Diagnosis of human prion disease

Jiri Safar

Michael Geschwind

Camille Deering

Svetlana Didorenko

Henry Baron+4 authors

^{Institute for Neurodegenerative Diseases,}^{Memory and Aging Center, and Departments of}^Neurology,^{Pathology, and}^{Biochemistry and Biophysics, University of California, San Francisco, CA 94143;}^{ZLB Behring, 35041 Marburg, Germany; and}^{ZLB Behring, 75601 Paris, France}

^{To whom correspondence should be addressed at: Institute for Neurodegenerative Diseases, University of California, Box 0518, San Francisco, CA 94143-0518. E-mail:}ude.fscu.dni@yelnats.

^{J.G.S., S.J.D., A.S., K.G., and S.B.P. have financial interest in InPro Biotechnology, Inc.}

Contributed by Stanley B. Prusiner, December 22, 2004

Abstract

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt–Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP^{) used limited proteolysis to digest the precursor cellular PrP (PrP}^{). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP}^{, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP}^{in all 10–24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, ≈90% of the total PrP}^{was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP}^{. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.}

Keywords: Creutzfeldt–Jakob disease, detection, endpoint titration, immunoassay, neurodegeneration

Abstract

Human prion diseases include Creutzfeldt–Jakob disease (CJD), kuru, and Gerstmann–Sträussler–Scheinker disease. Sporadic CJD (sCJD) accounts for 85% of all cases of human prion disease, familial CJD (fCJD) for 10–15%, and infection from exogenous, frequently iatrogenic CJD (iCJD) prions, for <1% (1). Prions consist solely of a disease-causing prion protein (PrP^{) that is derived from the cellular isoform (PrP}^{) (}2). During prion replication, PrP^{stimulates conversion of PrP}^{into nascent PrP}^.

Human prions from many cases of sCJD, fCJD, and iCJD were transmitted to apes and monkeys, but few titrations were performed, so there is little quantitative data on the levels of prions from these investigations (3). The development of mice expressing human prion protein (HuPrP) and chimeric mouse–human transgenes (MHu2M) (4–7) allowed us to measure the levels of prions in human brains as reported here. The incubation times of these mice were sufficiently abbreviated to allow endpoint titrations. Based on these endpoint titrations, we surmise that each of three cases of sCJD harbors a different strain of prion. We also used the titrations to calibrate PrP^{measurements that were determined by the conformation-dependent immunoassay (CDI). Full-length, protease-resistant PrP}^(rPrP^{) and previously unrecognized protease-sensitive forms of PrP}^(sPrP^{) can be detected by the CDI (}8). Most PrP^{accumulating in the frontal cortex and white matter of sCJD cases was sPrP}^{. Other immunoassays for PrP}^{detect the N-terminally truncated protein PrP 27–30 derived from PrP}^{; these include Western blotting, ELISA, and histoblotting. It is unclear what forms of PrP}^{are detected by immunohistochemistry (IHC) after hydrolytic autoclaving in the presence of formic acid.}

Because the CDI can readily detect PrP^{molecules comprising one ID}₅₀ unit, we examined the diagnostic sensitivity of the test by measuring PrP^{in many different brain regions. We performed these measurements on brains of 28 people who died of either sCJD, fCJD(E200K), or iCJD. Whereas the CDI registered a positive signal in every brain region examined in all of the cases, standard histopathology and IHC were much less effective in diagnosing CJD. Indeed, the poor performance of these histological techniques indicates that they should no longer be used to rule out prion disease in a brain biopsy from a single cortical site and must be applied to multiple cortical and subcortical brain samples at autopsy.}

Adjacent sections from eight sCJD patients were analyzed in parallel by all three methods.

Adjacent sections from two fCJD(E200K) patients were analyzed in parallel by all three methods.

Acknowledgments

We thank the staff of the Hunters Point Animal Facility for their expert mouse studies and ZLB Behring for making the MAR1 mAb available. This work was supported by National Institutes of Health Contract NS02328 and National Institutes of Health Grants AG02132, AG010770, and AG021601. M.D.G. is supported by the John Douglas French Foundation for Alzheimer's research, the McBean Foundation; National Institute on Aging Grants AG021989, AG019724, and AG023501; the Alzheimer's Disease Research Center of California; and National Institutes of Health Grant M01 RR00079 to the General Clinical Research Center. B.L.M. is supported by National Institute on Aging Grants AG019724 and AG023501.

Acknowledgments

Notes

Author contributions: J.G.S. and S.B.P. designed research; M.D.G., C.D., S.D., M.S., H.S., and S.J.D. performed research; A.S., M.V., and H.B. contributed new reagents/analytic tools; J.G.S., K.G., B.L.M., S.J.D., and S.B.P. analyzed data; and J.G.S., S.J.D., and S.B.P. wrote the paper.

Abbreviations: CDI, conformation-dependent immunoassay; CJD, Creutzfeldt–Jakob disease; sCJD, sporadic CJD; fCJD, familial CJD; iCJD, iatrogenic CJD; IHC, immunohistochemistry; PrP, prion protein; PrP^{, normal cellular PrP; PrP}^{, disease-causing PrP; HuPrP, human PrP; MHu2M, chimeric mouse–human transgene; sPrP}^{, protease-sensitive PrP}^{; rPrP}^{, protease-resistant PrP}^{; PTA, phosphotungstate; PK, proteinase K; H&E, hematoxylin and eosin; Tg, transgenic; (D - N), difference in Ab-binding between native and denatured samples.}

Notes

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