Introduction
Gerstmann-Sträussler-Scheinker disease (GSS) [
9] is a rare dominantly inherited prion protein (PrP) amyloidosis. GSS patients, from a large kindred, have been extensively studied in three generations [
13]; they carry a TTC to TCC DNA change at codon 198 of the prion protein gene (
PRNP) resulting in a phenylalanine to serine substitution (F198S) in the prion protein [
6,
7,
11,
12,
14,
16,
35]. Neuropathologic examinations in these patients have shown that the extracellular PrP amyloid coexists with a severe intraneuronal tau pathology, characterized by deposits of hyperphosphorylated tau and neurofibrillary tangles (NFT) in the cerebral gray matter, but not in the cerebellum [
10,
11,
14]. Clinically,
PRNP F198S mutation carriers present with cerebellar ataxia and dysarthria, with later bradykinesia and rigidity. These neurologic symptoms may be preceded by psychiatric manifestations including drug dependence, depression, and/or psychosis [
7]. As the disease progresses, memory impairment and cognitive dysfunction become severe [
35].
In the brain of individuals carrying the
PRNP F198S mutation, PrP amyloid deposits occur in the form of multicentric plaques and diffuse deposits throughout the cerebral cortex, subcortical nuclei, cerebellum, and brainstem [
10,
12]. Neuropathologically, the pattern of distribution of PrP amyloid differs substantially from that of the amyloid β (Aβ) peptide, which is the major component of the plaques in the dominantly inherited and sporadic forms of Alzheimer’s disease (AD). Limited neuropathologic data from non-symptomatic
PRNP F198S carriers suggest that extracellular PrP amyloid deposits precede the development of tau pathology [
11]. Deposition of tau occurs in the form of tau-immunoreactive intracytoplasmic deposits in neurons, NFT, and neuropil threads [
10,
12]. By transmission electron microscopy and Western blot analysis, the neurofibrillary tangles in GSS associated with the
PRNP F198S mutation are similar to those seen in AD [
33]. Tau deposition occurs in close proximity to the PrP amyloid deposits, and thus, the pattern of tau pathology in the cerebrum mirrors that of PrP amyloid. As a consequence, both tau spread and topography in this prion disease differ substantially from those observed in AD and other neurodegenerative diseases with tau pathology.
Neuroimaging studies of patients with GSS have employed structural magnetic resonance imagin (MRI), positron emission tomography (PET), and single photon emission computerized tomography (SPECT). To date, only a few studies have evaluated neuroimaging measures in
PRNP F198S GSS patients. Vitali et al. (2011) studied changes on fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted imaging (DWI) in patients with GSS, including two who carried the
PRNP F198S mutation and demonstrated hyperintense signal in
PRNP F198S individuals in limbic, neocortical, and subcortical regions [
36]. Kepe et al. (2010) evaluated alterations in GSS patients, including two symptomatic and two asymptomatic
PRNP F198S GSS patients from the Indiana kindred, on 2-(1-(6-[(2-[fluorine-18]fluoroethyl)(methyl)amino]-2-naphthyl)-ethylidene)malononitrile ([
18F]FDDNP) PET, [
18F]fluorodeoxyglucose (FDG) PET, and structural MRI [
19] images. In that report, the symptomatic
PRNP F198S GSS patients and one asymptomatic carrier had increased [
18F]FDDNP binding in the basal ganglia, thalamus, cerebral cortex, and cerebellum. Reduced metabolism and mild atrophy were also seen in similar regions in symptomatic
PRNP F198S GSS patients. Recently, another study demonstrated that [
11C]PiB, which is selective for Aβ deposition, showed no specific signal in asymptomatic and symptomatic individuals carrying the
PRNP P102L mutation or the
PRNP F198S mutation [
4]. Currently, no ligand is available to specifically demonstrate PrP amyloid deposition by PET.
In this study, we sought to: 1) determine the pattern of [
18F]flortaucipir uptake in
PRNP F198S GSS patients; 2) compare the tau distribution on [
18F]flortaucipir PET among the following three groups:
PRNP F198S GSS affected individuals, sporadic early onset AD patients (EOAD), cognitively normal older adults (CN); and, 3) compare the pattern of [
18F]flortaucipir uptake, in vivo
, with that of tau neuropathology,
post-mortem. Based on the neuropathological similarity of the tau NFT in
PRNP F198S GSS and AD [
33], we hypothesized that [
18F]flortaucipir, a recently developed PET tracer that is specifically sensitive to tau NFTs [
2,
37], would permit in vivo detection of tau deposits in
PRNP F198S GSS patients. In the present study, we report, for the first time, data showing [
18F]flortaucipir uptake in two symptomatic GSS individuals carrying the F198S
PRNP mutation and compare the uptake patterns in these individuals with patterns observed in cognitively normal older adults (CN) and in patients with EOAD. The [
18F]flortaucipir PET results are also validated by the neuropathologic demonstration of PrP amyloid and tau deposits in one of the two GSS patients, who died 9 months after the [
18F]flortaucipir PET scan.
Materials and methods
Clinical assessment
All participants were evaluated in the context of annual research visits to the Indiana Alzheimer Disease Center (IADC). The clinical assessments included neurological examinations, structured informant interviews for symptoms and function, and neuropsychological assessments. Diagnoses were made by consensus panel using research criteria. Assessments were compliant with National Alzheimer’s Coordinating Center (NACC) procedures at the time of the visits including: demographics, health histories, medications, family histories, Clinical Dementia Rating (CDR), Functional Assessment Scale (FAS), Geriatric Depression Scale (GDS), and Neuropsychiatric Interview Questionnaire (NPI-Q). At the time of the [18F]flortaucipir PET scans, cognitive testing with the UDS3 measures included: Montreal Cognitive Assessment (MoCA), Craft Stories immediate and delayed recall, Benson Complex Figure copy and delayed recall, the Multilingual Naming Test (MINT), Animal fluency, Vegetable fluency, Phonemic fluency (letters F and L), Trail Making Test Parts A and B (TMT-A and TMT-B), and Number Span forward and backward. The Rey Auditory Verbal Learning Test (RAVLT) and Digit Symbol Substitution Test were also given to all participants. Written informed consent was obtained from all participants in accordance with the Declaration of Helsinki and the Belmont Report. All procedures were approved by the Indiana University School of Medicine Institutional Review Board.
Genetics
DNA was extracted from fresh blood samples of patients A and B and the open-reading frame of the Prion Protein gene (
PRNP) was analyzed by direct sequencing. The same procedure was used to study DNA extracted from the brain tissue of patient B and several previously deceased family members [
16].
Structural MRI
A T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) structural MRI sequence was acquired at the time of the [
18F]flortaucipir PET scan on a 3 Tesla Siemens Prisma scanner for both patients. Automatic parcellation with Freesurfer version 5.1 (
https://surfer.nmr.mgh.harvard.edu/fswiki/FreeSurferWiki) was completed to create subject-specific regions of interest (ROIs) to use for extraction of mean [
18F]flortaucipir standardized uptake value ratio (SUVR; see [
18F]Flortaucipir section) from target regions. MPRAGE scans were also segmented in Statistical Parametric Mapping 8 (SPM8) to generate subject-specific spatial normalization parameters for use in PET scan processing.
[18F]Flortaucipir PET
Both patients were studied with [
18F]flortaucipir PET within two months of the nearest clinical visit (mean age in the sixth decade). They were injected intravenously with approximately 10 mCi of [
18F]flortaucipir and after a 75-min uptake period were scanned for 30 min on a Siemens mCT (six 5-min frames). Scans were reconstructed according to the Alzheimer’s Disease Neuroimaging Initiative (ADNI)-2 protocol (
http://adni.loni.usc.edu/wp-content/uploads/2015/02/01_DOD-ADNI_Tau-Addendum-Protocol_23Oct2014.pdf). Standard processing, including spatial alignment for motion and normalization to Montreal Neurologic Institute (MNI) space using parameters from the MRI segmentation, was completed in SPM8. Mean static images from 80 to 100 min post-injection were generated by averaging the appropriate frames and smoothed with an 8 mm full-width half maximum (FWHM) Gaussian kernel. Finally, [
18F]flortaucipir SUVR images were generated by intensity normalizing by mean cerebellar crus uptake. The [
18F]flortaucipir PET scans were qualitatively visualized using MRIcron (
http://www.mccauslandcenter.sc.edu/mricro/mricron/). Mean [
18F]flortaucipir SUVR values were extracted from subject-specific ROIs, including the caudate nucleus, putamen, pallidum, thalamus, insula, anterior cingulate gyrus, posterior cingulate gyrus, overall lobar regions (frontal, parietal, temporal, and occipital), the overall cingulate cortex, the sensory-motor cortices, and the global cortex.
[
18F]Flortaucipir PET scans from two CN (mean age of approximately 67.5 years) and two Aβ-positive EOAD patients (mean age of approximately 61 years; mean age of onset of approximately 59 years) were used as comparisons. The patients were selected to match the
PRNP F198S GSS patients by sex and, as closely as possible, by age and global cognition on the MoCA. All scans were processed as described above. Scans were visualized using MRIcron and mean SUVR was extracted from Freesurfer-generated subject-specific ROIs for the regions described above. For display purposes (Fig.
4), mean SUVR values were calculated for the target ROIs in both CN individuals and both EOAD patients as comparisons to the
PRNP F198S GSS patients.
Neuropathology
Patient B expired 9 months after the [18F]flortaucipir PET scan. The brain, harvested at Indiana University School of Medicine, was hemisected along the mid-sagittal plane. The left hemibrain was fixed in formalin. Following fixation in a 10% formalin solution, the left cerebral and cerebellar hemispheres, as well as the left half of the brainstem, were sliced and tissue samples were selected. In order to compare neuropathology with tau PET imaging, six hemispheric coronal slabs were selected that included areas of the frontal, insular, temporal, parietal, and occipital lobes. These were submitted in their entirety for histology and immunohistochemistry. In addition, blocks of the following CNS areas were also submitted: superior frontal gyrus, middle frontal gyrus, anterior cingulate gyrus, superior temporal gyrus, middle temporal gyrus, hippocampus at two levels, entorhinal cortex, precentral cortex, postcentral cortex, inferior parietal lobule, posterior cingulate gyrus and precuneus, calcarine cortex, caudate nucleus, putamen, globus pallidus, amygdala, claustrum, thalamus, subthalamic nucleus, cerebellar vermis, cerebellar cortex and dentate nucleus, midbrain, pons, medulla, and spinal cord at cervical, thoracic, lumbar, and sacral levels. The areas submited were representative of the following Brodmann Areas: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28, 31, 32, 36, 37, 38, 44, 47. The right hemibrain was sliced, frozen, and stored at − 70 °C for structural, biochemical and molecular genetic studies of PrP and tau and for the analysis of the seeding properties of PrP and tau in PRNP F198S GSS.
Brain tissue samples from the left hemibrain were dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin. Eight-micrometer-thick sections from multiple brain areas were stained using the histological and immunohistochemical methods described below. Hematoxylin and eosin (H&E) and Luxol fast blue-hematoxylin & eosin (LFB-H&E) were used to survey gray and white matters for neuronal loss, gliosis, vascular pathology, and other possible pathologic lesions. The Thioflavin S method was used to visualize amyloid deposits and neurofibrillary tangles. Prussian blue stain enhanced by DAB (Prussian Blue-DAB) visualized the ferric iron deposits in the tissue. Neurodegenerative pathology was further analyzed using antibodies raised against PrP (3F4, 1:800, Dr. Richard Kacsak, Staten-Island, New York, USA), tau (AT8, 1:300, Thermo Fisher Scientific, Waltham, MA, USA; PHF-1, 1:10, gift of Dr. P. Davies); 3-repeat tau (3R, 1:3000, Millipore, Billerica Massachusetts, USA), 4-repeat tau (4R, 1:100, Millipore, Billerica Massachusetts, USA), anti-phospho-TDP-43 (1:1000, Cosmo Biologicals, Carlsbad, CA, USA), α-synuclein (ASy119–137, 1:300, Dr. P. Piccardo, Dr. B. Ghetti) and amyloid β (Aβ 21F12, 1:1000, Janssen Research & Development, South San Francisco, CA, USA). AT8 recognizes tau phosphorylated at serine 202 and threonine 205, while PHF-1 recognizes tau phosphorylated at serine 396 and serine 404. The signal from polyclonal or monoclonal antibodies was visualized using avidin-biotin, with goat anti-rabbit immunoglobulin or goat anti-mouse as the secondary antibody as required, followed by horseradish peroxidase-conjugated streptavidin and the chromogen diaminobenzidine. Immunohistochemical sections were counterstained with hematoxylin.
Discussion
The PET tracer [
18F]flortaucipir was used to investigate the pattern of tau deposition in two GSS patients who carry the
PRNP F198S mutation and are members of a pedigree that has been previously studied extensively from the clinical and neuropathological points of view [
7,
11,
14,
35].
GSS caused by the
PRNP F198S mutation is a PrP amyloidosis associated with severe tau deposition in all regions of the cerebrum and brainstem in which misfolded PrP amyloid is observed. On the contrary, tau does not aggregate in the cerebellum, in spite of the heavy PrP burden. DNA changes in the
PRNP gene, including missense, nonsense, insertion, and deletion mutations, may be associated with a PrP amyloidosis that coexists with severe tau deposition [
1,
10,
14,
17,
20,
26,
31]. Patients with different forms of dominantly inherited PrP amyloidosis may present with neuropsychiatric manifestations including depression, personality changes, psychosis, and hallucinations, as well as with frontotemporal dementia-like phenotypes, similar to those previously observed in some
PRNP F198S mutation carriers [
7,
21,
30]. Therefore, it is notable that the two GSS
PRNP F198S carriers reported in the present study had neurological signs that were preceded by psychiatric symptoms.
This study reveals for the first time that there is a considerable uptake of [18F]flortaucipir in various brain regions of both GSS patients with the PRNP F198S mutation. Specifically, [18F]flortaucipir uptake is evident in the anterior and posterior cingulate gyri, insular cortex, caudate nucleus, nucleus accumbens, putamen, globus pallidus, thalamus, and entorhinal cortex. Furthermore, in comparing the more affected individual with the less affected one, the uptake in the former was greater than that in the latter in the majority of the regions mentioned above. This suggests that a correlation between clinical severity and [18F]flortaucipir uptake may exist and that greater [18F]flortaucipir uptake may correlate with a higher burden of aggregated tau.
Similar to what occurs in AD, the NFT present in symptomatic
PRNP F198S mutation carriers are made of 3R and 4R tau; however, the anatomical pattern of [
18F]flortaucipir PET uptake differs considerably from that seen in early- and late-onset AD [
5,
18,
28,
29,
32]. In AD, tau pathology as visualized by [
18F]flortaucipir PET, appears to be distributed over widespread brain areas in the amnestic, non-amnestic, behavioral, corticobasal, and posterior cortical atrophy variants of the disease [
29]. Further, tau PET patterns have been shown to reflect the variability of clinical syndromes and topography of pathologic regions [
29]. Many of the anatomical regions found to have high [
18F]flortaucipir uptake in
PRNP F198S GSS patients belong to the salience network, as well as to the reward system [
27]. Reduction in the function of these networks has been associated with numerous neuropsychiatric disorders, drug dependence, and psychosis. Taken together, our findings and those reported in the literature suggest that in GSS patients carrying the
PRNP F198S allele the behavioral, psychiatric, and neurological symptoms, as well the dementia that develops in the late stage, result from the interaction of misfolded PrP amyloid and tau. The spreading of tau throughout the entire cerebral cortex and subcortical nuclei contributes to the progression and severity of the psychiatric, neurologic, and cognitive dysfunctions [
7,
11,
12,
14].
The neuropathologic results observed in Patient B support the concept that [18F]flortaucipir uptake likely reflects the anatomical localization of misfolded tau and NFT that are distributed in the cerebral gray structures. In fact, there is a definite topographic correspondence between [18F]flortaucipir uptake and the extent of fluorescent NFT in Thioflavin S preparations of cingulate gyrus, caudate nucleus, putamen, and insular cortex. Immunohistochemical preparations of adjacent serially cut sections of these anatomical regions also revealed a strong immunoreactivity to monoclonal antibodies AT8 and PHF-1 in the cingulate gyrus, caudate nucleus, putamen, and insular cortex. It is also important to note that no Aβ immunoreactivity was present in any CNS area affected by PrP or tau pathology.
A finding that needs to be emphasized and that is relevant to the mechanisms of tau spread and to the correlations between [
18F]flortaucipir PET images with those obtained by neuropathologic studies is that many CNS gray matter regions demonstrated notable tau immunopositivity, but not an increased signal on PET images. This discrepancy may be due to differences in tau species in these regions, as [
18F]flortaucipir is known to recognize the NFT, similarly to what Thioflavin S recognizes in histological preparations. Whether [
18F]flortaucipir may detect states of tau aggregation that precede NFT formation needs further investigation. Thus, tau immunolabeled tissue preparations and those stained with Thioflavin S reveal presence of tau in different states of aggregation. AT8 or PHF-1 reveal the hyperphosphorylated tau burden, which is known to be more widespread than that represented by the fluorescent profiles detected in Thioflavin S preparations. The process of tau becoming hyperphosphorylated is not completely understood; however, this process is known to precede tangle formation. Thus, while Thioflavin S detects only the NFTs made of aggregated tau filament cores, by knowing which tau epitopes are recognized by AT8 or PHF-1, we can conclude that the labeling observed in the specific immunohistochemical preparations recognizes neurons containing not only NFT but also portions of the tau fuzzy coat. It is also possible that the inherent spatial and sensitivity limitations of PET imaging may lead to an inability to detect low levels of tau deposition in vivo. Novel tracers with greater specificity for particular tau filaments are needed [
15].
Another region lacking good correspondence between PET imaging and immunohistochemistry is the thalamus, where [
18F]flortaucipir uptake is relatively strong in both patients, but is weak in the immunohistochemical preparation for tau in GSS Patient B. It should be noted that the thalamus and cerebellum are strongly labeled by immunohistochemistry for PrP; however, no tau immunopositivity is seen in the cerebellum and only a weak immunopositivity is detected in the thalamus. The [
18F]flortaucipir PET signal in the thalamus may represent “off-target” binding similarly to what has been reported in other studies with [
18F]flortaucipir tracer showing binding to neuromelanin-containing cells and other targets [
22‐
24]. In view of published studies that showed high iron binding in GSS patients [
11] and non-specific binding of [
18F]flortaucipir to iron [
3], we studied neuropathologically Patient B using a method to detect iron deposits, and showed that iron deposits occur mostly in the globus pallidus and the substantia nigra, but not in the thalamus. Therefore, additional studies will be needed to clarify the significance of the uptake of [
18F]flortaucipir in the thalamus.
Overall, the present study shows for the first time that [
18F]flortaucipir detects in vivo the severe neurofibrillary pathology that is a significant neuropathologic phenotype in GSS patients carrying the
PRNP F198S mutation, which potentially has a profound impact on the pathogenesis of psychiatric and neurological symptoms of the disease [
11,
12,
14]. Based on evidence obtained in two unpublished presymptomatic mutation carriers, PrP amyloid deposits are detected prior to the occurrence of tau pathology and NFT formation; however, additional in vivo evidence is needed to confirm the PrP-tau relationship and its temporal evolution [
11].
In conclusion, it is shown that deposits of tau are detected in vivo by [18F]flortaucipir PET in patients who carry the PRNP F198S mutation and that tau accumulates with a pattern that is strikingly different from that seen in AD. The patterns of tau anatomical spread seen in GSS PRNP F198S and AD may reflect the mechanisms of PrP and Aβ distribution. The results of this study support the view that tau pathology, and not just PrP, contributes significantly to both the psychiatric and the motor symptoms that characterize the phenotype of GSS PRNP F198S and that the different clinical phenotypes of GSS PRNP F198S and AD correlate with the specific pattern of tau anatomical distribution seen in each disease.
Future studies, comparing in vivo longitudinal PET imaging with the post-mortem PrP and tau immunolabeled preparations may allow a precise assessment of the spread of the abnormally conformed proteins over time during the evolution of the PRNP F198S GSS disease process.
Acknowledgements
The authors thank Trina Bird, Christina Brown, Steve Brown, Madeline Cassidy, Ryan Crosbie, Su Gao, Bradley Glazier, Kala Hall, Lili Kyurkchiyska, Heather Polson, Dr. Adam Schwarz, and Wendy Territo for their contributions to this work. We would also like to thank Dr. Kimberly Quaid for her assistance and support with genetic counseling for the patients described in this manuscript and others in the Indiana Alzheimer Disease Center (IADC).