The primary role of acetylcholinesterase (AChE) is hydrolysis of acetylcholine (ACh). However, observations by numerous groups have suggested that AChE may have non-cholinergic functions. Furthermore, developmental roles for AChE and its related enzyme, butyrylcholinesterase (BuChE), which is also capable of ACh hydrolysis, have been postulated. One line of evidence to support a non-cholinergic role for AChE is the apparent disparity in several brain areas between the distribution of AChE and the cholinergic marker choline acetyltransferase. The substantia nigra (SN), an area of the ventral mesencephalon (VM), which contains the dopaminergic cells that degenerate in Parkinson's disease (PD), is an area that displays such a disparity. One approach to treating PD involves implantation of embryonic dopaminergic VM cells into the parkinsonian brain. This procedure, known as foetal transplantation, has met with limited success, in part due to degeneration of dopaminergic cells within the donor preparation. It is known that incorporation of trophic factors into the preparation for grafting improves dopaminergic cell survival. It has previously been shown that AChE enhances survival and neurite outgrowth of postnatal dopaminergic cells in organotypic cultures of the VM. The aim of the studies in this thesis was to establish the effects of BuChE and AChE on embryonic dopaminergic neurons in a preparation analogous to that used in the animal model of foetal transplantation as a treatment for PD. Addition of BuChE and monomeric (G_1-) and tetrameric (G_4-) forms of AChE enhanced dopaminergic neurite outgrowth. Inhibition of the active site of BuChE and AChE by echothiophate had no effect upon neurite outgrowth or cell survival, demonstrating that the trophic effects of BuChE and AChE on neurite outgrowth were not dependent upon ACh hydrolysis. In contrast, inhibition of the peripheral anionic site (PAS) of AChE by BW284c51 markedly decreased cell survival and neurite outgrowth. The mechanism of action of BW284c51 toxicity was subsequently investigated using a mixture of nicotinic ACh receptor antagonists in order to demonstrate that the chronic toxic effects of BW284c51 were not a consequence of elevated ACh resulting from inhibition of AChE. Finally, the technique of whole-cell patch-clamp electrophysiology revealed a novel inhibitory effect of BuChE and G_1- and G_4-AChE on voltage-dependent calcium currents. It was postulated that these actions underlie the trophic effects of BuChE and AChE on embryonic dopaminergic neurons, a suggestion that was supported by the findings that established inhibitors of voltage-dependent calcium currents enhanced dopaminergic neurite outgrowth. The findings of this thesis are discussed in the context of other studies and are related to both physiological and pathological functions of the central nervous system.

(Abstract shortened by UMI.) / The conclusion of the studies reported herein can be summarized as follows: (1) PI in TCD correlates well with WMC volume and helps to differentiate those with and without WMC in stroke patients. (2) Post-stroke cognitive complaints are not related to severity of WMC among lacunar stroke patients. (3) The ARWMC scale correlates with objective cognitive performances and the operational definitions of ARWMC scale improves inter-rater reliability on CT. (4) Cognitive impairment in patients with confluent WMC is mediated by global and frontal cortical atrophy. Predictors for cognitive progression are cortical atrophy, absence of hyperlipidemia, low BP, and low cognitive scores. / With an aging population, prevalence of dementia is expected to escalate in the coming decades. The burden is especially great in developing countries like China. Similar to Alzheimer's pathology (e.g. amyloid plaque), age-related white matter changes (WMC) are important substrates of dementia. Since WMC are considered to be of ischemic origin, dementia related to WMC is believed to be more preventable than Alzheimer's disease. Yet, studies focusing on WMC have been relatively few. The thesis will cover 4 aspects of WMC and cognitive impairment. / Xiong, Yunyun. / Adviser: Vincent Mok. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 198-244). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese; some appendixes in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.

Brain stroke is considered as one of the three diseases that threaten human health all over the world. Hypertension and cerebral arteriosclerosis are thought to be the most dangerous risk factors of brain stroke, and they frequently occur together, leading to ischemia of brain tissue. Unfortunately, it is not clear whether the pathological changes resulting from hypertension are related to those resulting from cerebral arteriosclerosis. There have been no ideal animal models mimicking the pathological changes in such a combined condition. In this thesis, an animal model of hypertension combined with cerebral arteriosclerosis in rats was established by occlusion of both the left and right common carotid arteries in spontaneous hypertension rats. Pien Tze Huang (PTH), a reputed traditional Chinese medicinal complex, contains Radix notoginseng, snake bile, calculus bovis, and musk and some other components that are known to protect vessels and cells from injuries. Since different tissue injuries share many common cellular mechanisms, the protection by PTH to in nerves and the circulation systems may also be benefical to cerebrovascular conditions as well. In present experiments, PTH was used to treat hypertension rats that also developed chronic brain ischemia as a result of the bilateral carotid occlusion, and its protective role for neurons and blood vessels was investiaged. / From the data above, more severe damage could be caused by hypertension combined with chronic ischemia. The model of SHR with bilaterally occluded common carotid artery can be used to study pathological changes resulted from hypertension combined with chronic ischemia. PTH was able to protect neurons in stroke. / In the initial part of the work, patients from clinics in two cities in South and North China were compared and analysed; they had been suffering from brain ischemic stroke. About two thirds of the stroke patients were found to have hypertension before the onset of stroke. Their prognosis was significantly worse than those stroke patients without hypertension. In the hypertensive rats with occluded arteries, mean of functional magnetic resonance imaging (fMRI) examination showed that brain blood flow was very weak or even transiently became undetectable at the beginning of the acute stage of brain ischemia, but was restored one hour after the occlusion surgery. In addition, pathological changes in brains of hypertensive rats with induced brain ischemia (carotid occlusion) were examined by Nissl staining, TUNEL staining, cell death ELISA and anti-oxidation enzymes. At day 15 after ischemia, a large number of pyramid cells in the hippocampus of SHR were lost and a great deal of apoptotic cells were found in the CA1 of the hippocampus, while activities of some enzyme including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were increased. At day 30 and 60, some degenerative changes appeared to have subsided and the cells appeared morphologically normal. The activities of the above enzymes were also decreased at day 60. In WKY control rats with normal blood pressure, neurons in the CA1 were found less damaged after the bilateral carotid occlusion. It was found that apoptotic and dead cells were significantly reduced in rats with hypertension combined with chronic brain ischemia if they had been pre-treated with PTH. Moreover, brain stroke damage was less severe in this pretreated rats. / Zhang, Lihong. / "March 2010." / Adviser: WH Kwong. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 116-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Reactive gliosis is known to have a beneficial and a degenerative effect following injury to neurons. Although many factors have been implicated in reactive gliosis, their role in regulating this change is still unclear. We investigated the role of bone morphogenetic proteins in reactive gliosis in vivo and in vitro. In vivo, IHC analysis indicated reactive gliosis in the 6 week Ins2Akita mouse and WPK rat retinas. Expression of BMP7 was upregulated in these models, leading to an increase in the phosphorylation of downstream SMAD1. In vitro, treatment of murine retinal astrocyte cells with a strong oxidizing agent such as sodium peroxynitrite regulated RNA levels of various markers, including GFAP, CSPGs, MMPs and TIMPs. BMP7 treatment also regulated RNA levels to a similar extent, suggesting reactive gliosis. Treatment with high glucose DMEM and BMP4, however, did not elicit increase in levels to a similar degree. Increase in SMAD levels and downstream targets of SMAD signaling such as ID1, ID3 and MSX2 was also observed following treatment with sodium peroxynitrite in vitro and in the 6 week Ins2Akita mouse retinas in vivo. These data concur with previously established data which show an increase in BMP7 levels following injury. It also demonstrates a role for BMP7 in gliosis following disease. Further, it suggests SMAD signaling to play a role in initiating reactivity in astrocytes as well as in remodeling the extracellular matrix following injury and in a disease condition.

Indiana University-Purdue University Indianapolis (IUPUI) / Posttraumatic epilepsy, the development of temporal lobe epilepsy (TLE) following traumatic brain injury, accounts for 20% of symptomatic epilepsy. Reorganization of mossy fibers within the hippocampus is a common pathological finding of TLE. Normal mossy fibers project into the CA3 region of the hippocampus where they form synapses with pyramidal cells. During TLE, mossy fibers are observed to innervate the inner molecular layer where they synapse onto the dendrites of other dentate granule cells, leading to the formation of recurrent excitatory circuits. To date, the molecular mechanisms contributing to mossy fiber sprouting are relatively unknown. Recent focus has centered on the involvement of tropomycin-related kinase receptor B (TrkB), which culminates in glycogen synthase kinase 3β (GSK3β) inactivation. As the neurite outgrowth promoting collapsin response mediator protein 2 (CRMP2) is rendered inactive by GSK3β phosphorylation, events leading to inactivation of GSK3β should therefore increase CRMP2 activity. To determine the involvement of CRMP2 in mossy fiber sprouting, I developed a novel tool ((S)-LCM) for selectively targeting the ability of CRMP2 to enhance tubulin polymerization. Using (S)-LCM, it was demonstrated that increased neurite outgrowth following GSK3β inactivation is CRMP2 dependent. Importantly, TBI led to a decrease in GSK3β-phosphorylated CRMP2 within 24 hours which was secondary to the inactivation of GSK3β. The loss of GSK3β-phosphorylated CRMP2 was maintained even at 4 weeks post-injury, despite the transience of GSK3β-inactivation. Based on previous work, it was hypothesized that activity-dependent mechanisms may be responsible for the sustained loss of CRMP2 phosphorylation. Activity-dependent regulation of GSK3β-phosphorylated CRMP2 levels was observed that was attributed to a loss of priming by cyclin dependent kinase 5 (CDK5), which is required for subsequent phosphorylation by GSK3β. It was confirmed that the loss of GSK3β-phosphorylated CRMP2 at 4 weeks post-injury was likely due to decreased phosphorylation by CDK5. As TBI resulted in a sustained increase in CRMP2 activity, I attempted to prevent mossy fiber sprouting by targeting CRMP2 in vivo following TBI. While (S)-LCM treatment dramatically reduced mossy fiber sprouting following TBI, it did not differ significantly from vehicle-treated animals. Therefore, the necessity of CRMP2 in mossy fiber sprouting following TBI remains unknown.