorsal root, smooth muscle cells and intramural nerves maintained their function after long-term bladder decentralization. Thus, preservation of at least some nerve activity may allow for successful surgical reinnervation after long-term injury. Following confirmation of smooth muscle and intramural nerve viability after decentralization, we sought to determine if nerve transfer after long-term decentralization restores bladder function in canines. In Chapter 3, we detail both decentralization and surgical reinnervation procedures used in our model. Briefly, decentralization of the bladder included bilateral transection of hypogastric nerves, as well as all spinal roots caudal to L7, with a subset of animals undergoing additional transection of the dorsal root of L7. One year after decentralization, animals that showed consistent loss of sensory and motor function underwent surgical reinnervation, which included the bilateral transfer of part of the obturator nerve to the anterior vesical branch of the pelvic nerve and the semimembranosus branch of the sciatic nerve to the pudendal nerve. Behavioral observations, in vivo stimulation of transferred nerves, and retrograde tracing studies were used to explore the efficacy of reinnervation on both sensory and motor components of bladder function. Ultimately, results showed that the new neuronal pathways created by nerve transfer can restore bladder sensation and possibly motor function in lower motor neuron-lesioned canines. Beyond the effects of surgical reinnervation on bladder function, we were interested in taking a closer look at the mechanisms that dictate function after decentralization and reinnervation (Chapter 3). Based on our previous work that found that transfer of somatic nerves resulted in bladder smooth muscle expression of a nicotinic receptor subunit thought to be expressed primarily in striated muscles, we were interested in assessing changes in the profile of nicotinic receptors responsible for bladder function. Ex vivo smooth muscle contractility studies showed that response to nicotinic receptor agonists were not altered after decentralization or reinnervation. Furthermore, the α1 nicotinic receptor subunit was expressed in bladder smooth muscle across all surgical groups. Future studies are necessary to better elicit the physiological relevance of these nicotinic receptors in the bladder. Additionally, due to the complexity of surgical reinnervation, it was important to understand all contributions to bladder innervation (Chapter 4). We previously identified that cells in the ventral horns of spinal cord levels rostral to the sacral cord can directly innervate the bladder via retrograde tracing. Because these direct inputs were not in proximity of the spinal root transections made during decentralization, we wanted to know how decentralization and reinnervation impacted their effects on the bladder when stimulated. L2-mediated detrusor contractions were significantly decreased by transection of the hypogastric nerves, suggesting that many of the nerves originating from the L2 cord are sympathetic in nature; however, treatment with phentolamine did not completely eliminate the increase in pressure in response to L2 stimulation. Therefore, the remaining inputs likely act upon the bladder through a yet undefined pathway. The quantity of positively labelled cells did not change in sections of the L2 ventral horn across all surgical groups, suggesting no change in the contribution of direct inputs to bladder innervation. Finally, anatomical feasibility of the obturator and semimembranosus branch of the sciatic nerve transfers has been assessed in an unembalmed cadaver, the results of which have not yet been published. Overall, this research gives us reason to believe that surgical reinnervation is a viable option for patients who develop lower neurogenic bladder after injury to the sacral cord, cauda equina, or peripheral nerves mediating bladder function. / Biomedical Sciences

Neonatal quinpirole (NQ) treatment to rats increases dopamine D2 receptor sensitivity persistent throughout the animal’s lifetime. In Experiment 1, we analyzed the role of α7 and α4β2 nicotinic receptors (nAChRs) in nicotine behavioral sensitization and on the brain-derived neurotrophic factor (BDNF) response to nicotine in NQ- and neonatally saline (NS)-treated rats. In Experiment 2, we analyzed changes in α7 and α4β2 nAChR density in the nucleus accumbens (NAcc) and dorsal striatum in NQ and NS animals sensitized to nicotine. Male and female Sprague-Dawley rats were neonatally treated with quinpirole (1mg/kg) or saline from postnatal days (P)1-21. Animals were given ip injections of either saline or nicotine (0.5mg/kg free base) every second day from P33 to P49 and tested on behavioral sensitization. Before each injection, animals were ip administered the α7 nAChR antagonist methyllycaconitine (MLA; 2 or 4mg/kg) or the α4β2 nAChR antagonist dihydro beta erythroidine (DhβE; 1 or 3mg/kg). Results revealed NQ enhanced nicotine sensitization that was blocked by DhβE. MLA blocked the enhanced nicotine sensitization in NQ animals, but did not block nicotine sensitization. NQ enhanced the NAcc BDNF response to nicotine which was blocked by both antagonists. In Experiment 2, NQ enhanced nicotine sensitization and enhanced α4β2, but not α7, nAChR upregulation in the NAcc. These results suggest a relationship between accumbal BDNF and α4β2 nAChRs and their role in the behavioral response to nicotine in the NQ model which has relevance to schizophrenia, a behavioral disorder with high rates of tobacco smoking.