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High Dose

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Scientific papers - High dose - page 2

Phosmet-induced covalent modification of prion protein

It is hypothesized that the high dose OP warblecide phosmet induces a hitherto unrecognized abnormal irreversible post-translational modification of CNS PrP, most probably on its PI glycolipid anchor. This impairs tertiary folding of PrP, resulting in the formation of a conformationally deranged isoform of PrP. Thus, the biochemical genesis of prion formation involves a two-stage process, implicating a primary covalent modification of PrP which causes a secondary conformational modification of PrP during the tertiary folding process.

Phosmet is classed as a 'neuropathic' type of OP (9). Like other types of OP in this class, high doses of phosmet have been shown to modify certain membrane proteins such as neurotoxic esterase by inducing a covalent modification which produces an irreversible conformational change (10) via both direct phosphorylation/aging and alkylation of various active sites (6,11). It is therefore feasible to propose that phosmet could also directly modify the conformation of prion protein and/or its glycolipid anchor in some way, or perhaps indirectly, by influencing other proteins, etc. which are integral to the activities of PrP in cells. For instance, subacute low doses of 0Ps such as phosmet can disrupt turnover of the phosphoinositide second messenger cycle (12) which is interlinked to the activities of PrP and its PI anchor, or phosmet can inhibit glycosidases (13) which could impair/block deglycosylation at linked glycosylation sites on PrP (14). 0Ps such as phosmet can overexcite NMDA receptors (15) which would generate free radicals (16) that peroxidize membrane lipids such as PrP's PI anchor or disrupt essential electron interactions with tyrosine radicals in the main body of PrP. The further effects of OP increased turnover of glutathione peroxidase, superoxide dismutase (17) or cytochrome P450 activity would further aid the proliferation of neurodegeneration created by the OP-induced generation of free radical chain reactions.

Hypothetically, once PrP is abnormally modified and 'aged', the extra negative charge corrupting PrP's molecular surface subsequently disrupts interaction with foldases/isomerases or Van der Waal's forces during tertiary folding, which could lead to an influx of chaperones that bond onto misfolded PrP, blocking proteolytic/phospholipase C cleavage sites, causing the development of the new variant misfolded PrP isoform.

Whilst potential sites for phosphorylation on PrP have been researched by Stanley Prusiner's team (18), the characteristic acid-labile nature of these sites made such an investigation impossible to conclude due to the acidic conditions employed in the highperformance liquid chromatography purification procedure. This work did not recognize and take into account the well-recognized protein kinase C endogenous phosphorylation sites on the phosphatidylinositol glycolipid anchor that is conjugated onto the C terminus of PrP. However, studies investigating the activities of phosphorylating and dephosphorylating enzymes in the TSE diseased brain have demonstrated abnormally elevated levels (2), suggesting that some aspect of phosphorylation may play a role in the pathogenesis of the disease.

Like other neuropathic 0Ps, phosmet can covalently modify and cause conformational changes upon a diverse array of membrane and cytoskeletal proteins such as neurofilament protein, tubulins and microtubule-associated proteases (10,19-22). Some of these proteins are modified indirectly, by the secondary messenger 'feedback' mediator protein kinase C as a result of the OP's disturbing turnover of the phosphoinositide cycle (see biochemical section) or by the OP and/or calmodulin kinase 2's phosphorylating high-affinity neurotoxic binding sites (21).

An unconventional autoantibody binding onto the phosmet modified prion protein-chaperone complex (or onto other types of modified membrane proteins) could also set up the final stages of pathogenic complications creating the so-called 'infectious' protein end product of the prion disease process. The pathology of conventional autoinimune diseases is characterized by inflammation and mononuclear infiltrations, but the lack of the inflammatory response in TSE pathology could be attributed to the failure of the abnormal prion protein to perform its normal function in the pathways of lymphoid activation (23). Thus, phosmet-modified brain protein is then artificially inoculated into a healthy, uncontaminated secondary host, then that organism will mount its own autoantibody assault against the exogenous challenge, which, in turn, creates spongiform degeneration.

Several studies have demonstrated how autoantibodies are raised against OP-protein modified complexes in humans who have been occupationally exposed to low doses of these chemicals (24,25).

Interestingly, analysis of the prion rods that hallmark the CNS pathology of BSE brain reveals that prion protein is not the sole component; for the microtubule-associated protease, 'tau', and 'neurofilament protein' (8), as well as other unidentified tightly bound proteins (26) (possibly chaperones) are also found in association with PrPbse in these rod structures. The presence of these other proteins besides PrP does not necessarily imply that they represent mere incidental artefacts of the prion disease process, as some researchers suggest. They may also be fulfilling some hitherto unrecognized primary or secondary aetiological input into the pathogenesis of prion disease, albeit without unleashing the same devastating degree of 'infectivity' and pathogenic propensity to initiate long-term delayed neurotoxicity as has been attributed to the abnormal PrP isoform.

However, recent French research (27) has demonstrated that TSEs can be artificially transmitted into the recipient animals whose resulting CNS pathology fails to demonstrate the presence of prions at post mortem. This could suggest that some particle or site on other membrane/cytoskeletal proteins implicated in TSEs could also carry an 'infectious' pathogenic property (e.g. a phosmet-induced abnormal charge or free radical) in common with the 'infected' prion, such as the OP-modified PI anchor complex. Once injected into the healthy organism, that OP-modified PI is then capable of conjugating onto other types of membrane protein besides prion protein, thereby invoking a similar mode of steric hindrance/ misfolding in the main body of those proteins which manifests similar aspects of pathology and symptomology that are expressed in true prion disease.

'BSE positive', whose CNS pathology is characterized by prion rods, amyloid plagues and spongiform degeneration, could implicate an aetiology of 'in utero' phosmet intoxication where abnormal isoforms of both PrP and various other hitherto unrecognized delayed neurotarget proteins are implicated in the pathogenesis. On the other hand, 'BSE negative', whose pathology is neither characterized by prion rods nor spongifonn degeneration, involves an aetiology of 'postnatal' phosmet intoxication where various types of neurotarget protein are abnormally modified - and conceivably rendered 'infectious' - yet PrP is spared. One could also argue that 'BSE negative' represents the phosmet or other OP-poisoned individual who carries a PrP genotype which does not express susceptibility to prion disease.

Extremely high doses of these neuropathic OPs, as ingested during a suicide attempt or accidental poisoning, have produced a characteristic irreversible neuropathy in such misfortunate victims. It is proposed that when lower, subacute doses of the unique phthalimido-formulated neuropathic OP, phosmet, is absorbed by the CNS (perhaps during embryogenesis), then a hitherto unrecognized subtle modification to the prion and other membrane/cytoskeletal proteins is initiated. Once other TSE promoting secondary cofactors come into the play, a cascade of infectious prions is created. These partially proteaseresistant prions and associated proteins start accumulating over the 'so-called' incubation period until they reach critical pathogenic levels; whence outward symptoms of disease start to manifest years after the initial trigger event.

The characteristic spongiform pathology of BSE is caused, in part, by the failure of the misfolded. prion to perform its normal regulatory role at specific voltage-sensitive calcium channels, which leads to a rise in intracellular free calcium, resulting in the generation of nitric oxide free radicals (16), apoptosis and cell death (28,29). An unconventional autoantibody assault on the phosmet modified PrP-chaperone complex may ensue, whereby a tightly bound antibody attaches to modified PrP, impairing tertiary folding. Pathology lacks the usual inflammation characteristic of autoimmune disease because the abnormal prion is no longer capable of performing its normal role of activating lymphocytes (23).

The specific CNS distribution of the spongiform pathology may also be partly reflected by the failure of the misfolded. prion to perform its assumed role in transportation of copper ions to the copper-dependent neuronal systems, such as the dopaminergic tracts. Copper deficiency in these regions would further inactivate copper-dependent superoxide dismutase leading to the further accumulation of neurotoxic free radicals. Interestingly, both of the copper chelating chemicals, cuprizone (30) and disulfiram (31) produce a 'short circuited' type of 'pseudo' SE without having to exert a direct modification upon PrP's copper carriage sites. Interestingly, a high prevalence of SE in humans occupationally involved with disulfiram in the vulcanizing process of auto tyre manufacture has been reported (31).

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