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Richard Bach

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Quantum Dots and Prion Proteins: Is This a New Challenge for Neurodegenerative Diseases Imaging?

Alzheimer disease (AD) is characterized by the formation of senile plaques composed of the amyloid-ß (Aß) peptide and neurofibrillary tangles composed of hyperphosphorylated Tau. However, it is the accumulation of Aß in the brain that appears to be critical for the pathogenesis of AD. The prion protein (PrP, Fig. 1) is involved in neurodegeneration via its conversion from the normal cellular form, PrPC, to the infectious form, PrPSc, which is the causative agent of the transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease (CJD) 1-3. While there is an established role for PrPC in TSEs, the physiological role of PrPC has still not been fully elucidated. Roles in metal homeostasis, neuroprotective signalling, lymphocyte activation, neurite growth, synaptogenesis, cellular signalling, cell viability, and cellular response to oxidative stress have all been proposed 4-9. There are many neuropathological similarities and genetic links between AD and prion diseases. The coexistence of AD pathology in CJD has been reported 10 and PrPC has been shown to co-localise with Aß in plaques. These PrPC-Aß plaques have been found in most CJD patients with associated AD-type pathology and it has been proposed that PrPC may promote Aß plaque formation 11, 12. A genetic correlation between PrPC and AD has also been reported 13, 14. A systematic meta-analysis of AD genetic association studies revealed that the gene encoding PrPC (PRNP) is a potential AD susceptibility gene and the Met/Val 129 polymorphism in PRNP has been determined as a risk factor for early-onset AD 15.

2. Current diagnostics of prion protein related diseases
Diagnostics of infectious diseases is a relatively well-established area. It can be done either by the immunologic methods or by the amplification of a nucleic acid specific to infectious agent using polymerase chain reaction (PCR) 16. However, in TSEs, the infectious agent, prion protein, has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrPSc detection is that the pathological form of PrP is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges 17 as that hosts can incubate infectious prion proteins for many months or even years. During this period, they exhibit no overt clinical symptoms. Incubation period for some human prion diseases can be as long as 40 years 18. The incubation time of the first human transmissible spongiform encephalopathy “Kuru” described by Prof. Gajdusek in the late 1950s was estimated within the range from 21 to 40 years 19, 20.

2.1 Post mortem immunological detection of PrPSc
Generally, the majority of analytical diagnostic methods rely on the proteolytic removal of endogenous PrPC prior to detection of PrPSc (Fig. 1). PrPSc is relatively resistant towards proteolytic degradation whereas PrPC is entirely digested by Proteinase K. Identical treatment leads to removal of a variable number of N-terminal amino acids in case of PrPSc. This results in appearance of three distinct bands, corresponding to the di-, mono-, and unglycosylated form of PrP, upon Western blotting. Several techniques have been developed to detect PrPSc in brain tissues, including Western blot or other immunoblot methods 21-23. Quantitative Western blot analysis revealed the highest expression of PrPC in cerebellum, obex, and spinal cord. Intermediate levels were detected in thymus, intestine, nervous, heart, and spleen; lower levels were found in lung, muscle, kidney, lymph node, skin, pancreas, and liver 24. Western blotting coupled with gel electrophoresis is one of the immunodetection methods successfully used for detection of PrPSc in tissue extracts 25, 26. After denaturation of the tissue extract by heating with sodium dodecyl sulfate, it is analyzed by polyacrylamide gel electrophoresis and the denatured protein is transferred to a solid support and detected with an enzyme-labeled antibody, often of goat, rabbit and mouse. The specificity of Western blotting is based on the fact that proteolysis with proteinase K characteristically alters the molecular mass (app. 5 kDa) of the PrPc, due to the partial degradation of the N-terminal part of the protein 27. Immunohistochemical analysis detected intense cellular-specific PrPC staining in neurons, thymocytes and lymphocytes. PrPC was also detected in the enteric wall, pancreatic islets of Langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli, and dermal epithelial cells 24.

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