Beta-amyloid (Aâ) remains to be the focus of research interest of the pathogenesis of Alzheimers disease (AD). Aâ is subject to oligomerization and its polymers are cytotoxic. Advanced aggregation leads to formation of senile plaques. Depositions of Aâ surrounding the cerebral vasculature, i.e. cerebral amyloid angiopathy (CAA), occur in most AD patients. The occurrence of Aâ aggregation in AD brains is not due to over-expression of amyloid precursor protein in most cases of AD. Factors influencing Aâ polymerization are yet to be established.<p> Aldehydes are highly reactive. They can cause protein crosslinkage. It is interesting to study whether endogenous aldehydes may be involved in Aâ polymerization process. In order to investigate the potential interaction of endogenous aldehydes with Aâ and their effects on its aggregation, various techniques including thioflavin T fluometry, dynamic light scattering, circular dichroism and atomic force microscopy were employed to assess Aâ aggregation at different stages. Formaldehyde, methylglyoxal, malondialdehyde and 4-hydroxyl-nonenal were found to enhance Aâ â-sheets formation, oligomerization and fibrillogenesis in vitro. The sizes of the oligomers are increased after interaction with the aldehydes. Lysine residues of Aâ were identified to be the primary site of interaction with aldehydes by forming Schiff bases, which may subsequently lead to intra- and inter-molecular crosslinkage. Aldehydes can also crosslink Aâ with other proteins such as apolipoprotein E and á2-macroglobulin (á2M), to form large complexes. Results suggest that aldehydes substantially increase the rate of Aâ oligomerization at each stage of fibrillogenesis.<p> The native and formaldehyde-modified Aâ oligomers were isolated by size exclusion chromatography and their cytotoxic effects towards SH-SY5Y neuroblastoma cells were assessed using MTT, LDH and caspase-3 activity assays. The aldehyde-modified oligomers are slightly but significantly more cytotoxic compared to the native oligomers. Since aldehydes significantly increase the production of Aâ oligomers, an increase in aldehydes would enhance the total cytotoxicity, suggesting that aldehydes may potentially exacerbate neurovascular damage and neurodegeneration caused by Aâ.<p> Low-density lipoprotein receptor related protein-1 (LRP-1) plays a crucial role in Aâ clearance via the cerebral vasculature. Semicarbazide-sensitive amine oxidase (SSAO) and LRP-1 are both richly expressed on the vascular smooth muscle cells (VSMCs). We demonstrated that SSAO-mediated deamination affects LRP-1 function using isolated VSMCs. Formaldehyde at low concentrations decreases LRP-1-mediated uptake of á2M, a substrate of LRP-1 and a carrier for Aâ. Methylamine, an SSAO substrate that is converted to formaldehyde, also inactivates LRP-1 function, but not in the presence of an SSAO inhibitor. Increased SSAO-mediated deamination can potentially impair Aâ clearance via LRP-1.<p> In conclusion, aldehydes derived from oxidative stress and SSAO-mediated deamination induce Aâ aggregation, enhance Aâ cytotoxicity and impair Aâ clearance. The exclusive localization of SSAO on the cerebral vasculature may be responsible for the perivascular deposition of Aâ, i.e. CAA, which is associated both with vascular dementia and with AD. Vascular surface SSAO may be a novel pharmacological target for the treatment of AD.

Beta-amyloid (Aâ) remains to be the focus of research interest of the pathogenesis of Alzheimers disease (AD). Aâ is subject to oligomerization and its polymers are cytotoxic. Advanced aggregation leads to formation of senile plaques. Depositions of Aâ surrounding the cerebral vasculature, i.e. cerebral amyloid angiopathy (CAA), occur in most AD patients. The occurrence of Aâ aggregation in AD brains is not due to over-expression of amyloid precursor protein in most cases of AD. Factors influencing Aâ polymerization are yet to be established.<p> Aldehydes are highly reactive. They can cause protein crosslinkage. It is interesting to study whether endogenous aldehydes may be involved in Aâ polymerization process. In order to investigate the potential interaction of endogenous aldehydes with Aâ and their effects on its aggregation, various techniques including thioflavin T fluometry, dynamic light scattering, circular dichroism and atomic force microscopy were employed to assess Aâ aggregation at different stages. Formaldehyde, methylglyoxal, malondialdehyde and 4-hydroxyl-nonenal were found to enhance Aâ â-sheets formation, oligomerization and fibrillogenesis in vitro. The sizes of the oligomers are increased after interaction with the aldehydes. Lysine residues of Aâ were identified to be the primary site of interaction with aldehydes by forming Schiff bases, which may subsequently lead to intra- and inter-molecular crosslinkage. Aldehydes can also crosslink Aâ with other proteins such as apolipoprotein E and á2-macroglobulin (á2M), to form large complexes. Results suggest that aldehydes substantially increase the rate of Aâ oligomerization at each stage of fibrillogenesis.<p> The native and formaldehyde-modified Aâ oligomers were isolated by size exclusion chromatography and their cytotoxic effects towards SH-SY5Y neuroblastoma cells were assessed using MTT, LDH and caspase-3 activity assays. The aldehyde-modified oligomers are slightly but significantly more cytotoxic compared to the native oligomers. Since aldehydes significantly increase the production of Aâ oligomers, an increase in aldehydes would enhance the total cytotoxicity, suggesting that aldehydes may potentially exacerbate neurovascular damage and neurodegeneration caused by Aâ.<p> Low-density lipoprotein receptor related protein-1 (LRP-1) plays a crucial role in Aâ clearance via the cerebral vasculature. Semicarbazide-sensitive amine oxidase (SSAO) and LRP-1 are both richly expressed on the vascular smooth muscle cells (VSMCs). We demonstrated that SSAO-mediated deamination affects LRP-1 function using isolated VSMCs. Formaldehyde at low concentrations decreases LRP-1-mediated uptake of á2M, a substrate of LRP-1 and a carrier for Aâ. Methylamine, an SSAO substrate that is converted to formaldehyde, also inactivates LRP-1 function, but not in the presence of an SSAO inhibitor. Increased SSAO-mediated deamination can potentially impair Aâ clearance via LRP-1.<p> In conclusion, aldehydes derived from oxidative stress and SSAO-mediated deamination induce Aâ aggregation, enhance Aâ cytotoxicity and impair Aâ clearance. The exclusive localization of SSAO on the cerebral vasculature may be responsible for the perivascular deposition of Aâ, i.e. CAA, which is associated both with vascular dementia and with AD. Vascular surface SSAO may be a novel pharmacological target for the treatment of AD.

No description available.

Phenelzine (PLZ) is a monoamine oxidase (MAO) inhibitor that also inhibits the activity of GABA-transaminase (GABA-T), causing significant and long-lasting increases in brain GABA levels. Inhibition of MAO prior to PLZ administration has been shown to prevent the GABAergic effects of the drug, strongly suggesting that a metabolite of PLZ formed by the action of MAO is responsible for the GABAergic effects. While PLZ has been used clinically for decades for its antidepressant and antipanic effects, it has more recently been shown to be neuroprotective in an animal model of ischemia. The aim of the experiments described in this thesis was to identify the active metabolite of PLZ, and to determine the neurochemical mechanisms by which PLZ and this metabolite exert their neuroprotective effects (with a particular focus on degenerative mechanisms observed in cerebral ischemia and Alzheimers disease (AD)). The development of an analytical assay for -phenylethylidenehydrazine (PEH) was a major breakthrough in this project and permitted the positive identification of this compound as the active metabolite of PLZ. Further experiments demonstrated that PLZ and PEH could be neuroprotective in cerebral ischemia and AD not only by reducing excitotoxicity via increased GABAergic transmission, but also by (a) increasing brain ornithine, which could potentially lead to a decrease in glutamate synthesis and/or a decrease in polyamines (whose metabolism produces toxic aldehydes); (b) inhibiting the activity of human semicarbazide-sensitive amine oxidase (SSAO), an enzyme whose activity is increased in AD producing excessive amounts of the toxic aldehyde formaldehyde (FA); (c) by sequestering FA in vitro, forming a non-reactive hydrazone product. Since PEH appears to mediate or share the neurochemical effects of PLZ, two propargylated analogs of PEH were synthesized and tested for their potential as PEH prodrugs. Surprisingly these analogs were not particularly effective prodrugs in vivo, but they possessed an interesting neurochemical properties on their own (the ability to elevate brain levels of glycine), and warrant further investigation as potential antipsychotic agents. Together, these results suggest that PLZ and its active metabolite, PEH, should be further investigated for their neuroprotective potential in cerebral ischemia and in AD. / Neurochemistry

<p>The parasite <i>Echinococcus granulosus</i> causes hydatid disease, a major zoonosis. A fatty-acid-binding protein, EgFABP1, is important for the parasite, as it must acquire almost all its lipids from its environment or the host. The structure of EgFABP1 has been solved and refined to 1.6 Å resolution. The structure reveals that EgFABP1 has the 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins. </p><p>Human semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6), also known as vascular adhesion protein-1, is a copper-containing monoamine oxidase that occurs both as a membrane-bound protein and in a soluble form in plasma. SSAO has been implicated in glucose transport in adipocytes, the differentiation of adipose cells and the leukocyte extravasation process. Toxic reaction products have been suggested to cause some of the vascular complications associated with diabetes and SSAO is therefore of pharmaceutical interest.</p><p>The structure of a truncated, soluble form of human SSAO has been determined to 2.5 Å resolution. The structure reveals that a leucine residue located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five out of six potential N-glycosylation sites. Carbohydrates attached to Asn 232 flank the active site entrance and might influence substrate specificity. The structure also reveals a vicinal disulfide bridge, which we hypothesise could act as a redox switch involved in the protein’s mechanism of action. The structure of a complex of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 Å resolution. Both structures together will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies. </p>

The parasite Echinococcus granulosus causes hydatid disease, a major zoonosis. A fatty-acid-binding protein, EgFABP1, is important for the parasite, as it must acquire almost all its lipids from its environment or the host. The structure of EgFABP1 has been solved and refined to 1.6 Å resolution. The structure reveals that EgFABP1 has the 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins. Human semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6), also known as vascular adhesion protein-1, is a copper-containing monoamine oxidase that occurs both as a membrane-bound protein and in a soluble form in plasma. SSAO has been implicated in glucose transport in adipocytes, the differentiation of adipose cells and the leukocyte extravasation process. Toxic reaction products have been suggested to cause some of the vascular complications associated with diabetes and SSAO is therefore of pharmaceutical interest. The structure of a truncated, soluble form of human SSAO has been determined to 2.5 Å resolution. The structure reveals that a leucine residue located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five out of six potential N-glycosylation sites. Carbohydrates attached to Asn 232 flank the active site entrance and might influence substrate specificity. The structure also reveals a vicinal disulfide bridge, which we hypothesise could act as a redox switch involved in the protein’s mechanism of action. The structure of a complex of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 Å resolution. Both structures together will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies.