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Autophagy is a degradative mechanism involved in recycling and turnover of cytoplasmic components, including organelles. Eukaryotic cells have two major protein degradation systems; one is the ubiqutin-proteasome system, the other is lysosomal mechanism. Proteins can be transported to lysosome following at least four pathways; endocytosis, macroautophagy, microautophagy and chaperone mediated-autophagy. Autophagy (macroautophagy) is mediated by an autophagosome, a double membrane organelle containing undigested proteins and damaged organelles. Autophagy is associated with several pathological conditions including neurodegenerating diseases. One of the main pathological hallmarks of neurodegeneration is loss of neurons. Apoptosis was reported in several neurodegeneration diseases to be not high enough to account for the loss of neurons so autophagy was thought to be involved in neurodegenerative processes. We studied the ultrastructural signs of autophagy in prion diseases: in brain biopsies from Creutzfeldt-Jakob disease and fatal familial insomnia patients and in experimental scrapie in hamsters. Our biopsy material consisted of brain biopsies obtained by open surgery from 1 fatal familial insomnia case, 1 case of variant Creutzfeldt-Jakob disease, 7 cases of sporadic Creutzfeldt-Jakob disease and 1 case of iatrogenic (human growth hormone) Creutzfeldt-Jakob disease as well as brains of hamsters inoculated with the 263 K or 22C-H strains of scrapie. For electron microscopy, approximately 2 cubic mm samples were immersion fixed in 2.5% glutaraldehyde for less than 24 hours, embedded in Epon and routinely processed. Grids were examined in a transmition electron microscope. In many cells a large area of the cytoplasm was transformed into a collection of autophagic vacuoles of different sizes. Autophagic vacuoles formed not only in neuronal perikarya but also in neurites and synapses of all categories of studied human transmissible encephalopathies and in experimental animals. Autophagic vesicles developed also within dystrophic axons. On a basis of ultrastructural studies, we suggest that autophagy plays a major role in transmissible spongiform encephalopathies and may even participate in a formation of spongiform change.

In Creutzfeldt-Jakob disease (CJD), the type (type 1 and type 2) of abnormal isoform of prion protein (PrP^Sc) in the brain and the genotype at codon 129 of the PrP gene are major determinants of the clinicopathological phenotype. Type 1 and type 2 PrP^Sc are distinguished by the size of proteinase K (PK) resistant core (21 and 19 kDa), reflecting differences in the PK-cleavage site. Moreover, type 2 PrP^Sc can be subclassified into type 2A and type 2B by the difference in ratio of glycoforms. However, little is known about the difference in biochemical properties between the two types of PrP^Sc, exept for the different PK-cleavage sites. On the basis of these findings, we hypothesized that (1) type 1 and type 2 PrP^Sc may have distinct aggregation sizes and (2) PrP^Sc from a patient with PrP amyloid plaques in the brain has a larger aggregation size than PrP^Sc from a patient with synaptic non-amyloid type PrP deposits in the brain.

To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant protein in patients affected with prion diseases and the control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by the incubation of secondary antibodies alone, i.e., in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that majority of the PK-resistant 37 kDa proteins in patients’ urine were outer membrane proteins (OMPs) of Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, non-specific binding of OMPs to antibodies could be mistaken for PrP^Sc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrP^Sc in clinical samples, in particular, urine.

Total tau protein (t-tau) in CSF is now considered as a diagnostic biochemical marker of CJD. Especially, analysis of t-tau protein level in CSF is semi-quantitative, compared to western blot analysis of 14-3-3 protein in CSF. Also, MRI is diagnostic procedure of CJD. Diffusion-weighted MRI (DWI) is more diagnostic in early phase of CJD. We report here the comparative analysis of t-tau protein and MR images, which have been serially performed in the clinical course of sporadic CJD patients.

To evaluate the usefulness of clinical examinations in the diagnosis of CJD, we examined duration between initial manifestation of CJD and detection of PSD, specific findings on MRI, 14-3-3 protein, or high NSE. We analyzed data collected by Japanese CJD surveillance committee statistically. Patients consisted of 405 sporadic CJD and 52 infectious CJD. Average duration between the onset and detection of PSD, specific findings on MRI, 14-3-3 protein in the CSF, or high NSE in the CSF in the total cases was 3.6, 4.4, 4.8, or 3.6 months, respectively. There was no significant difference among 4 examinations (P=0.12). Peak of duration until positive result on MRI, 14-3-3 protein, or NSE was 1 month while that on EEG was 2 months. Similar result was obtained when we analyzed the subgroup of sporadic CJD. The sensitivity of EEG, MRI, 14-3-3 protein, or NSE in the definite cases was 72.4, 77.1, 74.2, 50.0 %, and the performed rate in the total cases was 99.1, 75.9, 36.5, 44.0 %, respectively as we showed in the previous report partially. The sensitivity and performed rate of EEG were high and duration until positive result was short while the number of detection of PSD reached its peak 2 months after the onset in contrast with 1 month as in the other examinations. Conventionality in diagnosis of CJD and inclusion in the criteria fot CJD might influence on high performed rate and short average duration. Although some researches reported very early detection of specific findings on MRI, the average duration until the detection of specific MRI findings was not significantly shorter than those on the other examinations in Japanese CJD surveillance. This might be due to lower recognition rate of usefulness of DWI in diagnosis of CJD. The sensitivity of NSE in the definite cases was lowest although the difference of the sensitivity was not significant among 4 examination (p=0.063). Low performed rate of 14-3-3 protein or NSE was remarkable. In conclusion, analysis of data from Japanese CJD surveillance revealed no significant difference of duration between the onset and detection of positive results among 4 examinations. Further spread of recognition of usefulness of DWI in the diagnosis of CJD might shorten duration between the onset and detection of positive result on MRI.

Transmissible spongiform encephalopathies (TSE) are transmissible, neurodegenerative disorders affecting humans and animals. Although the etiological agent of TSE has yet to be conclusively identified, a unifying feature is the accumulation of an abnormal protease-resistant isomer (PrP^Sc) of the host encoded prion protein (PrP^C). PrP^C expression is required for the transmission and pathogenesis of TSE disease. Despite the ubiquitous organ distribution of PrP^C, TSE pathogenesis and infectivity is principally restricted to the central nervous and lymphoreticular systems. However, recent studies have highlighted the presence of low levels of PrP^Sc and TSE infectivity in some peripheral tissues including muscle and the possible transmission of variant Creutzfeldt Jakob disease through blood transfusion. This has raised concerns regarding the risk of iatrogenic transmission of TSE disease through surgical procedures and organ or blood donation.