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Fatal familial insomnia: a new Austrian family

G. Almer, J. A. Hainfellner, T. Brücke, K. Jellinger, R. Kleinert, G. Bayer, O. Windl, H. A. Kretzschmar, A. Hill, K. Sidle, J. Collinge, H. Budka
DOI: http://dx.doi.org/10.1093/brain/122.1.5 5-16 First published online: 1 January 1999

Summary

We present clinical, pathological and molecular features of the first Austrian family with fatal familial insomnia. Detailed clinical data are available in five patients and autopsy in four patients. Age at onset of disease ranged between 20 and 60 years, and disease duration between 8 and 20 months. Severe loss of weight was an early symptom in all five patients. Four patients developed insomnia and/or autonomic dysfunction, and all five patients developed motor abnormalities. Analysis of the prion protein (PrP) gene revealed the codon 178 point mutation and methionine homozygosity at position 129. In all brains, neuropathology showed widespread cortical astrogliosis, widespread brainstem nuclei and tract degeneration, and olivary `pseudohypertrophy' with vacuolated neurons, in addition to neuropathological features described previously, such as thalamic and olivary degeneration. Western blotting of one brain and immunocytochemistry in four brains revealed quantitative and regional dissociation between PrPres (the protease resistant form of PrP) deposition and histopathology. In the cerebellar cortex of one patient, PrPres deposits were prominent in the molecular layer and displayed a peculiar patchy and strip-like pattern with perpendicular orientation to the surface. In another patient, a single vacuolated neuron in the inferior olivary nuclei contained prominent intravacuolar granular PrPres deposits, resembling changes of brainstem neurons in bovine spongiform encephalopathy.

  • fatal familial insomnia
  • prion diseases
  • prion protein
  • transmissible spongiform encephalopathies
  • BSE = bovine spongiform encephalopathy
  • FFI = fatal familial insomnia
  • GFAP = glial fibrillary acidic protein
  • HE = haematoxylin–eosin
  • PCR = polymerase chain reaction
  • PRNP = PrP gene
  • PrP = prion protein
  • PrPres = protease resistant form of PrP
  • SSCP = single-strand conformational polymorphism

Introduction

Fatal familial insomnia (FFI) was first described in 1986 as an autosomal dominant heredopathy, clinically characterized by progressive untreatable insomnia, dysautonomia and motor signs (Lugaresi et al., 1986). Meanwhile, the disorder has been recognized as a prion disease, thus enlarging the spectrum of familial spongiform encephalopathies consisting of familial Creutzfeldt–Jakob disease and Gerstmann–Sträussler–Scheinker disease (Goldfarb et al., 1992; Medori et al., 1992b). The neuropathological hallmark of FFI is predominance of lesions in the thalamus (Manetto et al., 1992; Gambetti et al., 1995). Genetically, FFI is linked to a GAC to AAC point mutation (aspartic acid to asparagine substitution) at codon 178 of the prion protein (PrP) gene (PRNP) on chromosome 20 in conjunction with methionine at the polymorphic position 129 of the mutant allele (Goldfarb et al., 1992; Medori et al., 1992b).

We report here the first Austrian family with FFI in five consecutive generations. We present detailed clinical features of five patients, and neuropathological and molecular genetic analysis of four patients. Data on this new family have been published in part as abstracts (Almer et al., 1997; Budka et al., 1997; Hainfellner et al., 1997a).

Methods

Information on the pedigree was collected by reviewing all pertinent medical and non-medical records, and notably by interviews with family members. In patients III-5, III-13 and IV-13, clinical data were retrieved retrospectively from medical records. Patients IV-5 and IV-8 underwent personal (G.A. and T.B.) neurological examination, and had several EEGs, CT and MRI.

Neuropathological and molecular genetic analysis were performed in patients III-5, IV-5, IV-8 and IV-13. Autopsy of patients IV-5 and IV-8 was done after 20 h post-mortem and was restricted to the brain including the upper cervical spinal cord. Numerous tissue blocks of cerebral cortex, basal ganglia, brainstem and cerebellar cortex were sampled and frozen. The remaining brain was immersion-fixed in 4% formalin for 2 weeks and cut. Coronal slices of cerebrum, an axial whole-mount slice of cerebellum and pons, and numerous smaller tissue blocks including all major brain regions were embedded in paraffin. Archival paraffin blocks containing various brain regions of patient III-5, who succumbed in 1991, and patient IV-13, who succumbed in 1986, were retrieved from two municipal Austrian (neuro)pathology laboratories. In addition, paraffin embedded blocks of lungs, kidney, liver and spleen of patient IV-13 were available. Histological work-up was performed on 5 μm thick sections with conventional and immunocytochemical stains. Conventional stains comprised haematoxylin–eosin (HE), luxol fast blue/nuclear fast red, Kanzler method and Bielschowsky silver impregnation. Immunolabelling used a polyclonal antibody against glial fibrillary acidic protein (GFAP) (Dako, Glostrup, Denmark), and monoclonal antibodies against neurofilament protein (NFP; clone NE14) (Dako), microtubule associated protein-2 (MAP2; clone AP20) (Boehringer, Mannheim, Germany), synaptophysin (clone SY38) (Boehringer) and PrP (clone 3F4) (Dr R. Kascsak, Staten Island, NY, USA). For anti-PrP immunocytochemistry, sections were pre-treated with a three-tiered protocol of hydrated autoclaving, concentrated formic acid and guanidine isothiocyanate (Goodbrand et al., 1995). Antibodies were followed by the avidin–biotin complex method (for monoclonals) or the peroxidase–anti-peroxidase technique (for polyclonals), with diaminobenzidine as chromogen for visualization. Grey matter lesioning was evaluated on HE- and GFAP-stained sections by semiquantitative assessment of neuronal loss, spongiform change and astrogliosis (Table 1). Lesioning of tracts and white matter structures was evaluated by semiquantitative assessment of regional nerve fibre degeneration on luxol fast blue/nuclear fast red stained sections (Table 2).

Analysis of PRNP was performed on genomic DNA isolated from the blood of patients IV-5 and IV-8 according to standard procedures (Sambrook, 1989). One hundred nanograms of DNA was used for PCR (polymerase chain reaction) amplification of the coding region of PRNP using the primers 895W and 896W (Kretzschmar et al., 1986; Nicholl et al., 1995). The PCR product was inspected on a 1% agarose gel for potential insertion mutations and deletions. Potential point mutations were screened by the single-strand conformational polymorphism (SSCP) technique (Orita et al., 1989). For this purpose the coding region of PRNP was reamplified in four overlapping fragments which were analysed alongside the PRNP gene of patients with known mutations (Windl et al., 1996). The genotypes of codons 129 and 178 were examined by digestion with the restriction endonucleases NspI and Tth111I. The final sequence confirmation was obtained by solid-phase direct sequencing of the complete coding region of PRNP after reamplification and purification of single-stranded PCR products using 5′-biotinylated primers 895W and 896W and streptavidin-coupled Dynabeads M-280 (Dynal, Oslo, Norway). The sequencing reactions were performed with the SequiTherm EXCEL Long-Read Kit-LC (Epicentre Technologies, Madison, Wis., USA), according to the manufacturer's recommendation, and 5′-IRD-41 labelled oligonucleotides 5HUSEQ (5′-TCTCCTCTTCATTTTGCAGAGC-3′) or 3HUSEQ (5′-GAAAGATGGTGAAAACAGGAAG-3′). The reaction products were loaded on a 4.3% Long-Ranger gel (AT Biochem, Malvern, Pa., USA) and separated by denaturing electrophoresis on an automated system (Model 4000L; LI-COR, Lincoln, Nev., USA).

DNA from paraffin embedded brain tissue of patients III-5 and IV-13 was isolated using the QIAamp tissue kit (QIAGEN, Hilden, Germany). The DNA from this material was highly degraded. Therefore, two fragments of PRNP were amplified with two sets of primers. Primers 5CEN (5′-AGGTGGCACCCACAGTCAGT-3′) and 3CEN (5′-ACGGTCCTCATAGTCACTGCCG-3′) amplified a fragment encompassing the codons 93–148 of PRNP, whereas primers P33 (5′-CATGGATGAGTACAGCAACCAG-3′) and P34 (5′-TCTGGTAATAGGCCTGAGATTC-3′) amplified a fragment encompassing codons 166–228. PCR used identical conditions to those used for the complete PRNP coding region, but two successive rounds of 35 PCR cycles were necessary for a sufficient yield of PCR product for further examination. Codons 129 and 178 were examined by digestion with restriction endonucleases NspI and Tth111I as well as direct sequencing. Sequencing was performed as outlined above, but the 5′-biotinylated primers 5CEN and P33 were used for purification of single-stranded PCR products and the 5′-IRD-41 labelled primers 3CEN and P34 for the sequencing reactions.

Western immunoblotting was performed with samples of occipital and precentral cortex, thalamus, basal ganglia, cerebellar cortex, brainstem and cervical spinal cord of patient IV-5. The tissue samples were homogenized in 9 volumes of lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 10 mM Tris pH 7.4) by repeated passage through needles of decreasing diameter. The homogenates were spun at 3000 r.p.m. for 5 min and the supernatant removed to a fresh tube for analysis. Aliquots of the homogenates were incubated at 37°C for 1 h with proteinase K (at a final concentration ranging from 12.5–50 μg/ml). The reactions were terminated by the addition of Pefabloc (Boehringer) to 1 mM. Samples were electrophoresed on 16% Tris–glycine acrylamide gels and blotted as described previously (Collinge et al., 1996). The blots were developed using an enhanced chemifluorescent substrate (Amersham, UK), and analysed on a Storm 840 phosphoimager (Molecular Dynamics, Sunnyvale, Calif., USA).

Results

Clinical findings

The family pedigree is depicted in Fig. 1. The pedigree data were collected after FFI was genetically diagnosed in a young man in 1996 with clinical signs of a neurodegenerative disorder (patient IV-5, see below). Among more than 50 members in five generations, probable (according to medical and non-medical records) or definite (diagnosed by molecular genetics and neuropathology) FFI was identified in 13 cases.

Patient IV-5

At the age of 25 years, this male patient started suffering from progressive tiredness and lethargy. Episodes of diplopia and complex hallucinations followed (e.g. the patient performed movements of sawing with a virtual saw and stopped bewildered when told that there was no saw). Despite increased appetite, he had continuous loss of weight (>20 kg within 6 months) and chronic therapy-resistant constipation. Progressive change of personality with apathy became evident and endogenous depression was diagnosed. Several clinical check-ups including MRI and EEG were all inconclusive. Four months after onset, dysarthria, hypophonia and reduction of spontaneous speech developed. Speech disorder was followed by gait ataxia and myoclonus with perioral and periorbital predominance.

The patient was hospitalized at the Clinic of Neurology, University of Vienna, 6 months after onset of disease. On admission, he presented with markedly impaired vigilance, lack of spontaneous speech, dysarthria, severe gait ataxia, myoclonus and tremor. He had mild sleep disturbances. He was fully oriented, but had severe deficiencies in short-term memory, and psychomotor speed was reduced. During hospitalization, autonomic dysfunction manifested with hyperhidrosis, hyperthermia, tachycardia, recurrent flushes, dyspnoe and irregular breathing. Within several months, marked insomnia with nocturnal motor unrest and stereotype movements developed.

MRI showed mild supratentorial atrophy and discrete hyperintense white matter lesions. EEG displayed mild to moderate signs of diffuse non-specific parenchymal dysfunction. In the late phase of disease, epileptiform discharges were observed. Periodic or pseudoperiodic activity was not recorded. Other clinical tests, such as electro-oculography, visual evoked potentials and analysis of the CSF revealed no pathology. ACTH and cortisol levels were within normal range. DNA analysis of blood leukocytes detected the pathognomonic genotype of FFI (see PRNP analysis below). The patient died from pneumonia 13 months after onset of disease in a condition of severe cachexia and stupor.

Patient IV-8

An elder brother of patient IV-5 had onset of disease at the age of 36 years. He had severe loss of weight and chronic constipation, and developed later mild gait ataxia and dysphagia. Autonomic dysfunction followed, notably prominent hypersalivation and hyperthermia.

On admission to the Clinic of Neurology, University of Vienna, the patient reported a 2-week episode of severe sleep disturbance, which had improved 6 months previously under treatment with benzodiazepines. During hospitalization, the patient had mild difficulty in falling asleep. Neurological examination revealed myoclonus of face, tongue and upper limbs, and mild ataxia of limbs and gait. Mild spastic paraparesis was considered a residue of a car accident in 1983 which had resulted in a fracture of the lumbar spine and affection of spinal cord. There was mild cognitive impairment, and apathy and lethargy.

CSF analysis revealed no abnormalities. CT disclosed mild diffuse brain atrophy. EEG displayed signs of diffuse non-specific parenchymal dysfunction. Three months after discharge from the clinic the patient died from pneumonia, 11 months after onset of disease. No progression of sleep disorder was reported by his family doctor.

Patient III-5

This patient was the mother of patients IV-5 and IV-8. Disease manifested with initial insomnia and nocturnal motor unrest, memory impairment, and perioral and periorbital myoclonus. Severe loss of weight, progressive apathy, dysarthria and episodic irregular breathing and inspiratory stridor followed. EEG showed moderate to prominent signs of diffuse non-specific parenchymal dysfunction. CT revealed moderate brain atrophy with frontocerebellar accentuation. The patient died at the age of 58 years from bronchopneumonia 8 months after onset of disease.

Patient IV-13

Disease manifested in this second cousin of patients IV-5 and IV-8 at the age of 20 years with severe loss of weight. She then developed vertigo and ataxia. Diplopia, dysarthria, tremor, autonomic dysfunction (hyperthermia and chronic constipation), progressive apathy and amnestic deficiencies followed. Sleep disturbances or insomnia have not been recorded. EEG showed mild diffuse non-specific parenchymal dysfunction. CT was normal. She died severely cachectic from pneumonia 20 months after onset of disease.

Patient III-13

The mother of patient IV-13 had onset of disease at the age of 62 years. Initial symptoms comprised loss of weight, tiredness and short-term memory impairment. Within several months, nocturnal insomnia, dysarthria and episodes of diplopia developed. Her family then noticed progressive apathy and confusions, notably at night. In the late phase of disease, she developed progressive gait ataxia and perioral myoclonus. Dysautonomia has not been recorded. Haematological and biochemical findings were normal. EEG showed moderate signs of diffuse non-specific parenchymal dysfunction. CT disclosed pronounced cerebral and cerebellar atrophy. The patient died 18 months after onset of disease.

Neuropathology

The fresh brain of patient IV-5 weighed 1360 g. Brain weights of other autopsied patients are not on record. Grossly, the brains of patients IV-5 and IV-8 showed diffuse oedema with narrowing of external and internal CSF spaces. Brain sectioning revealed thalamic atrophy with a marbled aspect of the cut surface (Fig. 2).

Tables 1 and 2 summarize grey and white matter lesioning in patients III-5, IV-5, IV-8 and IV-13. Regional histopathology was similar in all patients. Cerebral cortex showed no unequivocal neuronal loss. Neuronal loss was moderate to conspicuous in thalamus (most prominent in medial and anterior thalamic nuclei) and inferior olivary nuclei, and slight to moderate in dorsal raphes and superior central nuclei, in hypothalamus, some brainstem nuclei and spinal grey matter. Cerebellar cortex displayed slight to moderate reduction of Purkinje cells and the granular layer contained some torpedoes. Astrogliosis involved all grey matter structures and was particularly conspicuous in thalamic nuclei (in anterior and medial nuclei more than in ventral lateral nuclei) (Fig. 3D), nucleus ruber, periaqueductal, tectal and tegmental grey, and raphe and olivary nuclei (Fig. 4A and B). Cerebral cortex showed bilaminar accentuation of astrogliosis (Fig. 3B). Spongiform change was discrete and detectable in thalamic nuclei (Fig. 3C) of all four brains, in pre/parasubiculum of two brains and in a small focus of frontal cortex of one brain (Fig. 3A). Deposits of protease resistant PrP (PrPres) detected by immunocytochemistry were discrete and occurred only in brains III-5, IV-5 and IV-8. In brain III-5, patchy and strip-like PrPres deposits with perpendicular orientation to the surface were localized in the molecular layer in one out of three blocks of cerebellar cortex (Fig. 5A and B); pre/parasubiculum harboured discrete, fine-granular synaptic type deposits. In brain IV-5, a single vacuolated neuron in the inferior olivary nuclei (see below) contained prominent intravacuolar granular PrPres deposits (Fig. 5C). A few other neurons displayed discrete granular PrPres deposits on the surface and/or in the vacuoles, but the majority was negative. In brain IV-8, a small area of frontal cerebral cortex showed spongiform change and synaptic type PrPres deposits with perivacuolar accentuation were detectable. A few patchy PrPres deposits were confined to a small focus in the molecular layer of cerebellar cortex. A few vacuolated neurons in the inferior olivary nuclei had discrete granular PrPres deposits on the surface and/or in the cytoplasmic vacuoles. Internal organs of patient III-13 were devoid of detectable PrPres deposits.

White matter of cerebrum and cerebellum showed scattered myelin balls indicating widespread Wallerian type of nerve fibre degeneration (Table 2). The nerve fibre degenerations are possibly the pathological substrate, as lesions accompanied by oedema, of discrete supratentorial MRI findings in patient IV-5 (see Clinical findings). Primary demyelination with preserved axons was not detectable. Flourishing nerve fibre breakdown was most conspicuous in brainstem tracts (Table 2, Fig. 4E). Alveus and hilus of inferior olives, and hilus of dentate nuclei showed prominent depletion of nerve fibres. Residual olivary neurons showed signs of transneuronal degeneration (Fig. 4C and D) with vacuolation of cell bodies and hypertrophied antler-like dendrites (olivary `pseudohypertrophy') in all brains.

Western blotting

At a final proteinase K concentration of 50 μg/ml, PrPres was not detectable in any of the seven investigated regions. Reduction of proteinase K concentration to 12.5 μg/ml resulted in a positive signal from the basal ganglia, precentral region and thalamus (Fig. 6). Glycoform ratios are similar to those previously reported for FFI, with the diglycosylated PrPres band being the most abundant (Gambetti et al., 1995; Parchi et al., 1995). In the basal ganglia the average ratios for the three PrPres glycoforms are: high: 58.43%, low: 33.40%, unglycosylated: 8.17%, taken from an average of four separate blots.

PRNP analysis

PCR amplification of the complete coding region of PRNP generated a single product of 874 bp, thus excluding an insertion mutation or deletion. SSCP analysis revealed an aberrant migration pattern indicating a point mutation in the C-terminal half of the gene. Close inspection of this region by digestion of the PCR product with enzyme Tth111I and direct sequencing defined this mutation as aspartic acid (GAC) to asparagine (AAC) substitution at codon 178 of PRNP (D178N). SSCP analysis, NspI digestion and direct sequencing revealed homozygous codon 129 for methionine in all four patients.

Discussion

Onset of disease was insidious in our FFI family, with initial or early loss of weight in all five patients with detailed histories. The first neuropsychiatric symptoms were insomnia in one patient, lethargy in one patient, cognitive impairment in one patient and ataxia in two patients. In the course of disease, four patients developed progressive insomnia, four patients autonomic dysfunction and all five patients motor abnormalities. Symptomatology of our patients is thus typical for FFI (Lugaresi et al., 1986; Manetto et al., 1992; Nagayama et al., 1996). According to medical records, patient IV-13 presented clinically with some unusual features. Age at onset of disease was 20 years. Together with a recent FFI patient from Australia (Silburn et al., 1996), this patient is the youngest reported so far. Insomnia was not recorded during the whole course of disease. However, evaluation of sleep patterns by polysomnography was not performed.

Molecular analysis of PRNP in our family revealed the codon 178 point mutation and methionine homozygosity at position 129 in all four patients examined. Codon 178 mutation in conjunction with methionine at position 129 of the mutant allele is the diagnostic genotype for FFI (Goldfarb et al., 1992; Medori et al., 1992b). It has been shown that the genotype of polymorphic codon 129 associates in FFI with characteristic neuropathological features (Gambetti et al., 1995; Parchi et al., 1995). Patients with homozygous codon 129 have prominent thalamic pathology, whereas lesioning of cerebral cortex is minor or absent (Gambetti et al., 1995; Parchi et al., 1995; Reder et al., 1995; Nagayama et al., 1996; Silburn et al., 1996). However, two recent patients of two different kindreds with homozygous codon 129 showed prominent lesioning of cerebral cortex (McLean et al., 1997; Rossi et al., 1998). Patients with heterozygous codon 129 have prominent lesioning of the cerebral cortex in addition to thalamic pathology (Gambetti et al., 1995; Parchi et al., 1995). Our patients were all homozygous at position 129; neuropathology showed prominent thalamic lesioning, whereas cerebral cortex displayed only minor histopathology with spongiform change confined to small areas. However, anti-GFAP immunocytochemistry detected widespread laminar astrogliosis. Astrogliosis also involved regions that are not supposed to receive thalamocortical projections, i.e. the occipital cortex. Widespread cortical astrogliosis in the absence of neuronal loss and spongiform change has been observed in members of other FFI kindreds (Manetto et al., 1992; Medori et al., 1992a). Cortical astrogliosis indicates submicroscopical lesioning of brain parenchyma. Possible targets of lesioning are neuronal subpopulations of the cortex. In Creutzfeldt–Jakob disease brains, subtotal loss of the parvalbumin positive subset of GABAergic neurons has been observed despite `normal' appearance of tissue (Guentchev et al., 1997).

Brainstem histopathology in FFI has been described as variable in distribution and limited in extent. Neuronal loss and gliosis are most conspicuous and frequent in inferior olives and minor in other nuclei (Manetto et al., 1992; Gambetti et al., 1995; Reder et al., 1995; Nagayama et al., 1996; Silburn et al., 1996; McLean et al., 1997; Rossi et al., 1998). In nine patients from five different FFI families, the periaqueductal grey matter showed slight to moderate gliosis (Parchi et al., 1995). In one out of four patients of another kindred, brainstem lesioning involved locus coeruleus, raphe nucleus and reticular formation (McLean et al., 1997). One FFI patient had prominent neuronal loss in the tectum (Reder et al., 1995). In contrast, all our neuropathologically analysed patients consistently had widespread and conspicuous brainstem histopathology, although disease duration was short to moderate (8, 11, 13 and 20 months, respectively).

In bovine spongiform encephalopathy (BSE) and scrapie of sheep, vacuolation of brainstem neurons is pathognomonic (Wells and Wilesmith, 1995; DeArmond and Prusiner, 1997). In human prion disease, conspicuous neuronal vacuolation has been observed only in kuru (Hadlow, 1959; Klatzo et al., 1959; Hainfellner et al., 1997b). The olives of our FFI patients showed severe neuronal loss, and residual neurons were hypertrophied and vacuolated. Neuronal vacuolation in our patients is reminiscent of that in BSE and scrapie. However, we interpret neuronal vacuolation in our patients as transneuronal degeneration because vacuolation was confined to the olives, central tegmental tracts showed conspicuous degeneration and hypertrophied antler-like dendrites were found as well. Olivary `pseudohypertrophy' with neuronal vacuolation is a well known pattern of transneuronal degeneration which has been described as a sequel of central tegmental tract lesioning, most commonly due to ischaemic infarction within the brainstem (Gautier and Blackwood, 1961).

In our hands, immunocytochemistry on numerous blocks detected PrPres deposits in only three out of four patients. In patient III-3, conspicuous PrPres deposits accumulated in one out of three blocks in the molecular layer of cerebellar cortex, and minor PrPres deposits were detectable in pre/parasubiculum. Patients IV-5 and IV-8 had discrete PrPres deposits in the inferior olivary nuclei. Patient IV-8 had, in addition, a small focus of PrPres deposition in frontal cortex and scant deposits in the molecular layer of cerebellar cortex. Thus, the diagnostic value of anti-PrP immunocytochemistry is limited in FFI, and immunocytochemical PrPres detection requires examination of numerous tissue blocks including areas with minor histopathology. In contrast to immunocytochemistry, Western blot analysis of brain IV-5 detected PrPres in three out of seven CNS regions after mild protease K digestion. This confirms that Western blotting is superior to immunocytochemistry in detecting PrPres in FFI.

Descriptions of the immunomorphology of PrPres in FFI are scant. In two out of four patients of an Australian kindred, a weak fine granular staining of the neutropil has been observed (McLean et al., 1997). In one of our patients, focal PrPres deposits in the frontal cortex showed a synaptic type pattern with perivacuolar accentuation. In two out of four of our patients, a few vacuolated neurons in the inferior olivary nuclei had discrete fine granular PrPres deposits on the surface and/or in the cytoplasmic vacuoles; a single neuron had prominent intravacuolar deposits (Fig. 5C). This is a unique observation in FFI, resembling changes of brainstem neurons in BSE (Wells and Wilesmith, 1995). In another of our patients, PrPres deposits in the molecular layer of cerebellar cortex showed a peculiar strip-like pattern with perpendicular orientation to the surface. A comparable pattern of PrPres deposition has not been described in FFI so far and has not been observed in sporadic Creutzfeldt–Jakob disease (Hainfellner and Budka, 1996), kuru (Hainfellner et al., 1997b) or Gerstmann–Sträussler–Scheinker syndrome (Hainfellner et al., 1995), but has been observed in some cases of familial Creutzfeldt–Jakob disease (J.A.H. and H.B., unpublished observation).

Western blot analysis of the regional distribution of PrPres in FFI has shown that histopathology is confined to brain areas with PrPres accumulation. Conversely, PrPres was detectable in areas with and without histopathology. On the basis of this observation, it has been hypothesized that tissue lesioning in FFI develops only in the presence of critical amounts of PrPres and that vulnerability of brain parenchyma to the presence of PrPres is regionally variable (Parchi et al., 1995). However, immunocytochemistry detected little PrPres, in spite of severe and widespread histopathology. Moreover, in our patient IV-5, Western blotting did not detect PrPres in the severely damaged brainstem. Thus, PrPres accumulation in our FFI patients dissociates not only quantitatively but also topographically from histopathology. With regard to the unresolved pathogenic role of PrP in prion diseases, this dissociation supports a loss of function model (Whittington et al., 1995) rather than neurotoxicity (Brown and Kretzschmar, 1997). Experimental data suggest that loss of functional PrP impairs the maintenance and normal function of synapses. Thus, synapses are a likely target of lesioning, following loss of functional PrP in prion disease (Whittington et al., 1995).

View this table:

Regional severity of brain tissue lesioning in four Austrian FFI patients

Patient
IV-5IV-8III-5IV-13
NLGLSPNLGLSPNLGLSPNLGLSP
0, 1, 2, 3 = no, slight, moderate, prominent neuronal loss, gliosis, spongiform change, respectively; – = not available. NL = neuronal loss; GL = astrogliosis; SP = spongiform change. *Loss of Purkinje cells; focal SP, colocalizes with PrPres deposition; two blocks of cerebral cortex, region unknown; two fragments of thalamus, most likely of anterior and medial nucleus; #two blocks of cerebellar cortex, most likely vermis.
Cerebral cortex
Frontal cortex020011020020
Precentral gyrus020010
Temporal cortex020010020
Parietal cortex020010020
Occipital cortex020020020
Cingulate gyrus020010
Insular cortex020010
Dentate gyrus010010010
Hippocampus020010020
Subiculum020010020
Pre/parasubiculum021020021
Basal ganglia
Putamen020010020020
Caudate nucleus020010
Globus pallidus020010020020
Claustrum020010
Amygdaloid nucleus complex020
Basal nucleus020020
Thalamus
Anterior nucleus331231231
Medial nucleus331231231231
Ventral lateral nucleus121111121
Pulvinar231
Lateral geniculate nucleus010
Subthalamic nucleus010010000
Hypothalamus
Paraventricular nucleus010
Supraoptic nucleus010
Lateral nucleus120
Anterior nucleus110
Cerebellum*
Neocerebellum110110110
Vermis2101101#1#0#
Archicerebellum110110
Dentate nucleus120110110
Midbrain
Nucleus ruber130120
Substantia nigra120110110
Central grey mesencephali130120120
Nucleus raphes dorsalis230120
Formatio reticularis220120120
Oculomotor nucleus010
Trochlear nucleus010
Mesencenphatic nucleus of trigeminal nerve020010
Superior colliculus120120
Inferior colliculus120
Pons
Locus coeruleus120110110
Superior central nucleus120220220
Formatio reticularis220220
Vestibular nuclei120
Pontine nuclei010010010010
Medulla oblongata
Vestibular nuclei020010
Nucleus cuneatus010110
Nucleus gracilis110010
Hypoglossal nucleus010010
Dorsal nucleus of vagus010120
Spinal trigeminal nucleus120010120
Nucleus ambiguus010010010010
Raphe nuclei220110220
Formatio reticularis220120220230
Inferior olivary nucleus230230220330
Accessory olivary nuclei230230330
Spinal cord
Posterior horn120120
Lateral horn120
Intermediate grey120120
Anterior horn120120
View this table:

Nerve fibre degeneration in the CNS of four Austrian FFI patients

Patient
IV-5IV-8III-5IV-13
0, 1, 2 no, single, multiple nerve fibre degenerations, respectively, as indicated by myelin balls in luxol fast blue/nuclear fast red stain; – = not available. *Two blocks of cerebral cortex, region unknown; two blocks of cerebellar cortex, most likely vermis.
Cerebrum
Frontal white matter0111*
Central white matter21
Temporal white matter111
Parietal white matter011
Occipital white matter011
Corpus callosum00
Internal capsule2111
Anterior commissure00
Fornix21
Mamillothalamic tract2
Optic chiasm00
Optic tract00
Cerebellum
Neocerebellar white matter111
White matter of vermis221
Archicerebellar white matter11
Brainstem/spinal cord
Crus cerebri111
Superior cerebellar peduncle211
Middle cerebellar peduncle111
Inferior cerebellar peduncle120
Medial longitudinal fasciculus2211
Central tegmental tract2222
Medial lemniscus0011
Lateral lemniscus101
Fasciculus gracilis111
Fasciculus cuneatus111
Pyramidal tract0111
Posterior spinal nerve root1
Fig. 1

The pedigree of the Austrian FFI family comprises more than 50 members in five generations.

Fig. 2

A coronal slice of brain IV-5 shows diffuse oedema with narrowing of external and internal CSF spaces. The thalami are atrophic and display a marbled aspect.

Fig. 3

Cerebral histopathology in the Austrian FFI family. Spongiform change (A) is detectable in a focus of frontal cortex (brain IV-8; HE; ×110), and (C) is discrete in thalamic nuclei (brain IV-5; HE, ×140). Anti-GFAP immunocytochemistry shows (B) cortical astrogliosis with bilaminar accentuation (brain IV-5; ×25) and (D) prominent astrogliosis in the thalamus (brain IV-5; ×170).

Fig. 4

Brainstem pathology in the Austrian FFI family (brain IV-5). (A) In the pons, gliosis (dark blue colour) is conspicuous in periaqueductal grey and raphe (Kanzler stain; ×3). (B) In medulla oblongata, gliosis is prominent in inferior olivary nuclei (Kanzler stain;×4). Residual olivary neurons show signs of transneuronal degeneration with (C) neuronal vacuolation (HE; ×400) and (D) hypertrophied antler-like dendrites (Bielschowsky; ×260). (E) flourishing nerve fibre breakdown with myelin balls in olivocerebellar tract (luxol fast blue/nuclear fast red; ×430).

Fig. 5

PrPres deposition patterns in the Austrian FFI family. In patient III-5 (A and B), patchy and strip-like PrPres deposits are confined to the molecular layer and show perpendicular orientation to the surface (anti-PrP) (A, ×50; B, ×145). (C) A single vacuolated neuron in the inferior olivary nuclei of patient IV-5 shows prominent intravacuolar accumulation of granular PrPres deposits. Other neurons are devoid of PrPres deposits (anti-PrP) (×160; inset: ×730).

Fig. 6

Western blot analysis of a normal brain and of FFI brain IV-5. Positive signals are present in FFI in basal ganglia, precentral and thalamus regions. The diglycosylated PrPres band is the most abundant. All samples were treated with proteinase K (PK) at a concentration of 12.5 μg/ml before electrophoresis. Molecular weight standards are shown on the left.

Acknowledgments

We wish to thank Dr G. R. Trabattoni for helping with neuropathological analysis and Mrs H. Flicker for excellent technical assistance. This work is part of the European Union Biomed-2 Concerted Action `Human transmissible spongiform encephalopathies (prion diseases): neuropathology and phenotypic variation' (project leader: H. Budka).

References

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