Intrinsic resistance of oligodendrocytes to prion infection.

Within the CNS, the normal form of cellular prion protein (PrP(C)) is expressed on neurons, oligodendrocytes, and astrocytes. The contribution of these cell types to prion replication and pathogenesis is unclear. To assess the role of oligodendrocytes, we expressed PrP(C) under the control of the myelin basic protein (MBP) promoter in mice lacking endogenous PrP(C). PrP(C) was detected in oligodendrocytes and Schwann cells but not in neurons and astrocytes. MBP-PrP mice never developed scrapie after intracerebral, intraperitoneal, or intraocular challenge with scrapie prions. Transgenic brains did not contain protease-resistant prion protein and did not transmit scrapie when inoculated into PrP(C)-overexpressing indicator mice. To investigate whether prion spread within the CNS depends on oligodendrocytic PrP(C), we implanted PrP(C)-overexpressing neuroectodermal grafts into MBP-PrP brains. After intraocular prion inoculation, none of the grafts showed spongiform encephalopathy or prion infectivity. Hence oligodendrocytes do not support cell-autonomous prion replication, establishment of subclinical disease, and neural spread of prions. Prion resistance sets oligodendrocytes aside from both neurons and astrocytes.

Accumulation of prion protein in muscle fibers of experimental chloroquine myopathy: in vivo model for deposition of prion protein in non-neuronal tissues.

Prion protein (PrP) is known to accumulate in some non-neuronal tissues under conditions unrelated to prion diseases. The biochemical and biological nature of such accumulated PrP molecules, however, has not been fully evaluated. In this study, we established experimental myopathy in hamsters by long-term administration of chloroquine, and we examined the nature of the PrP molecules that accumulated. PrP accumulation was immunohistochemically demonstrated in autophagic vacuoles in degenerated muscle fibers, and this was accompanied by the accumulation of other molecules related to the neuropathogenesis of prion diseases such as clathrin, cathepsin B, heparan sulfate, and apolipoprotein J. Accumulated PrP molecules were partially insoluble in detergent solution and were slightly less sensitive to proteinase K digestion than normal cellular PrP. Muscle homogenates containing these PrP molecules did not cause disease in inoculated hamsters. The findings indicate that the PrP molecules that accumulated in muscle fibers have distinct biochemical and biological properties. Therefore, experimental chloroquine myopathy is a novel and useful model to investigate the mechanism of deposition of PrP in non-neuronal tissues and might provide new insights in the pathogenesis of prion diseases.

A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins.

Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and 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. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) 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 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 the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. 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 PrPSc in clinical samples, in particular, urine.

Results of quinacrine administration to patients with Creutzfeldt-Jakob disease.

Several chemicals inhibit the accumulation of abnormal prion proteins in vitro. We administered one, the antimalarial agent quinacrine, to three patients with sporadic Creutzfeldt-Jakob disease (CJD) and to one with iatrogenic CJD. Quinacrine at 300 mg/day was given enterally for 3 months. Within 2 weeks of administration, the arousal level of the patient with akinetic mutism improved. The other 3 patients, insensible before treatment, had integrative responses such as eye contact or voluntary movement in response to verbal and/or visual stimuli restored. Clinical improvement was transient, lasting 1-2 months during treatment. Quinacrine was well tolerated, except for liver dysfunction and yellowish pigmentation. Although its antiprion activity in the human brain has yet to be proved, these modest effects of quinacrine suggest the possibility of using chemical intervention against prion diseases.

[Prospects of the therapeutic approaches to Creutzfeldt-Jakob disease: a clinical trial of antimalarial, quinacrine].

Prion diseases are lethal transmissible neurodegenerative illnesses that affect humans and many other animals. Since the accumulation of the pathogenic form of prion protein is a pivotal event in prion diseases, most of the therapeutic strategies are designed to prevent the conformational change of normal prion protein to that of the pathogenic form or to remove the accumulated prion protein. Quinacrine is one of the compounds that can inhibit the accumulation of pathogenic prion protein in cultured neuroblastoma cells. Here we report the results of clinical trials of quinacrine administration to the patients of Creutzfeldt-Jakob disease. In some patients, response to visual and auditory stimulations improved transiently. Lemon-yellow discolorization of skin and liver dysfunction were common side effects by quinacrine.