Intracellular transportation is an essential cellular event controlling neuronal development, morphology, function and survival. Kinesin-1 is the molecular motor conveying cargo along microtubule by utilizing the chemical energy from ATP hydrolysis. This motor consists of two heavy chains and two light chains. Both heavy and light chains are responsible for cargo bindings. There are three kinesin-1 heavy chains in eukaryotic cells. Kif5a and Kif5c are neuronal specific, while Kif5b is ubiquitously expressed. Kif5b carries various cargos essential for neuronal functions, and the early embryonic death of Kif5b null mice suggests the importance of Kif5b in vivo. N-methyl-d-aspartate receptors (NMDARs) are glutamate elicited channel, which is permeable to calcium and crucial for synaptic plasticity in the central nervous system. NMDARs are heteromeric assemblies consisting of NR1, NR2 and NR3 subunits. These transmembrane subunits contain three parts. Other than the transmembrane domain, the extracellular domain serves as the ligand binding site while the intracellular domain interacts with various partners regulating downstream signaling and receptor trafficking. Synaptic NMDAR activation regulates synaptic plasticity, while extrasynaptic NMDAR activation leads to excitotoxicity. In this project, I find that kinesin-1 directly interacts with NMDAR subunit, NR1, NR2A and NR2B in vivo. NMDAR colocalizes with kinesin-1 in the cell body and neurites. By GST-pull-down assays with different Kif5b fragments, the cytoplasmic domains of NR1, NR2A and NR2B are found to directly bind with Kif5b via a Kif5b C-terminal region independent of kinesin light chains. To examine the role of Kif5b in NMDAR trafficking, dominant negative Kif5b fragments are expressed in cell lines together with NR1-1a and GFP-NR2B. Overexpression of dominant negative Kif5b significantly disrupts GFP-NR2B forward trafficking and prevents it from entering into Golgi apparatus. Furthermore, the surface NR1 and NR2B levels are significantly reduced whilst the NR2A levels are not affected in Kif5b+/- mice in which the Kif5b protein level is reduced by 50% compared with the wild-type littermates. Consistent with this observation, the NR1 and NR2B levels are decreased in fractions containing synaptosomal membrane but not the one containing only postsynaptic densities, suggesting that the extrasynaptic NMDAR levels are affected in Kif5b+/- mice. NMDARs are highly permeable to calcium while activated, thereby activating neuronal nitric oxide synthases (nNOS) to produce nitric oxide (NO). It is found that NMDA triggered calcium influx is perturbed in Kif5b+/- neurons, while the synaptic NMDA receptor mediated calcium influx is normal. In Kif5b+/- slices, the production of NO reduces significantly. Calcium ionophore, A23187, rescue this NO defect, indicating insufficient supply of calcium as the main contribution to this defect. Therefore, Kif5b-dependent extrasynaptic localization of NMDA receptors mediates calcium influx upon NMDA stimulation and controls NO production. In the summary, above results suggest kinesin-1 as a novel motor involving in NMDA receptor trafficking. This interaction may contribute to the extrasynaptic distribution of NMDARs. By regulating NO production through interaction with NMDARs, Kif5b may mediate neuronal survival in cerebral ischemia and certain aggressive behaviors. This provides a novel target for therapy development against stroke and schizophrenia. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliographical references.

As the aging population continues to grow worldwide, age-related complications are becoming more apparent within the aging population. One of the first age-related complications to become apparent is age-associated memory impairment and it can make the elderly more dependent on caregivers early on. The N-methyl-D-aspartate (NMDA) receptor is important to learning and memory and appears to be especially vulnerable to the process of aging. The density of NMDA receptors declines with age more than any other ionotropic glutamate receptor. Both the density of NMDA receptors and the mRNA and protein expression of its subunits decline with age. In particular, the GluN2B subunit of the NMDA receptor shows the greatest age-related declines in expression across multiple brain regions, including the frontal lobe (including the prefrontal and frontal cortices), caudate nucleus and hippocampus. These declines are strongly correlated to age-related declines in spatial memory. Specifically, age-related decreases in the protein expression of the GluN2B subunit within crude synaptosomes of the frontal cortex of C57BL/6 mice show a relationship to the declines in performance in a long-term spatial memory task across age groups. However, within the population of aged mice, there was a subpopulation of aged mice in which higher expression of the GluN2B subunit within the synaptic membrane of the hippocampus was associated with poorer performance in the same task. Moreover, transgenic mice designed to express higher levels of the GluN2B subunit from birth also possess superior memory, including spatial memory, across adulthood to middle-age. Taken together, these data led to the hypothesis that increasing the expression of the GluN2B subunit within the aged brain could potentially alleviate age-related declines in memory. However, increasing its expression regionally was first examined since higher expression of the GluN2B subunit within the hippocampus has been associated with poorer memory in aged animals. Since age-related decreases in the protein expression of the GluN2B subunit within the frontal cortex show a relationship to impaired memory function, the first study was designed to determine if increasing GluN2B subunit expression in the frontal lobe would improve memory in aged mice. Mice received bilateral injections of either an adenoviral vector, containing cDNA specific for the GluN2B subunit and enhanced Green Fluorescent Protein (eGFP) (GluN2B vector); an adenoviral vector containing only the cDNA for eGFP (control vector); or vehicle into their frontal lobe. Spatial memory, cognitive flexibility and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression in the frontal lobe, exhibited improved long-term spatial memory, comparable to young mice, in the second day of training. Moreover, a higher concentration of the specific GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B subunit expression, as compared to aged controls. The requirement for greater antagonism in aged mice to block memory performance suggests that the number of GluN2B-containing receptors in their frontal lobe was enhanced and contributed to the improved memory. This study provides suggestive evidence that therapies that enhance GluN2B subunit expression within the aged brain could have the potential to ameliorate age-related memory loss. Since higher expression of the GluN2B subunit within the hippocampus of aged mice is associated with poorer memory, the second study was designed to determine if increasing GluN2B subunit expression in the hippocampus would improve or further impair memory in aged mice. This would help to determine if a therapy aimed at enhancing the GluN2B subunit expression or function of GluN2B-containing receptors throughout the aged brain could help ameliorate age-associated memory loss. Mice were injected bilaterally with either the GluN2B vector, a control vector or vehicle into the hippocampus. Spatial memory, cognitive flexibility and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression in the hippocampus, exhibited improved long-term spatial memory, comparable to young mice, early in training. However, there was a trend for impaired memory later in the long-term spatial memory trials. Still, these data suggest that enhancing GluN2B subunit expression in the aged hippocampus could be more beneficial to memory than harmful. In addition, the results of this study suggest that enhancing GluN2B subunit expression in different brain regions may improve memory at different phases of learning. Therefore, therapies that enhance GluN2B subunit expression throughout the aged brain could help ameliorate age-related memory loss. The first two studies demonstrated that enhancing the expression of the GluN2B subunit within either the frontal lobe or hippocampus of the aged brain has the potential to reduce age-related memory declines. However, the increase was not global nor specific to the synapse. Therefore, a third study was developed with the intent of garnering a more global increase in GluN2B subunit expression that was localized to the synapse. Cyclin dependent kinase 5 (Cdk5) enhances endocytosis of the GluN2B subunit-containing NMDA receptors from the synapse. Previous research has shown that inhibiting Cdk5 increases the number of GluN2B subunits at the synapse and within the whole cell and improves memory in young mice. This study was designed to determine if using antisense phosphorodiamidate morpholino oligomers (Morpholinos) to decrease the expression of Cdk5 protein within the brain would improve memory in aged mice. Morpholinos were conjugated to a cell penetrating peptide, which enhances cellular uptake, and delivered bilaterally to the lateral ventricles of both young and aged mice via acute stereotaxic injection. Treatments consisted of equivalent volumes and concentrations of either vehicle, control Morpholino or a Morpholino targeting the mRNA of Cdk5 (Cdk5 Morpholino). Memory was evaluated in the Morris water maze and using a novel object recognition task. Aged mice treated with the Cdk5 Morpholino exhibited improved early acquisition and spatial bias in the long-term spatial memory trials, as well as improved performance overall, compared to control Morpholino-treated aged animals. However, aged mice treated with the Cdk5 Morpholino performed similarly to vehicle-treated aged animals. The presence of the peptide-conjugated Morpholinos within the brain may have worsened performance in the Morris water maze task since control Morpholino-treated animals performed significantly worse than vehicle-treated animals. In concurrence, there was significantly greater gliosis in peptide-conjugated Morpholino-treated animals over vehicle-treated brains, suggesting it was neurotoxic. In contrast, young mice treated with the Cdk5 Morpholino showed impaired early acquisition and spatial bias but a trend for improved later learning in the long-term spatial memory task compared to control Morpholino-treated animals. Treatment with the Cdk5 Morpholino had no significant effect on cognitive flexibility, associative memory or novel object recognition for young or aged animals. Immunohistochemistry revealed increased GluN2B subunit expression within cells with characteristics of neurons and astroglia in regions of the frontal lobe, caudate nucleus and hippocampus of aged mice who received the Cdk5 Morpholino compared to control treatments. However, the increased GluN2B subunit expression appeared to be greater within the hippocampus. These results suggest that inhibiting the translation of Cdk5 using Morpholinos increased GluN2B subunit expression in both young and aged mice and may have contributed to the improved long-term spatial memory observed in aged mice, despite the Morpholino being administered at a presumably toxic concentration. An additional group of mice was used to determine a non-neurotoxic dosage of the peptide conjugated Morpholino. However, future studies are needed to determine the efficacy of the Cdk5 Morpholino at this dosage. Taken together, the studies presented here suggest that increasing expression of the GluN2B subunit within the aged brain does improve age-associated memory declines. In addition, cell penetrating peptide- conjugated Morpholinos show promise as tools for genetic manipulation within the brain and Cdk5 could prove to be a novel target for enhancing GluN2B subunit expression within the aged brain. Though future studies are needed, the studies presented here do suggest that therapies that enhance GluN2B subunit expression within the aged brain have the potential to help ameliorate memory loss. However, since enhanced GluN2B subunit expression itself can increase the potential for excitotoxicity, an optimal dose of such a therapeutic would need to be determined. / Graduation date: 2013

Not available / text

Dysfunction of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to the cognitive deficits of many neurodegenerative diseases and psychiatric disorders. Cognitive symptoms of Alzheimer's disease can be treated with NMDA receptor antagonists or drugs targeting the cholinergic system; however, there are no effective treatments for cognitive deficits of schizophrenia or Huntington's disease. With the discovery of a potent and selective allosteric modulator of the NMDA receptor, there is the possibility of new treatments based on NMDA receptor functional-enhancement through neuroactive steroids, closely related in structure to the endogenous neurosteroid, cerebrosterol. The aim of this thesis was to examine steroidal modulation of the NMDA receptor both in vitro and in vivo. In chapter 2, NMDA receptor enhancement of both the synthetic and endogenous neuroactive steroids was assessed in neurons maintained in cell culture using calcium imaging techniques. Sulphation of the steroids greatly increased the efficacy of NMDA receptor enhancement compared to the unsulphated steroids. Chapters 3 and 4 investigate the potential for neuroactive steroids to treat cognitive impairments of Huntington's disease. Using a mouse model, tests were selected that were analogous to those in which patients are impaired; however, no impairments were found in the mouse model. Chapter 5, therefore, used a different model of cognitive impairment – namely, rats with a set-shifting impairment, as is seen in many psychiatric and neurological disorders, including Huntington's disease – to assess the effect of the synthetic steroid administration. Unfortunately, the rats did not show the expected impairment. The lack of reliable animal models compromised testing the efficacy of these promising NMDA receptor positive allosteric modulators. Nevertheless, the promising in vitro results suggest that there could still be therapeutic potential. In addition, the compound is a useful research tool for exploring NMDA receptor function in health and disease.

The N-methyl-D-aspartate (NMDA) receptors are highly regulated ligand-gated ion channels, which are affected by many substrates. Overactivation of the NMDA receptor can lead to hyperexcitability and a number of neurotoxic effects and neurological diseases. Agmatine has been demonstrated to act allosterically as an inhibitory modulator at the polyamine recognition sites of the NMDA receptor complex. The present study synthesized and evaluated a library of agmatine analogs for their ability to displace tritiated MK-801 from NMDARs in P2 membrane preparations from rat brains at ligand concentrations of 1 mM and 50 uM. A full dose-response curve was generated for the most active compounds, in the presence and absence of a pathological level of spermidine (100 uM). A forty-five member subset of arylidenamino-guanidino compounds was synthesized and all were demonstrated to be NMDA receptor inhibitory modulators in the above assay. Three of these compounds generated biphasic curves, indicating activity at two binding sites: the postulated high-affinity agmatine binding site, and a low-affinity site (perhaps the channel itself). (4-Chlorobenzylidenamino)-guanidine hydrochloride demonstrated an IC50 of 3.6 uM at the former site and 124.5 uM at the latter. Several computer models were generated to direct further synthesis. Based on the structure-activity relationship of the arylidenamino-guanidino compounds, a pharmacophore model of the agmatine binding site of the NMDAR was proposed.

Chapter 1 gives an introduction on the physical and electronic properties of fluorine and the C-F bond. The application of fluorine in organic chemistry, which is mainly attributed to the electronic properties of fluorine is described. The role of fluorine in neuropsychiatric drug development and for influencing the conformational study of bioactive amines is also illustrated. Chapter 2 of the thesis describes the synthesis of the two fluorinated stereoisomers (2R, 3S) and (2S, 3S) 3-fluoro N-methyl–D-aspartate (NMDA). These were prepared as analogues to study the binding conformation of NMDA on the glutameric NMDA receptor. The (2S, 3S)-3-fluoro NMDA D-72 was successfully prepared from diethyl D-tartrate. The (2S,3R)- stereoisomer was prepared by separation of diastereoisomers generated by reaction of a meso- epoxide with an enantiomerically pure amine, followed by fluorination. Both the (2S,3R)- and (2R,3S)- enantiomers were prepared separately, however assignment of the absolute configuration to each enantiomer could not be unambiguously proven. The fluorinated 3F-NMDA stereoisomers were assessed by dose response analysis and TEVC analysis in the rat glutamate receptor. The biological results show that the (2S, 3S)-3F NMDA D-72 is a good agonist, whereas (2R, 3S)- and (2S, 3R)-3-fluoro NMDA are inactive stereoisomers. The result of this study indicates that (2S, 3S)-3F NMDA D-72 is the only relevant agonist that can access a conformation for binding to NMDA receptor. Chapter 3 describes the preparation of fluorinated analogues of the calcium receptor agonist Cinacalcet. The (2R,1’R)-123 and (2S,1’R)-124 fluoro Cinacalcet diastereoisomers were prepared from 3’-(trifluoromethyl)cinnamic acid and 3’’-SF₅-137 Cinacalcet was synthesized from pentafluorosulfanyl benzyl alcohol. The biological assessment in the calcium receptor (CaR) revealed that both (2R,1’R)-123 and (2S,1’R)-124 fluoro Cinacalcet is slightly lower in potency compared to the non-fluorinated Cinacalcet 117. This suggests that the Cinacalcet 117 adopts an extended conformation when bound to the receptor. The 3’’-SF₅-137 Cinacalcet possesses equipotent activity with Cinacalcet 117.

Reproductive senescence in females is a natural part of the aging process. However, the process by which it occurs, and the relative role of each level of the hypothalamic-pituitary-gonadal axis, remains largely unknown. The neural circuitry regulating the hypothalamic axis, including glutamate acting through N-Methyl-D-Aspartate receptors (NMDARs) on GnRH neurons, appears to be key to this process. The NMDAR is tetrameric and composed of an obligatory NR1 subunit together with NR2 subunits. The subunit composition determines the channel kinetics of the receptor and changes through the life span. This dissertation examines the physiological role of the NR2b subunit on LH pulsatile release and LH surge, both important for reproductive function. The expression of NR2b subunits in the anteroventral periventricular (AVPV) nucleus of the hypothalamus was also examined in aging rats. Experiment 1 showed that the NR2b-antagonist, ifenprodil, enhanced pulsatile LH release in estradioltreated females (both age groups). Experiment 2 showed that the LH surge in middle-aged animals was slightly accelerated and that results were dependent upon prior reproductive status of the animals. In Experiment 3, examination of the NR2b-immunoreactive cell population in young, middle-aged, and aged ovariectomized females given vehicle, estradiol, or estradiol with progesterone showed an age-associated decline in NR2b density. However, the immunofluorescent fraction volume of NR1 colocalized with NR2b increased with aging, and that of immunofluorescent fraction volume of NR2b increased with estradiol treatment. This is indicative of the amount of protein expressed in the AVPV. In total, NR2b cell density in the AVPV declines with age, but the amount of NR2b expressed in NR1-positive cells increases, suggesting a larger population of NR2b containing channels. This may translate to age-associated inhibition of GnRH/LH activity, which is relieved with blockade of NR2bcontaining NMDARs. Thus, this dissertation describes a novel way to examine the mechanism by which age-associated changes to neuromodulators of the HPG axis may affect the onset of reproductive senescence. / text

NMDA receptor antagonists such as Ketamine and PCP are potent psychoactive drugs used recreationally. This class of drug induces a number of phenomena in humans similar to those associated with schizophrenia including reduced selective attention, altered working memory, thought disorders and hallucinations. These psychotomimetic drugs have thus been used as a longstanding model to study this disease in animals. Importantly, such animal models allow for recording of brain activity using invasive techniques that are inappropriate in humans. Previous electrophysiological studies have shown that MK-801, a potent non-competitive NMDA receptor antagonist, increases gamma-frequency oscillations and produces a state of disinhibition in the prefrontal cortex of rats wherein the activity of putative excitatory pyramidal neurons increases while the activity of putative inhibitory interneurons decreases. These features are relevant to schizophrenia because molecular evidence suggests dysfunction of inhibitory cortical interneurons, while electroencephalographic recordings show altered gamma-frequency oscillations in this disease. It has been hypothesized that the disinhibited cortical state produces “noisy” information processing, but this has not been directly observed in the interaction of neuronal firing in either humans or animal models. We therefore tested this hypothesis by examining the synchronization of neural activity in the NMDA receptor antagonist model of schizophrenia. We used high-density electrophysiological recordings in the medial prefrontal cortex of freely moving rats before and after systemic injection of MK-801. Analysis of these recordings revealed that drug administration: (i) increases gamma power in field potentials in a manner dissociated from increased locomotion; (ii) does not change the gamma power in multi-unit activity; (iii) decreases spike synchronization among putative pyramidal neurons in the gamma range (30ms), and despite of this it (iv) does not change the synchronization between gamma-range field potentials or between sum-of-spikes and field potentials. These effects in synchronization may be revealing of potent cognitive effects associated with NMDA receptor antagonism, and may reflect impaired communication processing hypothesized to occur in schizophrenia. / xi, 42 leaves : ill. ; 29 cm