The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth.

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth. / Medicine, Faculty of / Graduate

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Selective ion and solute transport across cell membranes is a vital process occurring in all types of cells. Evolutionarily developed transport proteins work as membrane-embedded molecular machines, which alternately open a gate on each side of the membrane to bind and translocate specific ions. Sodium/proton exchange plays a crucial role in maintaining cytoplasmic pH and membrane potential, while, if not regulated, the process causes severe heart diseases in humans. Here I applied single-molecule force spectroscopy to investigate molecular interactions determining the structural stability of the sodium/proton antiporter NhaA of Escherichia coli, which serves as a model system for this class of proteins. Mechanical pulling of NhaA molecules embedded in the native lipid bilayer caused a step-wise unfolding of the protein and provided insights into its stability. Modified experiments allowed observing refolding of NhaA molecules and estimating folding kinetics for individual structural elements, as well as detecting eventual misfolded conformations of the protein. The activity of NhaA increases 2000fold upon switching pH from 6 to 8. Single-molecule force measurements revealed a reversible change in molecular interactions within the ligand-binding site of the transporter at pH 5.5. The effect was enhanced in the presence of sodium ions. The observation suggests an early activation stage of the protein and provides new insights into the functioning mechanism. When studying interactions of NhaA with the inhibitor 2-aminoperimidine, I exploited single-molecule force measurements to validate the binding mechanism and to describe quantitatively formation of the protein:inhibitor complex. The ability of single-molecule force measurements to probe structurally and functionally important interactions of membrane proteins opens new prospects for using the approach in protein science and applied research.

Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon reintroduction of the recipient circulation. This is reflected in Transplant Registry data showing increased primary allograft failure as a function of increasing ischaemic time. The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways by the addition of pharmacological agents to Celsior, a clinical preservation solution. Studies were conducted in an isolated working rat heart model of donor heart preservation. The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Maximum protective effect (after a 6 hour storage period) was observed when the PARP inhibitor was administered prior to cardiac arrest and storage and when the agent was added to the Celsior cardioplegic / storage solution. This protective affect was associated with activation of the Akt signalling pathway and could be prevented by inhibition of Akt phosphorylation and activation. The second study examined functional protection and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Here, cardiac protection was accompanied by activation of the ERK 1/2 pro-survival pathway as well as a decrease in apoptosis. The third study examined the cardioprotective effect of supplementation of Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Significant recovery of function was only observed in the triply supplemented hearts, being accompanied by activation of both the Akt and ERK pathways. These studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.

Selective ion and solute transport across cell membranes is a vital process occurring in all types of cells. Evolutionarily developed transport proteins work as membrane-embedded molecular machines, which alternately open a gate on each side of the membrane to bind and translocate specific ions. Sodium/proton exchange plays a crucial role in maintaining cytoplasmic pH and membrane potential, while, if not regulated, the process causes severe heart diseases in humans. Here I applied single-molecule force spectroscopy to investigate molecular interactions determining the structural stability of the sodium/proton antiporter NhaA of Escherichia coli, which serves as a model system for this class of proteins. Mechanical pulling of NhaA molecules embedded in the native lipid bilayer caused a step-wise unfolding of the protein and provided insights into its stability. Modified experiments allowed observing refolding of NhaA molecules and estimating folding kinetics for individual structural elements, as well as detecting eventual misfolded conformations of the protein. The activity of NhaA increases 2000fold upon switching pH from 6 to 8. Single-molecule force measurements revealed a reversible change in molecular interactions within the ligand-binding site of the transporter at pH 5.5. The effect was enhanced in the presence of sodium ions. The observation suggests an early activation stage of the protein and provides new insights into the functioning mechanism. When studying interactions of NhaA with the inhibitor 2-aminoperimidine, I exploited single-molecule force measurements to validate the binding mechanism and to describe quantitatively formation of the protein:inhibitor complex. The ability of single-molecule force measurements to probe structurally and functionally important interactions of membrane proteins opens new prospects for using the approach in protein science and applied research.