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-helical, is detergent soluble, and is readily digested by proteases. In contrast, the pathogenic isoform (PrPSc) has a substantially ß-sheet structure, is insoluble in nondenaturing detergents, and shows relative resistance to proteolytic digestion.2,3,4 The protease-resistant core of PrPSc, designated PrP27–30, is usually detectable in humans and animals with prion disease. The relative molecular mass of the protease-resistant segment varies among prion strains and appears to be coupled with or associated with disease-specific phenotypes.5,6 Understanding of the prion strains has been advanced by investigations of the genotype–phenotype correlations in inherited forms of prion disease, such as fatal familial insomnia. Eleven kindreds with fatal familial insomnia have been identified.7,8,9,10,11,12,13,14 Patients from these kindreds usually present with untreatable insomnia, followed by dysautonomia and ataxia, although some variation has been reported.15 Cognitive function is relatively spared until late in the course of the disease, when diffuse slowing of activity on the electroencephalogram becomes apparent.10 Neuronal loss and astrocytic gliosis within the thalamus and olives, and to a lesser degree the cerebellum, are seen. The average age at the onset of the disease is 48 years (range, 25 to 61), and the duration of disease is about 18 months (range, 7 to 33).8,16,17 PrP27–30 is detectable, though often at low levels, and is usually confined to the thalamus and temporal lobe.18
Fatal familial insomnia is caused by a mutation in the PRNP gene that results in the substitution of asparagine for aspartic acid at codon 178, in conjunction with the methionine at polymorphic codon 129. When the dominant D178N mutation is coupled with substitution of a valine at position 129, a dementing phenotype, characterized by diffuse spongiosis and widely distributed PrPSc, results. This illness is known as familial Creutzfeldt–Jakob disease.19 The relative molecular mass of deglycosylated PrP27–30 in familial Creutzfeldt–Jakob disease is approximately 21 kd, whereas that of PrP27–30 in fatal familial insomnia is approximately 19 kd.20 This difference in size reflects the different conformations adopted by PrPSc in familial Creutzfeldt–Jakob disease and fatal familial insomnia.
We studied a patient who presented with the typical features of fatal familial insomnia. Pathological examination, molecular genetic and PrPSc conformation studies, and transmission studies to susceptible transgenic mice revealed that this patient carried a strain of prions similar to that in fatal familial insomnia but that he did not carry the PRNP mutation that causes it. These findings argue that his disorder was the sporadic equivalent of fatal familial insomnia, which we term sporadic fatal insomnia, and thus support the concept that the prion strain that specifies the clinicopathological phenotypes of the naturally occurring prion disease in humans and of the experimental disorder in mice is determined not by the sequence of the prion protein but rather by the conformation of PrPSc.
Case Report
A previously healthy 44-year-old man had progressive insomnia. He sought medical advice after one week but was told it was "all in his head." Heat intolerance soon developed. During the next four months, as his insomnia worsened, he slept an average of one hour per night. Six months after the onset of symptoms, excessive lacrimation developed and he began to have difficulty walking. A neurologic evaluation revealed severe appendicular ataxia. His tandem gait was especially poor, but he could still walk independently. Four months later, he was treated with large doses of hypnotic drugs, which were only briefly beneficial. Loss of the cough reflex, resulting in repeated aspiration, and progressive dysarthria developed. Cognitive function was only slightly impaired, according to results on the Mini–Mental State Examination (score, 28 of 30),21 although psychomotor slowing was evident, suggesting that a subcortical disease was present. Over the next few months his short-term memory began to deteriorate, and his conversation became repetitious. One year after the onset of symptoms, he was wheelchair-bound because of severe ataxia, his speech was fluent but severely dysarthric, and he began to have difficulty separating dreams from reality. He was admitted to a long-term care facility. No similar neurologic disease was reported in the family.
The following tests were performed: lumbar puncture; electrolyte analysis; hematologic analysis; urinalysis; liver-function and thyroid-function tests; tests for treponemal antibodies; coagulation studies; tests for anti-Hu, anti-Ri, and anti-Yo antibodies and human immunodeficiency virus antibody; analysis of sedimentation rate; heavy-metal and copper screening; ceruloplasmin tests; serum protein electrophoresis; and a complete serologic study to rule out inflammation. All the results were negative. A magnetic resonance image of the head was normal. Examination of a cerebellar-biopsy sample was unrevealing. On a positron-emission tomogram of the brain, the uptake of [18F]fluorodeoxyglucose within the thalamus was greatly reduced (Figure 1A and Figure 1B), a characteristic feature of fatal familial insomnia.22 Sixteen months after the onset of symptoms, myoclonic jerks developed, the patient became severely hallucinatory and delusional, and he died as a result of aspiration pneumonia. Postmortem studies were performed.
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Genetic Analysis
Genomic DNA was extracted from blood leukocytes by routine methods.23 A denaturing gradient gel-electrophoresis system that has been previously described was used to screen for mutations in PRNP.24 This system uses five sets of primers to circumvent the problem of single-allele amplification, which could result if a polymorphism in a noncoding region, although rare, was present.25 In addition, the entire coding region of PRNP was sequenced by using the 7-deaza-2'-deoxyguanosine triphosphate method (Sequenase version 2.0, U.S. Biochemical, Cleveland), as described previously.24
Neuropathological Analysis
The left hemisphere of the brain of the patient with sporadic fatal insomnia was removed and coronally sectioned. The right hemisphere was frozen on dry ice for subsequent studies. Formalin-fixed sections were prepared from several brain regions and analyzed by staining with hematoxylin and eosin, Bielschowsky's method, Nissl's method, and Luxol fast blue periodic acid–Schiff histochemical stain. Immunoperoxidase staining for amyloid beta-protein and glial fibrillary acidic protein was performed with standard techniques.
Protein Analysis
Western immunoblotting was performed as described previously,24 with minor modifications. A 10 percent (wt/vol) homogenate of either human or mouse brain was prepared in 0.5 percent Nonidet P-40 and 0.5 percent sodium deoxycholate; the homogenate was digested with 20 µg of proteinase K per milliliter for one hour at 37°C, and then the reaction was terminated by the addition of 2 mM phenylmethylsulfonyl fluoride. For deglycosylation of PrPSc, sodium dodecyl sulfate (SDS) was added to the digested samples to a final concentration of 1 percent, and the protein was denatured by heating to 100°C for 10 minutes. An aliquot of this preparation was diluted to a final concentration of 0.5 percent Nonidet P-40 and 0.5 percent SDS; N-glycosidase F (Boehringer Mannheim, Mannheim, Germany) was added according to the manufacturer's instructions, and the preparation was then incubated overnight at 37°C. PrPSc was recovered by centrifugation at 100,000xg for one hour at 4°C. Samples were subjected to SDS–polyacrylamide-gel electrophoresis (SDS-PAGE) in a 16 percent polyacrylamide slab gel. Prion protein was detected with the use of the anti–prion-protein 3F4 monoclonal antibody diluted 1:500026 and an enhanced chemiluminescence system (Amersham, Buckinghamshire, United Kingdom).
Immunoblotting and Histologic Examination of Transgenic Animals
Transgenic mice expressing chimeric mouse–human PrP were deficient for mouse PrP.27,28 When inoculated intracerebrally with human prions, these mice generate chimeric mouse–human prions in their brains. Nontransgenic mice are generally poor hosts for human prions. Coronal sections of the brain from transgenic animals inoculated with brain extracts from the patient with sporadic fatal insomnia or from patients with fatal familial insomnia (final wt/vol, 1 percent) were prepared from several regions of the brain for immunoblotting, as previously described.29 Blots were immunostained with anti–prion-protein monoclonal antibody 3F426 after limited digestion with proteinase K for detection of PrPSc.
A semiquantitative method was used to estimate the degree and pattern of spongiform degeneration in the brains of the transgenic mice.30 Two animals that had been inoculated with brain extract from the patient with sporadic fatal insomnia were examined; one had been inoculated with extract from the thalamus, and one with extract from the frontal cortex.
Results
Neuropathological Findings
The pathological findings on examination of the patient's brain were characteristic of those described for fatal familial insomnia in every detail.10,17,18 Severe loss of neuronal fibers was observed in the anteroventral and mediodorsal nuclei of the thalamus (Figure 2C and Figure 2F) and, to a much lesser extent, in the ventrolateral nuclei (Figure 2H and Figure 2I). Only slight vacuolation of the mediodorsal nuclear gray matter was observed on staining with hematoxylin and eosin, and no spongiosis typical of Creutzfeldt–Jakob disease was observed. Profound loss of neurons, proliferation of microglia, and astrocytic gliosis were also evident within the olives (data not shown). Slight vacuolation was present in the white matter of the temporal and occipital lobes, cingulate gyrus, basal ganglia, hippocampus, and midbrain, but vacuolation was notably absent from the white matter of the cerebellum. These changes were suggestive of neuronal-fiber loss rather than typical Creutzfeldt–Jakob disease. Astrocytic gliosis was associated with white-matter vacuolation in the cingulate gyrus, basal ganglia, and hippocampus. There was thinning of the molecular layer and moderate, focal loss of nerve cells from the granular-cell layer of the cerebellum. Axonal degeneration of Purkinje cells was moderate, as evidenced by torpedo formation visualized with silver staining by Bielschowsky's method.
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Denaturing gradient gel electrophoresis was initially used to screen the entire open reading frame of the PRNP gene, which revealed a wild-type sequence and coding for methionine at polymorphic codon 129. DNA sequencing of the complete open reading frame of PRNP confirmed the presence of a normal GAC trinucleotide, which encodes aspartic acid, at codon 178, and an ATG trinucleotide, which encodes methionine, at codon 129. DNA was also extracted from the thalamus in an attempt to identify a site-specific mutation of PRNP that would account for the focal pathological changes; the sequence of the thalamic DNA was also wild type.
PrPSc Distribution and Conformation
Western immunoblot analysis was performed on tissues obtained from several regions of the brain (Figure 3A, Figure 3B, and Figure 3C). Although PrPSc was not detected in an initial examination of the frontal cortex, it was subsequently detected focally within the thalamus. A five-fold concentration of samples allowed the detection of low levels of PrPSc in extrathalamic regions (Figure 3A). The highest extrathalamic levels of PrPSc were detected within the subthalamic region, suggesting that the abnormal protein had accumulated focally; however, lower levels were detected within forebrain structures and the lower medulla, in the region of the olives. The extremely low levels of PrPSc outside the thalamic region suggest that axonal spread in the anterograde direction may account for its distribution. In general, the level of PrPSc in this patient, even in the thalamus, was lower than that in an equivalent sample from a patient with sporadic Creutzfeldt–Jakob disease. Similarly, the levels of PrPSc in patients with fatal familial insomnia have been reported to be lower than those typically found in patients with sporadic Creutzfeldt–Jakob disease.18
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On SDS-PAGE, the molecular mass of the deglycosylated form of PrP27–30 in patients with sporadic Creutzfeldt–Jakob disease with homozygosity for methionine at residue 129 is typically 21 kd, whereas in those with fatal familial insomnia its molecular mass is about 19 kd.18,20 A 19-kd fragment is also common in patients with sporadic Creutzfeldt–Jakob disease who are heterozygous (methionine and valine) or homozygous for valine at residue 129, but it is rare in patients homozygous for methionine at that residue.31 We therefore compared the molecular mass of fractions of deglycosylated PrP27–30 isolated from patients with sporadic Creutzfeldt–Jakob disease who were homozygous for methionine at position 129, a patient with fatal familial insomnia with D178N and methionine at position 129, and the patient with sporadic fatal insomnia who was homozygous for methionine at position 129; we found that the latter two fractions comigrated at approximately 19 kd, whereas that of the former migrated at approximately 21 kd (Figure 3B). These results suggest that PrPSc from sporadic fatal insomnia and PrPSc from familial fatal insomnia possess similar protease-susceptible cleavage sites, which result in the generation of the 19-kd fragment in each. Since the pattern of digestion depends on the tertiary structure of PrPSc, these observations argue that the conformations of wild-type PrPSc in sporadic fatal insomnia and of mutant PrPSc in fatal familial insomnia are similar. Because the wild-type and mutant PrPSc molecules differ in sequence, these findings indicate that the tertiary structure of PrPSc dictates both the clinical and the pathological phenotypes of prion disease.
Transmission Studies
Transgenic mice that expressed chimeric mouse–human PrP and that were deficient for mouse PrP were inoculated with extracts from the cortex or the thalamus of the patient with sporadic fatal insomnia. A few mice died before clear signs of central nervous system dysfunction were identified. The four mice that were inoculated with extracts from the thalamus exhibited signs of neurologic impairment a mean (±SD) of 180±2 days later; the eight inoculated with extracts from the cortex had central nervous system dysfunction after 221±6 days. Brains from two of the mice that had been inoculated with extracts from the patient with sporadic fatal insomnia (one received cortex and the other received thalamus) and four that had been inoculated with extracts from patients with fatal familial insomnia were examined histologically in a blinded fashion.
Vacuolation and reactive astrocytic gliosis were present in virtually identical distributions (Figure 4). Vacuolation was most intense in the thalamus and in the inner half of the neocortex, where thalamocortical afferent fibers terminate. Vacuolation also was present, to a lesser degree, in the outer half of the neocortex and in the caudate nucleus. There was no vacuolation in the cerebellar cortex or brain-stem nuclei. Intense reactive astrocytic gliosis was present in the same locations as vacuolar degeneration. No amyloid plaques were found. PrPSc deposition in the brains of these animals was predominantly in the thalamus and inner half of the neocortex; the brain stem and cerebellum were spared (Figure 5). The hypothalamus was also relatively unaffected. The corpus callosum and anterior commissure showed a marked deposition of PrPSc.
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Analysis of the molecular mass of PrPSc from the transgenic mice that had been inoculated with brain extracts from patients with sporadic fatal insomnia or fatal familial insomnia showed that similar conformations were transferred to nascent PrPSc. From the brains of mice inoculated with the human brain extracts, 19-kd fragments of deglycosylated PrP27–30 (Figure 3C) were found by SDS-PAGE; in contrast, the 21-kd fragments were observed in fractions prepared from mice inoculated with brain extract from a patient with sporadic Creutzfeldt–Jakob disease (Figure 3B). Thus, the conformation of chimeric mouse–human PrPSc derived from the transgenic mice reflected that of human PrPSc in the brain extracts used as inoculum.
Discussion
Our results provide evidence that both the clinical and the pathological phenotypes of prion disease are determined by the conformation of PrPSc. The patient with sporadic fatal insomnia had the distinctive clinical and neuropathological features of fatal familial insomnia, yet both of his PRNP alleles were wild type. Not only are the clinical manifestations of fatal familial insomnia and sporadic fatal insomnia similar, but PrPSc localizes almost exclusively to the thalamus in both disorders. Furthermore, the molecular mass of PrPSc from the patient with sporadic fatal insomnia was about 19 kd, after limited protease digestion, the same size as that in fatal familial insomnia.18,20,31,32
When extracts prepared from the brain of the patient with sporadic fatal insomnia were inoculated into transgenic mice, signs of prion disease developed in the animals. Pathological examination of their brains showed deposition of chimeric mouse–human PrPSc that was confined largely to the thalamus, as was deposition in the transgenic Prnp0/0 mice that were inoculated with extracts of brain affected with fatal familial insomnia.6 A 19-kd protease-resistant, deglycosylated fragment of chimeric PrPSc was detected in transgenic mice inoculated with brain extracts from patients with either sporadic or familial insomnia, whereas a 21-kd fragment was observed in transgenic mice inoculated with brain extract from a patient with sporadic Creutzfeldt–Jakob disease who was homozygous for methionine at position 129. These studies demonstrate that the strain of human prions associated with sporadic fatal insomnia and containing wild-type PrPSc is indistinguishable from the strain associated with fatal familial insomnia and containing mutant PrPSc; therefore, sporadic fatal insomnia is a phenocopy and not a genocopy of fatal familial insomnia. Moreover, the same strain of prions can be propagated in a human host who expresses either mutant prion protein (D178N) and methionine at codon 129 or wild-type prion protein and methionine (by homozygosity) at position 129 or in a murine host that expresses chimeric mouse–human prion protein. Since both the disease phenotype and the conformation of PrPSc remained similar even when host sequences of prion protein varied, these findings suggest that the biologic properties of prion strains result from the conformation of PrPSc.
Sporadic fatal insomnia in our patient might have been caused by a somatic D178N mutation in the PRNP gene that generated mutant PrPSc, which in turn functioned as a conformational template in the conversion of wild-type PrPC into wild-type PrPSc. Alternatively, a sufficient number of wild-type PrPC molecules may have been spontaneously transformed into PrPSc molecules with the prion conformation that characterizes sporadic fatal insomnia,33 resulting in sustained propagation. Whether wild-type PrPSc initially forms in the thalamus or in another region of the central nervous system, or even in another organ, presumably it folds into wild-type PrPSc with the 19-kd conformation that is adopted by the mutant PrPSc that causes fatal familial insomnia. It remains to be determined whether the glycosylation of PrPC in the thalamus34 or a region-specific chaperone, such as protein X,28,35 is involved in the folding of PrPSc into the 19-kd conformation. The intense deposition of PrPSc observed in the thalamus of experimental mice after transmission of both fatal familial and sporadic fatal insomnia argues for a thalamus-specific targeting mechanism. It seems likely, on the basis of our experience with this patient with sporadic fatal insomnia, that most or all nonfamilial cases of pure thalamic dementia consist of sporadic fatal insomnia, whereas the familial form consists of fatal familial insomnia.7,9,20,36
In summary, sporadic fatal insomnia represents the natural occurrence of fatal insomnia in the absence of a mutation in PRNP and provides evidence that the conformation of PrPSc is an important determinant of disease phenotype. Whereas conversion of PrPC to PrPSc involves an increase in ß-sheet content, more subtle but clinically important conformational changes appear to underlie the generation of prion strains. Development of sensitive assays to identify these subtle conformational differences37 will be important in defining the specific phenotypic determinants of PrPSc.
Supported by grants (NS14069, AG08967, AG02132, NS22786, and AG10770) from the National Institutes of Health and by a gift from the Leila Y. and G. Harold Mathers Foundation. Dr. Mastrianni is the recipient of a Clinical Investigator Award (NS01913) from the National Institutes of Health.
We are indebted to Dr. Pierluigi Gambetti (Case Western Reserve University) for the gracious donation of a brain sample from a patient with fatal familial insomnia; to Dr. William Jagust (University of California, Davis) for assistance in performing and interpreting the positron-emission tomographic study; to Juliana Cayetano for the preparation of histopathological specimens; and to Thomas Lisse for histologic blot preparations.
Source Information
From the Institute for Neurodegenerative Diseases, Department of Neurology (J.A.M., R.N., R.L., G.C.T., D.H., S.J.D., S.B.P.), and the Departments of Pathology (S.J.D.) and Biochemistry and Biophysics (S.B.P.), University of California, San Francisco; and the Department of Neurology, University of Chicago, Chicago (J.A.M.).
Address reprint requests to Dr. Prusiner at the Department of Neurology, Box 0518, University of California, San Francisco, CA 94143-0518.
References
-helices into ß-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A 1993;90:10962-10966.
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