Reduced transferrin binding in Down syndrome:a route to senile plaque formation and dementia

Plasma transferrin binding in Down syndrome and Alzheimer's disease is significantly reduced compared with age matched controls and it was thought this may help elucidate a pathological time sequence for the onset of dementia in Down syndrome. In Down syndrome, there was a reduction in gallium and aluminium transferrin binding both with age and the onset of dementia. Non-transferrin bound gallium species were identified as non-transportable phosphate or silicate. Thus, the route of entry of metals into the brain must be via a transferrin mediated complex only. A clear sequence of pathological events has been demonstrated in Down syndrome which shows the pathway to development of plaques and dementia and this is believed to have an immunological origin.

Gallium-transferrin binding in treated and untreated Parkinson's disease

The binding of gallium (Ga) to transferrin (Tf) was studied in plasma from control patients, in patients with untreated Parkinson's disease (PD) and in patients with PD treated either with levodopa (L-dopa) alone or in combination with selegiline. Mean percentage Ga-Tf binding was significantly reduced in untreated and treated PD compared with controls. Binding, however, was significantly greater in treated than in untreated patients. There was no difference in binding between patients treated with L-dopa alone and those treated with L-dopa and selegiline. The data support the hypothesis that oxidation reactions may be of pathogenic significance in PD.

Serum neopterin levels and gallium transferrin binding in Parkinson's disease

The unconjugated pterin neopterin is secreted by macrophages activated by interferon-gamma and hence, the level of neopterin in serum may be used as a marker of a cellular immune response in a patient. Serum neopterin levels were measured by high performance liquid chromatography (HPLC) in 28 Parkinson's disease (PD) patients and 28 age and sex matched controls. The level of serum neopterin was significantly elevated in PD compared with controls suggesting immune activation in these patients. The level of neopterin was negatively correlated with the level of binding of gallium to transferrin (Tf) but unrelated to the level of iron binding. Hence, in PD, it is possible that a cellular immune response may be important in the pathogenesis of the disease. One effect of the cellular immune response may be a reduction in the binding of metals other than iron to Tf and this could also be a factor in PD.

Metal binding to transferrin and immune reactions in Parkinson's disease

The binding of iron (59Fe) and gallium (67Ga) to the plasma protein transferrin (Tf) was investigated by G75 gel filtration chromatography in control patients and treated and untreated patients with Parkinson's disease (PD). Fe-Tf binding was 100% in all controls and PD patients suggesting that a defect in Fe-Tf binding was not involved in the aetiology of PD. Ga-Tf binding was significantly reduced in both untreated and treated PD patients compared to controls. In addition, treated PD patients had significantly higher Ga-Tf binding than untreated patients. A reduction in metal binding to Tf could result in the increase of a low molecular weight species which may more readily enter the CNS. Alternatively, it could lead to a decrease in the transport of essential metals into the brain via the Tf receptor system. A significant elevation in neopterin was demonstrated within the plasma of untreated PD patients compared to controls suggesting the activation of a cellular immune response. Furthermore, plasma neopterin was lower in treated compared to untreated PD patients, although the difference was not significant. There was no evidence for the activation of the humoral immune response in untreated or treated PD patients as measured by circulating immune complex (CIC) levels within the plasma. An inverse relationship between Ga-Tf binding and neopterin was observed in untreated PD patients. The addition of oxidants in the form of potassium permanganate and activated manganese dioxide reduced Ga-Tf binding in control plasma. However, relatively little response was observed using monocyte preparations. The results suggest that oxidants produced by activation of the cellular immune system could damage the Tf molecule thereby reducing its ability to bind metals.

Reduced metal transferrin binding in neurological diseases

By employing G75 gel-filtration chromotography, it has been demonstrated that human plasma gallium speciation (and by implication, Al speciation) is bimodal. Normally, gallium was predominantly bound to a high molecular weight fraction which was presumably transferrin. Literature reviews and experimental work throughout this thesis provided evidence to support this idea. An aluminium-transferrin species was assumed to be relatively non-toxic and a protective function for this complex has been suggested. A second, low molecular weight species of gallium was observed and its identity has been suggested to be citrate. The results of this thesis support the concept citrate was a gallium binding ligand present in the plasma, but there was another species (tentatively identified as phosphate) which bound gallium to a much greater degree than did citrate in the majority of samples studied. The consequence of a low molecular weight species of aluminium is the possibility that this leads to a more rapid, uncontrolled deposition of the metal in the brain compared to a transferrin mediated mechanism. Plasma speciation studies in Alzheimer's disease, Parkinson's disease, Down's syndrome, and neonates has revealed an altered ratio of the two gallium species found in control subjects. In all groups there was an increase in the potentially more neurotoxic low molecular weight species. These observations have led to a suggested mechanism of accumulation of metals in the brain, which is known to occur in the first three groups. Possible pathogenic mechanisms are described. The results can also offer an explanation to the reported increased sensitivity to the toxic effects of aluminium in the neonate. Speciation studies on normal plasma has shown the balance between high and low molecular weight species of gallium to be influenced by many physiological factors. There appears to be a fine equilibrium between both species which can be altered without any great difficulty. Therefore, in the diseased groups studied, it is possible that there are subtle biochemical changes within the circulatory system to affect the equilibrium which results in an increased low molecular weight species of aluminium. Furthermore, it has been demonstrated that there is a group of normal controls with no clinical signs of Alzheimer's or Parkinson's disease which have reduced transferrin binding. This indicates there is a population of healthy people who are at risk to the development of either disease.

Terminal galactose residues on transferrin are increased in mid-life adults compared to young adults

Humans undergo biological ageing at different rates. This associates with functional decline in a number of physiological systems and increasing incidence of age-related pathologies. The discovery of robust biomarkers of ageing could be used to identify early divergence from a path of healthy ageing towards age-related disease. In the present study, we undertook proteomic analysis of plasma from healthy young men (mean age = 21.4 ± 1.5 years) and healthy mid-life men (mean age = 57.0 ±1.6 years). We identified twelve spots including transferrin, complement C3b and transthyretin that differed in abundance between the age groups. Transferrin spots showed an acidic pI shift in older males. Sandwich ELISAs were used to investigate the changes further. C3b levels were below the level of detection by ELISA and plasma concentrations of total transferrin or transthyretin were not different between the age groups studied here. However, analysis of transferrin N-glycan structures showed an increase in terminal galactose residues in older men, suggesting that the loss of terminal N-acetyl neuraminic acid residues contributes to the more acid pI of transferrin spots observed with age. Terminal galactosylation of transferrin may be a biomarker of healthy ageing and is now under investigation in the MARKAGE study.

Role of fibroblast growth factor receptor in regulation of membrane traffic

Several studies show that membrane transport mechanisms are regulated by signalling molecules. Recently, genome-wide screen analyses in C.elegans have enabled scientists to identify novel regulators in membrane trafficking and also signalling molecules which are found to couple with this machinery. Fibroblast growth factor (FGF) via binding to fibroblast growth factor receptor (FGFR) mediate signals which are essential in the development of an organism, patterning, cell migration and tissue homeostasis. Impaired FGFR-mediated signalling has been associated with various developmental, neoplastic, metabolic and neurological diseases and cancer. In this study, the potential role of FGFR-mediated signalling pathway as a regulator of membrane trafficking was investigated. The GFP-tagged yolk protein YP170-GFP trafficking was analysed in worms where 1) FGFR signalling cascade components were depleted by RNAi and 2) in mutant animals. From these results, it was found that the disruption of the genes egl-15 (FGFR), egl-17(FGF), let-756(FGF), sem-5, let-60, lin-45, mek-2, mpk-1 and plc-3 lead to abnormal localization of YP170-GFP, suggesting that signalling downstream of FGFR via activation of MAPK and PLC-γ pathway is regulating membrane transport. The route of trafficking was further investigated, to pinpoint which membrane step is regulated by worm FGFR, by analysing a number of GFP-tagged intracellular membrane markers in the intestine of Wild Type (WT) and FGFR mutant worms. FGFR mutant worms showed a significant difference in the localisation of several endosomal membrane markers, suggesting its regulatory role in early and recycling steps of endocytosis. Finally, the trafficking of transferrin in a mammalian NIH/3T3 cell line was investigated to identify the conservation of these membrane trafficking regulatory mechanisms between organisms. Results showed no significant changes in transferrin trafficking upon FGFR stimulation or inhibition.

Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalytic

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Tetrahydrobiopterin metabolism in mental disorders

Changes in DHPR activity in those aged 12 and under with a variety of mental disorders were investigated using dried blood spots on Guthrie cards. DHPR activity was found to be lowered in autism and Rett's syndrome. DHPR activity was unaffected in non specific mental retardation suggesting that the deficit seen in autism and Rett's syndrome does not arise secondary to the mental dysfunction. In Down's syndrome blood biopterin levels correlated with blood spot DHPR activity. Human brain BH4 synthetic activity was investigated in aging and senile dementia of the Alzheimer type (SDAT). BH4 synthetic activity and DHPR activity decline with age in non-demented controls. In SDAT, decreases in BH4 synthetic activity were seen in temporal and visual cortices and locus coeruleus. The site of the defect is probably at 6-pyruvoyl-tetrahydropterin synthase. Aluminium inhibits human brain BH4 synthesis in vitro and produces an `Alzheimeresque' pattern of abnormalities in rats chronically exposed to the acetate salt in drinking water. Aluminium appears to chiefly affect enzymes requiring a metal ion cofactor. Aluminium induced inhibition of BH4 synthesis can be reversed by treatment with transferrin, an aluminium chelator. Transferrin treatment improves BH4 synthetic activity in SDAT brains whilst having no effect on controls, further implicating aluminium as the key neurotoxin in SDAT. Lithium inhibits human brain BH4 synthesis in vitro and lowers rat brain total biopterins and inhibits rat brain BH4 synthesis on chronic exposure to the carbonate salt in drinking water. A possible mechanism for the anti-manic actions of lithium is suggested. Monoamine oxidase inhibitors decrease human brain BH4 synthetic activity in vitro. 5-methyl-tetrahydrofolate had no effect on human brain BH4 synthesis in vitro but methionine increased BH4 synthesis in vitro. Oxotremorine is a potent inhibitor of BH4 synthesis in man and the rat. This may prove useful as a tool for modelling BH4 deficiency.

Influence of iron on bacterial infections in leukaemia

The influence of iron metabolism, both on the invading bacterial pathogen and in the host is widespread and often appears to be crucial in determining the outcome of an infection. This study involved the investigation of leukaemia, a clinical disease where abnormal availability of iron may play a part in predisposing patients to bacterial infection. The iron status throughout a Gram-negative septicaemia and in 20 random, newly diagnosed leukaemic patients was assessed. The results revealed that the majority of the patients exhibited high serum iron levels and serum transferrin saturation often at 100%, with an inability to reduce the latter to within normal values during an infection episode. The antibody response to P.aeruginosa, E.coli and K.pneumoniae outer membrane protein (OMP) antigens were investigated by immunoblotting with sequential serum samples during infection in the leukaemic host. Antibodies to all the major OMPs, were observed, although recognition of iron-regulated membrane proteins (IRMPs) was in many cases weak. Results from the enzyme-linked immunosorbent assay indicated that in all patients antibody titre in response to infection was poor. Sub-MICs of mitomycin C significantly altered the surface characteristics of P.aeruginosa. The silver-stained SDS-PAGE gels of proteinase K digested whole cell lysates of strains PAO1, 6750, M7 and PAJ indicated that core LPS was affected in the presence of mitomycin C. In contrast, the rough strain AK1012 showed no observable differences. Results obtained using quantitative gas-liquid chromatographic analysis showed the amount of LPS fatty acids to be unaffected, however, the KDO and carbohydrate content in strains PAO1, 6750 and M7 under Fe+ and Fe- growth conditions were decreased by up to 4-fold in the presence of mitomycin C, indicating perturbed expression of LPS. The cell surface became significantly more hydrophobic in the P.aeruginosa strains, except AK1012 which was comparatively unaffected. The induction of protein G (OprG) in P.aeruginosa was found to be a sensitive indicator of media iron. The data indicated that expression of OprG can be modulated by growth rate/phase, availability of iron and by the presence of ciprofloxacin in the growth medium.

Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides

BACKGROUND & AIMS: The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunolglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. METHODS: We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgACD71 complexes and intestinal permeability to the gliadin 3H-p3149 peptide were analyzed in polarized monolayers of Caco-2 cells. RESULTS: Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgACD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p3149, transport of intact 3H-p3149 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. CONCLUSIONS: Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apicalbasal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

The mammalian phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) regulates endosome-to-TGN retrograde transport

The yeast gene fab1 and its mammalian orthologue Pip5k3 encode the phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinases Fab1p and PIKfyve, respectively, enzymes that generates phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. A shared feature of fab1Delta yeast cells and mammalian cells overexpressing a kinase-dead PIKfyve mutant is the formation of a swollen vacuolar phenotype: a phenotype that is suggestive of a conserved function for these enzymes and their product, PtdIns(3,5)P(2), in the regulation of endomembrane homeostasis. In the current study, fixed and live cell imaging has established that, when overexpressed at low levels in HeLa cells, PIKfyve is predominantly associated with dynamic tubular and vesicular elements of the early endosomal compartment. Moreover, through the use of small interfering RNA, it has been shown that suppression of PIKfyve induces the formation of swollen endosomal structures that maintain their early and late endosomal identity. Although internalisation, recycling and degradative sorting of receptors for epidermal growth factor and transferrin was unperturbed in PIKfyve suppressed cells, a clear defect in endosome to trans-Golgi-network (TGN) retrograde traffic was observed. These data argue that PIKfyve is predominantly associated with the early endosome, from where it regulates retrograde membrane trafficking to the TGN. It follows that the swollen endosomal phenotype observed in PIKfyve-suppressed cells results primarily from a reduction in retrograde membrane fission rather than a defect in multivesicular body biogenesis.

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.