An autopsy report of three kindred in a Gerstmann–Sträussler–Scheinker disease P105L family with a special reference to prion protein, tau, and beta‐amyloid

Abstract Introduction Gerstmann–Sträussler–Scheinker disease P105L (GSS105) is a rare variant of GSS caused by a point mutation of the prion protein (PrP) gene at codon 105 (proline to leucine substitution). It is clinically characterized by spastic paraparesis and dementia and histopathologically defined by PrP‐plaques in the brain. This report describes a clinicopathological analysis of three autopsied kindred from a Japanese GSS105 family, plus a topological analysis of PrP, hyperphosphorylated tau (p‐tau), and beta‐amyloid (Aβ). Methods Using paraffin‐embedded sections, we applied histology and single‐ and multiple‐labeling immunohistochemistry for PrP, p‐tau, and Aβ to the three cases. Comparative semi‐quantitative analyses of tissue injuries and PrP‐plaques were also employed. Results Case 1 (45 years old (yo)) and Case 2 (56 yo) are sisters, and Case 3 (49 yo) is the son of Case 2. Case 1 and Case 2 presented with spastic paraparesis followed by dementia, whereas Case 3 presented, not with spastic paraparesis, but with psychiatric symptoms. In Case 1 and Case 2, the brain showed tissue injuries with many PrP‐plaques in the cerebral cortices, and the pyramidal tract showed myelin loss/pallor. In Case 3, the brain was least degenerated with a number of PrP‐plaques; however, the pyramidal tract remained intact. In addition, p‐tau was deposited in all cases, where p‐tau was present in or around PrP‐plaques. By double‐labeling immunohistochemistry, the colocalization of p‐tau with PrP‐plaques was confirmed. Moreover in Case 2, Aβ was deposited in the cerebral cortices. Interestingly, not only p‐tau but also Aβ was colocalized with PrP‐plaques. In all cases, both three repeat tau and four repeat tau were associated with PrP‐plaques. Conclusions The clinicopathological diversity of GSS105, which is possible even in the same family, was ascertained. Not only p‐tau but also Aβ could be induced by PrP (“secondary degeneration”), facilitating the kaleidoscopic symptoms of GSS.

More than 20 years ago, two members of a Japanese GSS105 family were reported as separate case reports in Japanese (Isshiki et al., 1994;Nakazato et al., 1991). Both of these cases presented with spastic paraparesis and later with dementia and so on, and were subjected to autopsy. Neuropathological examination disclosed numerous PrP-plaques in the cerebrum and cerebellum, and pyramidal tract degeneration was noted in the brain stem and spinal cord (Isshiki et al., 1994;Nakazato et al., 1991). Recently, we had the opportunity to learn of the third patient from this GSS105 family. This case was also subjected to autopsy limited to the brain, and a detailed neuropathological assessment was possible.
The paraffin blocks of the previous two cases had been stored in whole in our institution (Saitama Medical University). Given such a situation, we decided to undertake a comprehensive study that aims to compare the clinicopathological profiles among the three cases.

| Case materials and clinical history
The family tree of the three kindred is shown in Figure 1. Case 1 and Case 2 are sisters, and Case 3 is the son of Case 2. The parents of Case 1 and Case 2 are a married couple between cousins. Although the details are unknown, there is another sibling of Case 1 and Case 2, who was affected with spastic paraparesis, dementia, and cerebellar dysfunction (Figure 1). The clinicopathological and genetic profiles of Case 1 (Isshiki et al., 1994;Kitamoto, Amano, et al., 1993;Nakazato et al., 1991) and Case 2 (Isshiki et al., 1994;Kitamoto, Amano, et al., 1993) were previously reported in Japanese (Isshiki et al., 1994;Nakazato et al., 1991) and English (Kitamoto, Amano, et al., 1993). The genetic analysis confirmed a diagnosis of GSS P105L for this family (Patient No. 1 and Patient No. 2 designated by Kitamoto, et al (Kitamoto, Amano, et al., 1993) correspond to Case 1 and Case 2 in this report, respectively). Case 3 is the virgin case that remains to be reported.

| Case 1
Since the clinical history of this patient, a 45-year-old woman at death, is already available elsewhere in Japanese (Nakazato et al., 1991), this report only describes the outline of it. At the age of 38, she manifested with difficulty in walking. A neurological examination 6 months after the onset disclosed spastic gait coupled with spasticity and pyramidal signs in both legs. A clinical diagnosis of "familial spastic paraparesis" was made. Two years after the onset, she became unable to walk. Soon dysarthria, emotional incontinence, and tremor of the tongue and fingers appeared. Eventually, she became severely demented and died of aspiration pneumonia 6 years after the onset.

| Case 2
Since the clinical history of this patient, a 56-year-old woman at death is already available elsewhere in Japanese (Isshiki et al., 1994), autopsy, beta-amyloid, Gerstmann-Sträussler-Scheinker disease P105L, prion protein, spastic paraparesis, tau this report only describes the outline of it. When she was 5 years old, she was affected with poliomyelitis. Since then her right hand remained paralytic, but there was no problem in her daily life. At the age of 44, she manifested with difficulty in walking, and 4 months later, she became almost unable to walk. Soon dysarthria became evident. About 2 years after the onset, a clinical diagnosis of "familial spastic paraparesis" was made, and thereafter, she became bedridden. Then, she was admitted to a psychiatric hospital for the management of severe emotional disturbance. The pyramidal signs in the upper and lower limbs, chorea-like movement in the left upper limb, apathy, and dementia followed, and she died at the age of 56, 12 years after the onset.

| Case 3
This patient, a 49-year-old man at death, is the son of Case 2. When he was 47 years old, he suffered from hemorrhoids and had them surgically removed. But after the operation, he repeatedly complained of anal pain and kept on consulting several hospitals. Soon, the family members found his way of walking somewhat clumsy.
About 1 year later, he presented with tremor in his fingers. About 2 years after the onset, his stereotyped behavior and speech became more apparent; he walked around the same place at the same time each day; and he dropped in at the same store and bought the same foods (bread and cola). There was an episode where he complained of lucency of his teeth, and he consulted a dentist three days in a row. He began to make comments like "I have been deceived," or "I have been robbed." He became restless and easily agitated, which culminated in an attempted strangulation of his wife. He was hospitalized and remained conscious but did not utter any words. There was a tremor in the upper limbs, and there was myoclonus, which disappeared later, in the lower limbs. In all extremities, deep tendon reflexes were exaggerated but there was no paresis. There were no signs of sensory and cerebellar impairment. During the hospitalization, he relentlessly complained of anal pain and repeatedly ate the same foods (hamburgers and cola). The brain magnetic resonance imaging (MRI) showed mild frontotemporal atrophy, but did not disclose any signal abnormalities on diffusion MRI. The complaint of anal pain was so tenacious that oral morphine was introduced. But he unexpectedly passed away due to paralytic intestinal obstruction leading to septic shock. The whole clinical course was about 2 years (2 years and 3 months). The P105L point mutation of PRNP coupled with codon 129 polymorphism (Val/Met), which is identical to that of Case 1 and Case 2 (Kitamoto, Amano, et al., 1993), was detected by a genetic analysis using blood samples. An autopsy limited to the brain was performed. Table 1 summarizes representative symptoms and some laboratory data of the three cases with the help of previous papers (Isshiki et al., 1994;Kitamoto, Amano, et al., 1993;Nakazato et al., 1991) and medical records available in our institution.

| Preparation of paraffin blocks and histological evaluation
The paraffin blocks of Case 1 and Case 2, which had been stored in Saitama Medical University, were retrieved for re-evaluation of histology and immunohistochemistry; however, as PrP in these blocks had not been detoxicated before, the detoxication step by formic F I G U R E 1 The family tree of the three cases is shown. Other than the three cases analyzed in this study (arrows), there is another sibling (*), a 55-year-old woman at death, who showed spastic paraparesis, dementia, and cerebellar dysfunction. Square, male; circle, female; diamond, an individual whose information of sex is unavailable; +, dead. Closed symbols indicate an individual with definite or possible GSS105-associated symptoms acid treatment was necessary. The paraffin blocks were immersed several times in xylene for deparaffinization then were immersed in ethanol, and finally in tap water. The deparaffinized blocks were immersed in 100% formic acid for 1 hr. After this disinfection step, the blocks were paraffinized again. With respect to Case 3, the brain was fixed in 10% buffered formalin, and representative sections were sliced. The sections were immersed in concentrated formic acid (98%) for 1 hr, washed in tap water, and embedded in paraffin.
Gallyas silver stain was applied to all cases to evaluate neurofibrillary pathology.
After washing in PBS, the sections were incubated with a secondary antibody kit (Dako ChemMate EnVision kit/HRP (DAB)) (room temperature, 30 min). After washing in PBS, the sections were visualized with diaminobenzidine (DAB).
Double-labeling immunohistochemistry was carried out with the following combination of primary antibodies (a mouse monoclonal antibody and a rabbit polyclonal antibody): AT8 and a rabbit polyclonal anti-PrP antibody (PrP-N; 1:2,000; gifted from Dr. Kitamoto, Muramoto, Hilbich, Beyreuther, & Tateishi, 1991), 4G8 and PrP-N, and/or a rabbit polyclonal anti-tau antibody (1:1,000, A 024, Dako) and 4G8. PrP-N is an excellent antibody, as is 3F4, which can be used to visualize PrP-plaques . The use of PrP-N and 3F4 in immunohistochemistry is exchangeable, and both antibodies were successfully applied to single-and multiplelabeling immunohistochemistry in a previous study of GSS (Ishizawa TA   Biosciences), which was then followed by visualization with a peroxidase substrate (HistoGreen; E109, Cosmo Bio, Tokyo, Japan).

| Comparative semi-quantitative analyses of tissue injuries and 3F4-immunoreactive PrP-plaques among the three cases
To compare the three cases, the neuroanatomic structures, including the cerebral cortices and white matter, basal ganglia (putamen, globus pallidus, and/or caudate nucleus), thalamus, hippocampus, brain stem, cerebellar cortices and white matter, and spinal cord, were semi-quantified on HE, KB, and HE-LFB sections for tissue injuries (neuronal loss, gliosis, spongiform change, and/or myelin loss/pallor) in the most affected portion as ± (none or few: minimal), + (mild), ++ (moderate), and +++ (severe). As for PrP-plaques visualized by 3F4-immunohistochemistry, they were semi-quantified in the portion showing the highest density of immunoproducts as -(none), + (sparse), ++ (moderate), and +++ (frequent) with a reference to the semi-quantification strategy for senile plaques in Alzheimer's disease (AD) (CERAD) (Fillenbaum et al., 2008;Mirra et al., 1991). In the cerebral cortices, as the PrP-plaques were distributed in a layer-dependent manner, the semi-quantification was subdivided into the superficial (layers I and II), middle (III and IV), and deep (V and VI) cortical layers.

| Case 1
The brain weighed 1,060 g. Grossly, the brain was mildly atrophic in the

| Case 2
The brain, whose weight was 975 g, was considerably atrophic, par-

| Case 3
The brain, which weighed 1,560 g, was mildly atrophic in the frontal and temporal lobes ( Figure 2I and II). In the cerebral cortices, neuronal loss and gliosis as well as spongiform change were only minimal ( Figure 2III). PrP-plaques were identified, particularly along the deep cortical layers ( Figure 2III-V). In the basal ganglia, hippocampus, and thalamus, a variable number of PrP-plaques were visualized by PrP-immunohistochemistry ( Figure 2VI,VII). The cerebellum was unremarkable with no deposition of PrP-plaques ( Figure 2VIII). The cerebral white matter showed mild myelin loss/ pallor ( Figure 2IX). The brain stem, including the pyramidal tract, was unremarkable ( Figure 2X).

| Comparative semi-quantitative analyses of tissue injuries and PrP-plaques visualized by 3F4immunohistochemistry
The tissue injuries and PrP-plaques visualized by 3F4-immunohistochemistry, which were semi-quantified for the three cases, are presented in Tables 2 and 3, respectively. The tissue injuries were the most prominent in Case 2, which was followed by Case 1 and then by Case 3.
The pyramidal tract in Case 1 and Case 2 was severely affected, while that in Case 3 remained intact. Similarly, the PrP-plaques visualized by 3F4-immunohistochemistry were the most numerous in Case 2, which was followed by Case 1 and then by Case 3. Particularly in Case 2, a large number of PrP-plaques were noted not only in the cerebrum but also in the cerebellum. Even in Case 3, who was the youngest and had the shortest clinical course, there were a fair number of PrP-plaques in the cerebrum, especially along the deep cortical layers.
In Case 1 (Figure 3), PrP-plaques and p-tau were scattered in the cerebral cortices (Figure 3a Similarly to Case 1, p-tau was by far the most numerous in the temporal lobe and hippocampus, but was absent in the cerebellum. In this case, the deposition of Aβ was also noted in the cerebral cortices ( Figure 4c), but not in the hippocampus ( Figure 4f) and cerebellum.
Notably, the majority of Aβ had an immuno-negative central core PrP, with or without p-tau, could be present without Aβ, Aβ, with or without p-tau, could not be present without PrP (Figure 4l), suggesting that PrP deposition is likely a precursor event to Aβ deposition.
In Case 3 ( Figure 5), a minimal amount of p-tau was noted in the cerebral cortices (Figure 5a), all of which, by double-labeling immunohistochemistry, was colocalized with PrP-plaques (Figure 5b). Aβ was totally absent in all the areas studied.

| Single-labeling immunohistochemistry for three repeat tau (RD3) and four repeat tau (RD4)
In all cases, both three repeat tau (RD3) and four repeat tau (RD4) were associated with PrP-plaques (Case 1, Figure 6a

| D ISCUSS I ON
Clinically, Case 1 and Case 2, who were female siblings, manifested with spastic paraparesis, which was then followed by dysarthria, psychiatric symptoms, and dementia. Case 3, who was the son of Case 2, manifested with psychiatric symptoms, such as stereotyped speech and behavior; but in contrast to Case 1 and Case 2, there were no signs of spastic paraparesis during the whole clinical course.
Although the clinical picture of GSS105 is usually predominated by gait disturbance (spastic paraparesis), psychiatric symptoms, and dementia (Amano et al., 1992;Isshiki et al., 1994;Itoh et al., 1994;Kitamoto, Amano, et al., 1993;Kubo et al., 1995;Nakazato et al., 1991;Yamada et al., 1993), there is a substantial variation to it even within the same family (Iwasaki, Kizawa, Hori, Kitamoto, & Sobue, 2009;Koshi Mano et al., 2016;Shiraishi, Mizusawa, & Yamada, 2002;Yamada et al., 1999Yamada et al., , 1995Yamazaki et al., 1999). In a GSS105 family reported elsewhere (Shiraishi et al., 2002;Yamazaki et al., 1999), one member presented with gait disturbance (gait apraxia) that was followed by mutism (Yamazaki et al., 1999), and the other presented with sensory and psychiatric symptoms, including a persistent complaint of pains in various parts of the body, which were then followed by memory disturbance, delusion, and gait disturbance (Shiraishi et al., 2002). A recent paper described three GSS105 families (Families 1, three of the four affected individuals manifested with gait disturbance, and the remaining one manifested with dysesthesia and gait disturbance. Not only parkinsonism but also other neurological signs, such as spasticity, ataxia, involuntary movement, dementia, or emotional instability, were variably noted in all families. Taken together with these reports and others (Amano et al., 1992;Iwasaki et al., 2009;Kubo et al., 1995;Yamada et al., 1999Yamada et al., , 1995, the present study asserts a wide spectrum of symptoms associated with GSS105 even within the same family. The same is true for other mutations of GSS (Giovagnoli et al., 2008;Hsiao et al., 1991;Kovacs et al., 2001;Majtenyi, Brown, Cervenakova, Goldfarb, & Tateishi, 2000;Mastrianni et al., 1995;Nochlin et al., 1989;Piccardo et al., 1998;Popova et al., 2012;Webb et al., 2008). In this respect, though one point merits mention about the clinical profile of the present Case 3: this patient died of paralytic intestinal obstruction leading to septic shock about 2 years after the onset. Based on his clinical history, this comorbidity is more than likely the result, not of GSS105 itself, but of the oral administration of morphine. In fact, intestinal symptoms have rarely been reported in GSS105 (Iwasaki et al., 2009;Koshi Mano et al., 2016). The death of the patient is rather unexpected, and his clinical course, about 2 years in all, is much shorter than those expected for GSS105 (Iwasaki et al., 2009). This could have contributed to the lack of some clinical features compared to the other two cases. For example, although spastic paraparesis was not evident in this patient, F I G U R E 4 Case 2: (a-c, d-f) The distribution of PrP (a, d), p-tau (b, e), and Aβ (c, f) in the temporal cortex (a-c) and hippocampus (CA1 to subiculum, d-f) is shown. The photos are taken from an identical area for a-c and d-f. In the temporal cortex (a-c), p-tau (b, AT8immunostain) and Aβ (c, 4G8-immunostain) seem considerably overlapped with PrP (a, 3F4-immunostain). Notably, most deposits of Aβ have an immuno-negative central core (arrows, c). In the hippocampus (d-f), p-tau (e, AT8-immunostain) seems considerably overlapped with PrP (d, 3F4-immunostai), whereas Aβ (f, 4G8-immunostain) is totally absent. (Original magnification: a-c, ×40; d-f, ×40). (g) P-tau-positive dystrophic neurites (DNs) around PrP-plaques (arrows), neurofibrillary tangles (arrowheads), and neuropil threads are commonly seen (AT8immunostain, temporal cortex, ×400). (h) A small fraction of DNs around PrP-plaques are argyrophilic (Gallyas silver stain, frontal cortex, x600). (i) By double-immunostaining with AT8 and PrP-N, the colocalization of p-tau with PrP-plaques is confirmed. (p-tau, red; PrP, brown. Temporal cortex, x400). (j) By double-immunostaining with 4G8 and PrP-N, the deposition of Aβ around PrP-plaques is confirmed. Note that most Aβ is colocalized with PrP. (Aβ, red; PrP, brown. Temporal cortex, ×200). (k) By triple-immunostaining with AT8, 4G8, and PrP-N, the colocalization of p-tau, Aβ, and PrP is confirmed (p-tau, red; Aβ, brown; PrP, green. Temporal cortex, ×600). (l) By triple-immunostaining with AT8, 4G8 and PrP-N, it is also shown that PrP, with or without p-tau, can be present without Aβ (arrows); on the other hand, Aβ, with or without p-tau, cannot be present without PrP (arrowheads), suggesting that PrP deposition is likely a precursor event to Aβ deposition. (p-tau, red; Aβ, brown; PrP, green. Temporal cortex, ×600) it was recorded that his way of walking was clumsy and deep tendon reflexes were exaggerated. These neurological signs could have been a prelude to spastic paraparesis that could have been possible later if the patient had not prematurely passed away.
Pathologically, in Case 1 and Case 2, the cerebral cortices and pyramidal tract were severely affected. In contrast, in Case 3, the cerebral cortices were relatively preserved, and the pyramidal tract remained uninvolved.
The clinicopathological study of GSS105 with a focus of comparison between individuals within the same family is so far available in two families; one is no other than the present family, the two members of which were previously reported (Isshiki et al., 1994;Kitamoto, Amano, et al., 1993;Nakazato et al., 1991), and the other is the one reported by (Yamada et al. 1999;Itoh et al., 1994). The latter family is comprised of two siblings affected by the disease. One was a male patient whose initial symptom was clumsiness of the right hand at the age of 42, which was followed by spastic paraparesis. Subsequently, he showed signs of ataxia of the extremities, memory impairment, dysarthria, and apraxia.
He died at the age of 53. At autopsy, the brain weighed 1,150 g and showed frontal atrophy. In the cerebral cortices, various sizes of compact and amorphous PrP-plaques were noted, and the pyramidal tract was degenerated from the brain stem to the spinal cord. The other was the sister of the first patient, who initially presented with difficulty in writing because of tremulous movements of the upper extremities, which was followed by gait disturbance, involuntary movement of the legs, speech disturbance, and character changes. When she was 56 years old, she showed emotional and intellectual disturbance. Her speech was scanning with a small voice, and action myoclonus was prominent in the extremities.
There was hyperreflexia of the legs. She died at the age of 58.
At autopsy, the brain weighed 1,200 g. The histopathology of this patient was similar to that of her brother; in the cerebral cortices, various sizes of compact and amorphous PrP-plaques were noted, and the pyramidal tract was degenerated from the brain stem to the spinal cord. Taken together with these reports, the present study indicates that the histopathology of GSS105 can considerably differ from individual to individual within the same family; and yet, it is a good reflection of the symptoms of each patient.
Previously, a full-blown pathology comprised of PrP, p-tau, and Aβ was reported in a GSS102 patient, a 44-year-old man with a 7-year history of dementia (Ishizawa et al., 2002). Numerous p-tau lesions, including NFTs, DNs, and NTs, were found in or around PrP-plaques. An interesting feature of this case was that although a considerable amount of Aβ was found, the majority of it was not colocalized with PrP-plaques. A computer-assisted image analysis targeting the cerebral cortices disclosed a positive and significant correlation between PrP and p-tau, but not between PrP and Aβ. A similar observation, where PrP was associated with p-tau, but not with Aβ, was also reported in a fraction of GSS patients (Amano et al., 1992;Colucci et al., 2006;Ichimiya et al., 1994;Itoh et al., 1994;Kitamoto, Lizuka, et al., 1993;Yamada et al., 1999). In an in vitro experiment, the molecular interaction between tau and PrP was shown; the N-terminus and repeat region of tau are actively involved in its interaction with PrP, and more specifically, the GSS-related mutant of PrP, PrP102, was shown to have a higher tau-binding activity than wild-type PrP (Wang et al., 2008). It is plausible that PrP in GSS possesses an intrinsic ability to induce p-tau deposition without the help of Aβ (Ishizawa et al., 2002;Reiniger et al., 2011). Concerning biochemical properties of tau in GSS, the paired helical filaments, which are morphologically identical to those of AD, are reported in GSS145, 198, and 217 (Ghetti et al., 1995, 1996. Antigenic profiles of NFTs in GSS145 and 198 are shown to be similar, if not identical, to those in AD by immnocytochemistry and immunoblot analysis (Ghetti et al., 1995(Ghetti et al., , 1996(Ghetti et al., , 1994Giaccone et al., 1990;Tagliavini et al., 1993). In fact, the present study showed that p-tau lesions associated with PrPplaques contained both three repeat tau and four repeat tau, just as in AD (Siddiqua & Margittai, 2010). Supposing PrP deposition were the primary pathology of GSS, p-tau deposition in GSS could be regarded as a "secondary degeneration" due to PrP deposition, just as p-tau deposition is likely a "secondary degeneration" due to Aβ deposition in AD (Hardy, Duff, Hardy, Perez-Tur, & Hutton, 1998).

ACK N OWLED G M ENT
We thank Mr. T. Nagai and Mr. T. Honma for their expert technical assistance.

CO N FLI C T O F I NTE R E S T
This study has no conflict of interest.