Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests antibody-mediated platelet destruction occurs in the spleen via Fcγ receptors (FcγR). However, it has been demonstrated that anti-GPIbα-mediated ITP is often refractory to therapies targeting FcγR pathways. Utilizing a panel of murine monoclonal antibodies (mAbs) against murine and human GPIIbIIIa and GPIbα, it was found that anti-GPIbα induces not only platelet activation to a much greater extent than anti-GPIIbIIIa antibodies, but also significant surface expression of neuraminidase 1 and platelet desialylation. Utilizing inhibitors of platelet activation and desialylation, it was found that these two processes are not mutually exclusive, but rather exist in a positive feedback loop, leading to FcγR-independent platelet clearance in the liver likely via Ashwell-Morell receptors. Furthermore, in a murine model of ITP, sialidase inhibitor treatment rescued platelet counts in predominantly anti-GPIbα -mediated thrombocytopenia.

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests antibody-mediated platelet destruction occurs in the spleen via Fcγ receptors (FcγR). However, it has been demonstrated that anti-GPIbα-mediated ITP is often refractory to therapies targeting FcγR pathways. Utilizing a panel of murine monoclonal antibodies (mAbs) against murine and human GPIIbIIIa and GPIbα, it was found that anti-GPIbα induces not only platelet activation to a much greater extent than anti-GPIIbIIIa antibodies, but also significant surface expression of neuraminidase 1 and platelet desialylation. Utilizing inhibitors of platelet activation and desialylation, it was found that these two processes are not mutually exclusive, but rather exist in a positive feedback loop, leading to FcγR-independent platelet clearance in the liver likely via Ashwell-Morell receptors. Furthermore, in a murine model of ITP, sialidase inhibitor treatment rescued platelet counts in predominantly anti-GPIbα -mediated thrombocytopenia.

There is a large sex difference in the prevalence of attention deficit disorder; yet, little is known about sex differences in prefrontal attention circuitry. We investigated sex differences in the developmental nicotinic excitation of corticothalamic layer VI neurons, which play an important role in attention. Using whole cell recording in prefrontal brain slices, we examined the inward currents elicited by nicotinic stimulation in rodents. We found a prominent sex difference in the currents during the first postnatal month when males had significantly greater α4β2* nicotinic currents. Immunohistochemical analysis of α4YFP mice revealed no sex difference in the pattern or proportion of YFP-positive neurons in layer VI. Further electrophysiological experiments revealed that progesterone is able to rapidly and significantly suppress nicotinic currents in layer VI neurons. This is the first illustration at a cellular level that prefrontal attention circuitry is differently excited by nicotinic stimulation in males and females during development.

Some animals have adapted to hypoxia by increasing their haemoglobin affinity for oxygen, but in vitro studies have not shown any change of haemoglobin affinity for oxygen in human high altitude natives or lowlanders acutely acclimatized to high altitude. We conducted the first in vivo study of the oxyhaemoglobin dissociation curve by progressively reducing arterial PO2 while maintaining normocapnia in lowlanders at sea level, lowlanders sojourning at 3600m for two weeks and native Andeans at the same altitude. We found that the in vivo PO2 at which haemoglobin is half-saturated (P50) is higher in lowlanders at sea level (32 mmHg) than that measured in vitro (27 mmHg) and that lowlanders and highlanders do significantly increase the in vivo affinity of their haemoglobin for oxygen with exposure to high altitude. These results indicate the value of an in vivo approach for studying the oxyhaemoglobin dissociation curve.

Parkinson’s disease (PD) is characterized by the loss of dopamine in the basal ganglia and leads to paucity of movements, rigidity of the limbs, and rest tremor. Synaptic plasticity was characterized in the substantia nigra pars reticulata (SNr), a basal ganglia output structure, in 18 PD patients undergoing implantation of deep brain stimulating electrodes. Field evoked potentials (fEPs) in SNr were measured with one microelectrode using single pulses from a second microelectrode ~ 1 mm away. High frequency stimulation (HFS – 4 trains of 2s at 100Hz) in the SNr failed to induce a lasting change in test fEPs amplitudes in patients OFF medication. Following L-Dopa, HFS induced a potentiation of the fEPs that lasted more than 150s. Our findings suggest that extrastriatal dopamine modulates activity dependent synaptic plasticity at basal ganglia output neurons. Dopamine medication state clearly impacts fEP amplitude, and the lasting nature of the increase is reminiscent of LTP-like changes, indicating that aberrant synaptic plasticity may play a role in the pathophysiology of PD.

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.

The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.

Activity of the NMDA receptor is crucial for CA1 plasticity. Functional modification of the receptor is one way to modulate synaptic plasticity and affect hippocampus dependent behaviours. Two GPCRs, the dopamine receptor D1 and the PACAP38 receptor PAC1, have been shown to enhance NMDA activity via Gq and Gs signaling pathways respectively. Enhancement of NMDAR activity by the D1/Gs pathway depends on phosphorylation of the NR2B subunit by Fyn kinase. Conversely, enhancement by the PAC1/Gq pathway depends on phosphorylation of the NR2A subunit by Src kinase. SKF81297, a D1 agonist, was shown to enhance LTD whereas PACAP38, through the PAC1 pathway, was shown to lower the threshold for LTP. Both effects were blocked by specific antagonists and shown to be dependent on NR2 subunit phosphorylation. Ultimately, physiological metaplasticity at the CA1 synapse may be mediated by the relative activation of many GPCR signaling pathways via modification of the NR2 subunit.

Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.

Hypothalamic AMP-activated protein kinase (AMPK) regulates energy homeostasis in response to nutritional and hormonal signals. However, its role in glucose production regulation remains to be elucidated. Here, we tested the hypothesis that bidirectional changes in hypothalamic AMPK activity alter glucose production in rodents. First, we found that knocking down hypothalamic AMPK activity in an in vivo rat model led to a significant suppression of glucose production independent of changes in food intake and body weight. Second, we showed that activation of hypothalamic AMPK negated the ability of hypothalamic glucose- and lactate- sensing to lower glucose production. Collectively, these data indicate that changes in hypothalamic AMPK activity are sufficient and necessary for hypothalamic nutrient-sensing mechanisms to alter glucose production in vivo, and highlight the novel role of hypothalamic AMPK in the maintenance of glucose homeostasis in addition to energy balance.