Thesis (Ph.D. in Microbiology) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 145-167). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Dopaminergic systems regulate many brain functions and dysfunction of dopaminergic neurotransmission is thought to underlie numerous disorders, including schizophrenia, attention deficit hyperactivity disorder (ADHD), depression and Alzheimer’s disease. In the hippocampus, a dopaminergic projection from the ventral tegmental area (VTA) is proposed to be essential for controlling entry of sensory information into long-term memory through novelty and salience detection. However, the effects of the VTA-dopamine system on hippocampal synaptic transmission are largely under-explored and the underlying mechanisms are unclear. The goal of this project was to investigate mechanisms involved in dopaminergic modulation of hippocampal neurophysiology. Specifically, I (1) examined if dopamine modulates hippocampal synaptic transmission in a region- and input-specific manner, and (2) studied the signaling mechanisms underlying such modulation. In the first aim for the study, I tested whether SKF38393, a dopamine D1-like receptor agonist, differentially affects excitatory synaptic transmission in perforant path synapses onto dentate gyrus granule cells and whether such effects differ from those at area CA1 synapses. I found that SKF38393 produced a concentration-dependent increase in field excitatory postsynaptic potential (fEPSP) in both subregions, but that higher concentrations were needed in the dentate gyrus to produce comparable effects. This synaptic enhancement was long-lasting and largely irreversible which suggests it may be a form of long term enhancement (LTP). Also, the increase in synaptic transmission at medial perforant path synapses was larger than in the lateral perforant path. Importantly, effects in the dentate gyrus, unlike those in CA1, differed substantially along the dorsoventral axis, with effects being significantly larger at the dorsal compared to the ventral pole. In the second aim, various combinations of D1 and D2-like receptor agonists and antagonists as well as inhibitors of second messenger systems, demonstrated that differential mechanisms were required for initiation and maintenance of SKF38393-mediated early and late-phase enhancement and that a novel non-canonical phospholipase-C (PLC) dependent signaling pathway may be involved. Based on recent discoveries in other brain regions, we hypothesized that multiple subcellular signaling pathways may contribute to PLC activation which may include but are not limited to D1(5)-D2 heteromers and Gβγ complex. In conclusion, this work uncovers novel dopaminergic signaling pathways regulating hippocampal physiology, which will lead to development of better (functionally selective) therapeutic agents.

The influence of the membrane environment on the α1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the α1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic α1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the α1-adrenoceptor.

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTPγS and carbachol/GTPγS-stimulated PPI hydrolysis was linear for at least 20 min. GTPγS-stimulated [3H] inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT - 147%). SAX-HPLC seperation of [3H] inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP3) was Ins(1,4,5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca2+ increased PPI hydrolysis progressively from 100 nM through 50 μM and synergistically enhanced carbachol/GTPγS stimulation. Comparisons of age-matched controls with Alzheimer's patients indicated that GTPγS, carbachol/GTPγS, and 5HT/GTPγS-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimer's patients. Ca2+ stimulation of PPI hydrolysis was not different between controls and Alzheimer's patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimer's disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimer's disease. Further, this method can be used to study signal transduction events in postmortem human brain.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is typified by a robust microglial-mediated immune response. Genetic studies have demonstrated that many genes that alter AD risk are involved in the innate immune response and are primarily expressed in microglia. Among these genes is phospholipase C gamma 2 (PLCG2), a critical element for various immune receptors and a key regulatory hub for immune signaling. PLCG2 genetic variants are associated with altered AD risk. The primary objective of this thesis was to determine the role of PLCG2 in AD pathogenesis. We observed significant upregulation of PLCG2 expression in three brain regions of late-onset AD (LOAD) patients and a significant positive correlation of PLCG2 expression with amyloid plaque density. Furthermore, the differential gene expression analysis highlighted inflammatory response-related pathways. These results suggest that PLCG2 plays an important role in AD. We systematically investigated the impact of PLCG2 haploinsufficiency on the microglial response and amyloid pathology in the amyloidogenic 5xFAD mouse model. The results demonstrated that Plcg2 haploinsufficiency altered the phenotype of plaqueassociated microglia, suppressed cytokine levels, increased compact X34-positive plaque deposition, and downregulated the expression of microglial genes associated with immune cell activation and phagocytosis. Our study highlights the role of PLCG2 in immune responses; loss of function of PLCG2 exacerbates the amyloid pathology of AD. Genetic studies demonstrated that the hypermorphic P522R variant is protective and that the loss of function M28L variant confers an elevated risk for AD. Our results demonstrated that PLCG2 variants modulate disease pathologies through specific transcriptional programs. In the presence of amyloid pathology, the M28L risk variant impaired microglial response to plaques, suppressed cytokine release, downregulated disease-associated microglial genes, and increased plaque deposition. However, microglia harboring the P522R variant exhibit a transcriptional response endowing them with a protective immune response signature linked to their association with plaques and Aβ clearance, attenuating disease pathogenesis in an amyloidogenic mouse model of AD. Collectively, our study provides evidence that the M28L variant is associated with accelerated and exacerbated disease-related pathology, and conversely, the P522R variant appeared to attenuate disease severity and progression. / 2024-10-03

No description available.

Phospholipase C enzymes are important cell signalling enzymes that catalyse the cleavage of phosphatidylinositol 4,5-bisphophate PI(4,5)P₂ into two biologically active second messenger molecules. These are the inositol 1,4,5-trisphosphate which initiates Ca²⁺ release from the endoplasmic reticulum and the diacylglycerol that activates protein kinase C. Although this basic function is shared between the different isoforms, the PLC family encompasses a diverse collection of proteins with various domain structures in addition to the PLC-specific domains. The neuron-specific “6th family” of these enzymes, PLCηs have most recently been identified with two members, PLCη1 and PLCη2. The aim of the thesis is to characterise the PLCη2 variant from several aspects. Firstly, it describes that PLCη2 possesses a high sensitivity towards Ca²⁺. Secondly, it investigates how the Ca²⁺-induced enzymatic activity of PLCη2 is controlled by its different domains. Also it provides evidence that the pleckstrin homology domain targets PLCη2 to membranes by recognising PI(3,4,5)P₃. Moreover, the uniquely structured EF-hand is responsible for the Ca²⁺-sensitivity of the enzyme. Finally, it is demonstrated that the C2 domain is important for activity. The initial biochemical characterisation is followed by the description of a physiological role for PLCη2. It is shown using a neuroblast model that PLCη2 is crucial for neuronal differentiation and neurite growth. Further efforts were made to assess how PLCη2 is responsible for this effect. It was revealed that it might be involved in regulating intracellular Ca²⁺ dynamics, transcriptional activity and actin reorganisation in differentiating neurons. As the functions of PLCη2 are just beginning to come to light, more aspects for future research are also suggested in the thesis. Hopefully, this and the data presented within the thesis will stimulate even greater interest in this fascinating new field of research.

Thesis advisor: Mary F. Roberts / Thesis advisor: Eranthie Weerapana / Peripheral proteins from mammals often exhibit multi-domain structures and require metal ions such as calcium as co-factors. This dissertation investigates two types of such proteins -- pulmonary collectins (surfactant proteins A and D) and phosphatidylinositol-specific phospholipase C (PLC) delta1 -- and their interactions with model membranes. One approach to work around the complexity brought upon by such multi-domain protein structure is to use a truncated construct or an isolated single domain. For pulmonary collectins, homotrimers consisting of the neck domain and the carbohydrate recognition domain were used in a novel NMR assay for better understanding of their lipid-specific interactions with the membranes. For PLC delta1, we were particularly interested in the role of the EF-hand domain. The isolated EF-hand domain of PLC delta1 was first used to characterize its interactions with membranes and identify key residues responsible for such interactions. These key residues in the N terminal lobe of the EF-hand domain, either cationic or hydrophobic, were then found to affect the hydrolysis activity of the full-length enzyme. A common role for this region of the PLC in facilitating proper membrane association was thus proposed. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Secreted phosphatidylinositol-specific phospholipase Cs (PI-PLCs) are often virulence factors in pathogenic bacteria. Understanding how these enzymes interact with target membranes may provide novel methods to control bacterial infections. In this work, two typical PI-PLC enzymes, from Bacillus thuringiensis (Bt) and Staphylococcus aureus (Sa), were studied and their membrane binding properties were examined and correlated with enzymatic activity. BtPI-PLC is kinetically activated by allosteric binding of a phosphatidylcholine (PC) molecule. MD simulations of the protein in solution suggested correlated loop and helix motions around the active site could regulate BtPI-PLC activity. Vesicle binding and enzymatic studies of variants of two proline residues, Pro245 and Pro254, that were associated with these motions showed that loss of the correlated motions between the two halves of PI-PLC were more critical for enzymatic activity than for vesicle binding. Furthermore, loss of enzyme activity could be rescued to a large extent with PC present in a vesicle. This suggests that binding to PC changes the enzyme conformation to keep the active site accessible. SaPI-PLC shows 41.3% sequence similarity with BtPI-PLC but has very different ways its activity is regulated. While it is kinetically activated by PC it does not in fact bind to that phospholipid. Enzymatic and membrane interaction assays showed that SaPI-PLC has evolved a complex, apparently unique way to control its access to PI or GPI-anchored substrate. (i) An intramolecular cation-pi latch facilitates soluble product release under acidic conditions without dissociation from the membrane. (ii) There is a cationic pocket on the surface of enzyme that likely modulates the location of the protein. (iii) Dimerization of protein is enhanced in membranes containing phosphatidylcholine (PC), which acts not by specifically binding to the protein, but by reducing anionic lipid interactions with the cationic pocket that stabilizes monomeric protein. SaPI-PLC activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane depleted in anionic phospholipids. This protein also served as a way to test the hypothesis that a cation-pi box provides for PC recognition site. This structural motif was engineered into SaPI-PLC by forming N254Y/H258Y. This variant selectively binds PC-enriched vesicles and the enzyme binding behavior mimics that of BtPI-PLC. Itaconic acid (ITA) is a metabolite synthesized in macrophages and related cell lines by a cis-aconitate decarboxylase (cADC). cADC activity is dramatically increased upon macrophage stimulation. In this work, the cell line RAW264.7 was used to show that cADC activity upon stimulation requires de novo protein synthesis. MS analyses of partially purified RAW264.7 protein extracts from stimulated cells show a large increase for immunoresponsive gene 1 protein (IRG1) and siRNA knockdown of the IRG1 reduces cADC activity upon stimulation. Suspected active site residues of IRG1 were identified by mutagenesis studies of the recombinant protein based on a homology structure model of fungal cADC. The cloning and overexpression of this enzyme should help clarify the cofactor-independent decarboxylation mechanism of this mammalian enzyme as well as open up future studies into the specific role of ITA in the mammalian immune system and cancers. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Thesis advisor: Steven D. Bruner / The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from <italic>Bacillus thuringiensis</italic> is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling <super>31</super>P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.