Zinc, Carnosine, and Neurodegenerative Diseases.

Zinc (Zn) is abundantly present in the brain, and accumulates in the synaptic vesicles. Synaptic Zn is released with neuronal excitation, and plays essential roles in learning and memory. Increasing evidence suggests that the disruption of Zn homeostasis is involved in various neurodegenerative diseases including Alzheimer's disease, a vascular type of dementia, and prion diseases. Our and other numerous studies suggest that carnosine (ß-alanyl histidine) is protective against these neurodegenerative diseases. Carnosine is an endogenous dipeptide abundantly present in the skeletal muscles and in the brain, and has numerous beneficial effects such as antioxidant, metal chelating, anti-crosslinking, and anti-glycation activities. The complex of carnosine and Zn, termed polaprezinc, is widely used for Zn supplementation therapy and for the treatment of ulcers. Here, we review the link between Zn and these neurodegenerative diseases, and focus on the neuroprotective effects of carnosine. We also discuss the carnosine level in various foodstuffs and beneficial effects of dietary supplementation of carnosine.

[Innovation of therapeutics and prophylaxis for prion diseases].

There is no established treatment for prion diseases; however, recently several drug candidates, including pentosan polysulfate and doxycycline, have been clinically used on a trial basis to prevent accumulation of abnormal prion protein in the brain. So far the outcome of the trials is still very far from the goal where a complete cure of the diseases is expected. In order to bridge the gap between the reality and the ideal, the followings are suggested. First, combination therapy needs to be developed against multi-targets: inhibition of prion replication; degradation and scavengery of prion; inhibition of prion-related neurodegeneration. Secondly, preclinical diagnostic means, by which healthy prion-carriers can be revealed before the onset of the diseases, should be explored for earlier therapeutic interventions. The last is to disclose intrinsic disease susceptibility factors and environmental factors, both of which could solely or jointly facilitate in suppressing prion replication and disease progress. Exploitation of these items should be tough but will be deserved for overcoming the fatal diseases.

Prionet Canada: a network of centres of excellence for research into prions and prion diseases.

PrioNet Canada's strength in basic, applied, and social research is helping to solve the food, health safety, and socioeconomic problems associated with prion diseases. Prion diseases are transmissible, fatal neurodegenerative diseases of humans and animals. Examples of prion diseases include bovine spongiform encephalopathy (BSE, commonly known as "mad cow" disease), Creutzfeldt-Jakob disease in humans, and chronic wasting disease (CWD) in deer and elk. As of March 31, 2008, PrioNet's interdisciplinary network included 62 scientific members, 5 international collaborators, and more than 150 students and young professionals working in partnership with 25 different government, nongovernment, and industry partners. PrioNet's activities are developing strategies based on a sustained, rational approach that will mitigate, and ultimately control, prion diseases in Canada.

Antiprion properties of prion protein-derived cell-penetrating peptides.

In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.

The effect of high concentration potassium permanganate on protein contamination from metallic and synthetic rubber airway equipment.

We tested the hypothesis that supplementary cleaning using potassium permanganate 8 mg.l(-1) eliminates protein deposits from the reusable metallic and synthetic rubber airway equipment. Twenty Macintosh laryngoscope blades (surgical steel), 20 pairs of Magill's forceps (surgical steel) and 20 Guedel airways (synthetic rubber) were allocated to two groups for supplementary cleaning. In group A, the device was immersed in potassium permanganate 8 mg.l(-1). In group B (controls), the device was immersed in sterile water. The devices were then immersed in a protein staining solution, rinsed and the severity of staining was scored. In addition, the devices were inspected for tissue and then tested for occult blood. Protein contamination was lower in the potassium permanganate group for all devices (each device: p < 0.0001). There was no staining detected in the permanganate group. In the permanganate group, dried tissue was detected in the teeth of one pair of forceps, which was not detected following supplementary cleaning. Additionally, occult blood was detected on two pairs of forceps and a laryngoscope blade, which was not detected following supplementary cleaning. In the control group, no tissue was detected but one pair of forceps and two laryngoscope blades tested positive for occult blood before and after supplementary cleaning. We conclude that supplementary cleaning using potassium permanganate 8 mg.l(-1) eliminates protein deposits from re-usable metallic and synthetic rubber airway equipment.

Prophylactic effect of dietary seaweed Fucoidan against enteral prion infection.

Dietary seaweed fucoidan delays the onset of disease of enterally infected mice with scrapie when given orally for 6 days after infection, but not when given before the infection. This effect was not modified at a tested fucoidan dose range and appeared to reach the maximum level at a concentration of 2.5% or less in feed. Daily uptake of fucoidan might be prophylactic against prion diseases caused by ingestion of prion-contaminated materials, although further evaluation of its pharmacology remains to be done.

Chelating compound, chrysoidine, is more effective in both antiprion activity and brain endothelial permeability than quinacrine.

1. As an extension of our previous study of quinacrine and its derivatives, chelating chemicals were screened to obtain more effective, better brain-permeable antiprion compounds using either prion-infected neuroblastoma cells or brain capillary endothelial cells.2. Eleven chemicals were found to have antiprion activity. Most of them shared a common structure consisting of benzene or naphthalene at either end of an azo bond. Structure-activity data suggest that chelating activity is not necessary but might contribute to the antiprion action.3. Chrysoidine, a representative compound found here, was about 27 times more effective in the antiprion activity and five times more efficiently permeable through the brain capillary endothelial cells than quinacrine was.4. These chemicals might be useful as compounds for development of therapeutics for prion diseases.

The public health impact of prion diseases.

Several prion disease-related human health risks from an exogenous source can be identified in the United States, including the iatrogenic transmission of Creutzfeldt-Jakob disease (CJD), the possible occurrence of variant CJD (vCJD), and potential zoonotic transmission of chronic wasting disease (CWD). Although cross-species transmission of prion diseases seems to be limited by an apparent "species barrier," the occurrence of bovine spongiform encephalopathy (BSE) and its transmission to humans indicate that animal prion diseases can pose a significant public health risk. Recent reports of secondary person-to-person spread of vCJD via blood products and detection of vCJD transmission in a patient heterozygous at codon 129 further illustrate the potential public health impacts of BSE.

GC-MS detection of central nervous tissues as TSE risk material in meat products: analytical quality and strategy.

The detection of central nervous system (CNS) tissue (i.e. brain and spinal cord) by the use of GC-MS and certain fatty acids (FAs) as their methyl esters (FAMEs) was previously shown to be a very promising approach towards identification of CNS tissue as a specified risk material (SRM) in meat products, contrasting available immunochemical methods. This GC-MS method promised to allow quantification of CNS material as low as 0.01%. Here, we show that the CNS-relevant FAMEs C22:6, C24:1omega9, C24:1omega7, C24:0 and C24-OH are present in pure muscle and adipose tissue samples in detectable amounts. Thus, limits of detection are not feasible as quality parameters in this analytical GC-MS approach. Instead, cut-off values have to be applied as calculated from the baseline content of the respective FAME in CNS-free samples and its variation for a given statistical security. Furthermore, the FAs used for quantification of the CNS showed distinct differences depending on species and age. This finding is in accordance with previous studies where it had been concluded that species and age differentiation of CNS might be possible with GC-MS. However, it was not taken into account that it also necessitates a strict analytical strategy for quantification of the CNS content: identification of the presence of CNS (step 1); identification of species and age (step 2); and quantification by use of a species- and age-specific CNS calibration (step 3). Differences between the FA content of the CNS used for calibrating and the CNS in the sample will cause up to fivefold deviation from the true CNS content. Our results show that the FA best suited for identification (step 1) and quantification (step 3) purposes is cerebronic acid C24-OH after silylation. Further in-depth studies are needed in order to elucidate variability of brain FA content and to determine analytical limits. However, the present GC-MS approach is already a highly promising supplement to existing immunochemical methods for the detection of traces of CNS material in meat products.

Copper chelation delays the onset of prion disease.

The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrPSc). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(-)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p = 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p = 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p = 0.08) or blood (p = 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelator-based therapy.

Approaches to prophylaxis and therapy.

Despite important progress in experimental treatment of neurodegenerative diseases, no therapeutic strategy has today proven its capability to cure or even to stabilise human TSEs. Pathogenesis experiments performed in rodent TSE models have shown that central nervous system damages are detectable long before the appearance of the clinical symptoms. At the time of disease onset, PrP(Sc) accumulation has almost reached its highest level, and the neuropathological lesions (spongiosis, gliosis) are as intense as they are at the time of death. Therefore, the neurodegeneration that is present at the onset of the disease is beyond therapy, and, in theory, only a preclinical diagnosis of TSEs would permit the prevention (or delay) of neurodegeneration. Unfortunately, there are no diagnostic tests that can be used to show TSE agent infection during the preclinical phase of the disease. Nevertheless, since the appearance of variant Creutzfeldt-Jakob disease (vCJD), those in the scientific community working on experimental therapy have increased their efforts. Tens of drugs have been tested in several experimental models, and there are some high-output screening platforms being used in Europe and in the US. Any rational therapeutic strategy needs to be based on pathogenesis data and/or knowledge on the nature of the causative agent. Therefore, progress in therapy is tightly linked to a better understanding of the basic science of TSE.

Pentosan polysulfate as a prophylactic and therapeutic agent against prion disease.

Pentosan polysulfate (PPS) acts by imitating the physiological roles of the heparans. It binds to heparan binding sites on proteins and alters the physiological actions of these proteins. PPS acts as a prophylactic agent against infection with prions both in vivo and in vitro. Low concentrations (10 mg/ml) are needed extracellularly for this effect to be seen but, due to cellular uptake, it is believed that a much higher concentration is found intracellularly. The prophylactic effect of PPS is observed if the drug is administered to mice between 3 months before and approximately 30 days after the inoculation of the disease. After that point it is considered that the infection has entered the nervous system, and that the drug cannot penetrate the blood-brain barrier. The prophylaxis of humans with oral PPS and the current therapeutic activity of the drug when given by intracerebroventricular infusion to symptomatic, prion-infected animals are discussed.

[Handling of unconventional pathogens]. / Zum Umgang mit unkonventionellen Erregern.

This article summarizes decontamination problems in handling transmissible spongiform encephalopathies (TSE) in the field of human pathology. The combination of chemical (i.e. 1 M NaOH, 1 h, room temperature) and physical (i.e. autoclaving for 30 min at 131 degrees C) decontamination methods was proven to be suitable for instruments and other materials. Als for the autopsy of suspected TSE patients, safety measures have to be taken. The brain preparation, in particular, represents a considerable risk which must be minimized by appropriate safety measures. Conventional formaldehyde fixation does not decontaminate tissues! Embedding, cutting, deparaffinization and processing or staining have to be done with cautions; members of staff must be aware of the danger. After the handling of suspect materials, the hands should be washed without brushing the skin. In the case of percutaneous inoculation the injury must be decontaminated immediately using iodine- or phenol-containing preparations, 0.5 M NaOH or 1:3000 potassium permanganate.