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Accumulation of multiple neurodegenerative disease-related proteins in familial frontotemporal lobar degeneration associated with granulin mutation

In 2006, mutations in the granulin gene were identified in patients with familial Frontotemporal Lobar Degeneration. Granulin transcript haploinsufficiency has been proposed as a disease mechanism that leads to the loss of functional progranulin protein. Granulin mutations were initially found in tau-negative patients, though recent findings indicate that these mutations are associated with other neurodegenerative disorders with tau pathology, including Alzheimer’s disease and corticobasal degeneration. Moreover, a reduction in progranulin in tau transgenic mice is associated with increasing tau accumulation. To investigate the influence of a decline in progranulin protein on other forms of neurodegenerative-related protein accumulation, human granulin mutation cases were investigated by histochemical and biochemical analyses. Results showed a neuronal and glial tau accumulation in granulin mutation cases. Tau staining revealed neuronal pretangle forms and glial tau in both astrocytes and oligodendrocytes. Furthermore, phosphorylated α-synuclein-positive structures were also found in oligodendrocytes and the neuropil. Immunoblot analysis of fresh frozen brain tissues revealed that tau was present in the sarkosyl-insoluble fraction, and composed of three- and four-repeat tau isoforms, resembling Alzheimer’s disease. Our data suggest that progranulin reduction might be the cause of multiple proteinopathies due to the accelerating accumulation of abnormal proteins including TDP-43 proteinopathy, tauopathy and α-synucleinopathy.

Interestingly, loss-of-function GRN mutations have been identified in patients clinically diagnosed with Alzheimer's disease (AD) [12][13][14][15][16][17][18][19] . For example, p.Gly35Arg (c.103G > A) 19 , and a single base pair deletion (c. 154delA) were found in AD, and the latter was shown to cause a frame shift (p.Thr52HisfsX2) creating a premature stop codon 20 . The rs5848 (3′ UTR + 78C > T) variant was also found in AD 21 and associated with an increased risk of this disease 22 . In addition, GRN mutations were found in the accelerating accumulation of abnormal proteins in corticobasal syndrome 10,[23][24][25][26] . Furthermore, tau pathology, in addition to TAR-DNA binding protein of 43 kDa (TDP-43) pathology, was found in most members of two families harboring a GRN mutation 27 . These findings suggest that a decline in, or dysfunction of, PGRN may cause tau abnormalities, leading to the formation of tau pathology by activation of cyclin-dependent kinases in a P301L tau/GRN +/− mouse model 28 . To explore these issues, we performed immunohistochemical staining and biochemical analyses on human familial GRN mutation cases and examined whether GRN reduction accelerates the accumulation of neurodegenerative-related proteins other than TDP-43.
In this study, using a novel, highly sensitive immunohistochemical method employing free-floating sections, we noted massive phosphorylated-tau-positive staining in some familial GRN mutation cases. Notably, in these same cases, we also observed significant phosphorylated α-synuclein positive staining. Additionally, detergent-insoluble tau and α-synuclein proteins were detected by immunoblot analysis. Similar tau pathology was not seen in other GRN mutation cases when employing standard immunohistochemistry based on paraffin-embedded sections. Our results suggest that at least some cases with GRN mutations may show a hitherto unrecognized accelerated pathological accumulation of tau and α-synuclein.

Materials and Methods
Ethics Statement. All patients, or in some cases in which the patient had died, next of kin, provided written consent for autopsy and postmortem analyses for research purposes. Written informed consent was obtained from all patients. This study was approved by the Ethics Committee of the Tokyo Metropolitan Institute of Medical Science (permission No. 15-1 and 15-5(1)), the Banner Sun Health Research Institute and University of Manchester. The study was performed in accordance with the ethical standards laid down in the 1964 declaration of Helsinki and its later amendments.
Cases. The brain tissues used in Study A from four patients with GRN and three controls were from the Banner Sun Health Research Institute (Sun City, AZ), Brain and Body Donation Program 29,30 . The additional nine GRN mutation cases in Study B were from the Manchester Brain Bank (UK). Ten control cases in Study B were registered in the autopsy archives of Dementia Research Project, Tokyo Metropolitan Institute of Medical Science. Case details are presented in Table 1. Seven different GRN mutations were recorded. Briefly, Case 1 had a c.1252C > T mutation resulting in p.Arg418X. Cases 2, 4, 9 and 12 had a c.1477C > T mutation resulting in p.Arg493X. A point mutation in a translation initiation codon (c.1A > C) predicted reduced mRNA levels in case 3. Three patients (cases 8, 14 and 16) shared c.1355delG mutation resulting in p.V452WfsX38. Case 10 had a c.1402C > T mutation resulting in p.Q468X and case 13 had a c.90_91insCTGC mutation resulting in p.C31LfsX34. Two patients (cases 11 and 15) shared c.388_391delCAGT mutation resulting in p.Q130SfsX124.
Study B: As we could not obtain sections in cases 8-26 that had been fixed and preserved under the same conditions as cases 1-7, we used formalin-fixed, paraffin-embedded sections instead. Therefore, sections from cases 8-26 were cut at 10 μm thickness, deparaffinized, incubated with 1% H 2 O 2 for 30 min to eliminate endogenous peroxidase activity in the tissue, then pretreated for 10 min in 10 mM sodium citrate buffer, pH6.0 at 110 °C. Sections were then treated with formic acid for 10 min (for α-synuclein staining) or 30 min (for tau staining). For tau immunostaining, sections were incubated in 10 μg/ml of trypsin (Sigma-Aldrich) at 37 °C for 10 min. They were also incubated with AT8 and anti-phosphorylated α-synuclein antibody (1175), overnight, as in Study A. Antibody labeling was performed by incubation with goat anti-rabbit IgG (1:1,000, Vector Laboratories, Burlingame, CA, USA) or horse anti-mouse IgG (1:1,000, Vector Laboratories) for 3 hours. The antibody labeling was visualized by incubation with avidin-biotinylated horseradish peroxidase complex (ABC Elite, Vector Laboratories, 1:1,000) for 3 hours, followed by incubation with a solution containing 0.01% 3,3′-diaminobenzidine, 1% nickel ammonium sulfate, 0.05 M imidazole and 0.00015% H 2 O 2 in 0.05 M Tris-HCl buffer, pH 7.6. Counter nuclear staining was performed with Kernechtrot stain solution (Merck, Darmstadt, Germany) or hematoxylin (Muto Pure Chemicals, Tokyo, Japan). The sections were then rinsed with distilled water, mounted on glass slides, treated with xylene, and coverslipped with Entellan (Merck). Tissue sections (cases 1-4) were also stained using a modified Gallyas-Braak method. Photographs were taken with a BX53 microscope (Olympus, Tokyo, Japan).
Histopathological assessments. Age-related plaque scores were determined using the Braak staging 31 .
For purpose of this protocol, the letter corresponds to the following assessment: 0 = No Aβ deposits, A = initial Aβ deposits can be found in basal portions of the isocortex, B = Aβ deposits can be shown in virtually all isocortical association areas, C = Aβ deposits can be seen in all areas of the isocortex, including the sensory and motor core fields. For the evaluation of neurofibrillary changes (tau deposition), Braak staging was applied 31 . In this protocol, the number corresponds to the following assessment of the area of tau deposition: Stage I = transentorhinal cortex, Stage II = entorhinal cortex, Stage III = hippocampus-subiculum, Stage IV = temporal cortex, Stage V = parietal cortex, and Stage VI = occipital cortex. The degree of accumulation of tau and α-synuclein was also evaluated qualitatively and a score ranging from -(negative) to +++ (severe) was assigned.

Results
GRN mutation cases used in the present study. The age, gender, clinical and pathological diagnoses and genetic information on the familial GRN mutation cases and control cases used in this study is summarized in Table 1. None of the GRN mutation cases examined in this study had MAPT mutation, and the MAPT haplotype was determined to be H1/H1 in cases 3, 4, 10 and 11, H1/H2 in cases 1 and 16, H2/H2 in cases 8, 12 and 14 ( Table 1). The MAPT haplotype of cases 2, 9, 13 and 15 were unknown.
Tau accumulation in GRN mutation cases. We observed a considerable number of tau-positive neurons, astrocytes and oligodendrocytes in all 4 Study A GRN mutation cases by either AT8 immunostaining (Fig. 2, and Table 2) or Gallyas silver staining (data not shown). In particular, case 2 showed massive AT8-positive structures in the entorhinal cortex, hippocampus ( Fig. 2A), amygdala (Fig. 2B), temporal cortex (Fig. 2C), insula. In the temporal lobe, the majority of tau-positive neuronal cytoplasmic staining appeared as pretangle-like forms ( Fig. 2A,F,G,H). In the neuropil, fine tau-positive granules were abundant (Fig. 2C). The size of most of these granules appeared smaller than the tau-positive grains observed in argyrophilic grain disease (AGD) brains, and they were negative for Gallyas-silver staining (data not shown). Furthermore, tau-positive astrocytic structures, resembling "bush-like" astrocytes previously reported in AGD 33 , were found in the cortex in all cases in Study A. Their morphology was significantly different from the tufted-astrocytes in progressive non-fluent aphasia (PSP) patients, the astrocytic plaques in corticobasal degeneration (CBD) or the ramified astrocytes in Pick's disease (Fig. 2D). In the white matter, tau-positive oligodendroglial coiled bodies were observed (Fig. 2E).
Gallyas silver staining also revealed structures similar to those stained with AT8, including neurofibrillary tangles (NFTs), threads, and astrocytic and oligodendrocytic structures (data not shown). In the hippocampal region, many NFTs were found in cases 2 and 4 by both AT8 immunostaining and Gallyas silver staining (data not shown). The control cases of Study A showed mild to moderate AT8-positive structures and less glial tau deposition compared to GRN mutation cases ( Table 2). Case 1, 2, 4 and 5 exhibited tau deposition that corresponded to Braak stage IV, Case 3 corresponded to Braak stage V and Case 6 and 7 corresponded to Braak stage I, respectively ( Table 2). The degree of accumulation of tau was evaluated qualitatively and a score ranging from -(negative) to +++ (severe) was assigned (Supplementary Figure 1 and Table 2).
In Study B, eight of nine GRN mutation cases exhibited some AT8 immunoreactivity (Supplementary Figure 2  and Table 3), but the levels of phosphorylated tau deposition were up to Braak stage II except for Case 9 (Table 3), dissimilar to that seen in GRN mutation cases of Study A. No tau deposition or only Braak stage I-II were observed in the control cases of study B ( Table 3). The tau pathology in the GRN mutation cases (Study A) was also detected by 3-repeat (3R)-tau (RD3) and 4-repeat (4R)-tau (anti-4R) specific antibodies indicating that both 3R and 4R tau accumulation was present (Data not shown).
α-synuclein accumulation in GRN mutation cases. Immunohistochemistry using an antibody to phosphorylated α-synuclein, revealed small round or dot-like structures and short thread-like structures in the  temporal lobe (Fig. 3A-F), and oligodendroglial coiled body-like structures in the temporal white matter (data not shown) in cases 1-4 of Study A. The degree of accumulation of α-synuclein was evaluated qualitatively and a score ranging from -(negative) to +++ (severe) was assigned. In particular, case 4 exhibited atypical α-synuclein deposition in the temporal cortex (Fig. 3A,B and Table 2). Phosphorylated α-synuclein positive structures were not found in Study B using paraffin-embedded sections of GRN mutation cases (cases 8-16, Table 3) and control cases (cases 17-26, Table 3).
Amyloid β deposition in GRN mutation cases. Aβ deposition was found in the temporal lobe in three of four GRN mutation cases in Study A ( Fig. 4 and Table 2). In the cases 1 and 4, Aβ pathology was present mostly as diffuse plaques, corresponding to Braak stage A ( Fig. 4A and D). In case 2, there was no Aβ pathology ( Fig. 4B) but case 3 corresponded with Braak stage C (Fig. 4C). Of the control cases in Study A, three were similar to Braak stage A, but one (case 5) corresponded to Braak stage B. In Study B, Aβ accumulation in almost all cases corresponded to Braak stage 0, the others showing Braak stage A (Table 3). and AD (lane 5) by immunoblot analysis of the sarkosyl-insoluble fraction using C-terminal tau antibody (T46) (Fig. 5). The major tau band pattern in GRN mutation cases was triplets of 68, 64 and 60 kDa, similar to that in AD, but different from that in CBD and PSP (Fig. 5). GRN mutation cases were also detected by 3R-tau (RD3) and 4R-tau (anti-4R) specific antibodies indicating both 3R and 4R tau accumulation (Supplementary Figure 3). GRN mutation cases were also studied with anti-phosphorylated α-synuclein antibodies (1175 and pSyn#64) for cases 1-4. Very faint bands of phosphorylated α-synuclein were observed at 16 kDa. (Supplementary Figure 4).

Immunoblot analyses. Biochemical features of accumulated tau in cases of
Fluorescence immunohistochemistry. Fluorescent double-staining of the temporal lobe of the GRN mutation case 4 was performed to examine whether TDP-43/tau (Fig. 6A), TDP-43/α-synuclein (Fig. 6B) or tau/α-synuclein (Fig. 6C) were co-localized in the abnormal structures. Colocalization of these proteins was very infrequent in most abnormal structures.

Discussion
The results of the present study show that GRN mutations causing PGRN reduction may accelerate the intracellular accumulation of not only TDP-43 but also tau and α-synuclein in the brains of familial FTD patients with GRN mutations. This suggests that GRN mutations causing PGRN reduction may be causative or represent risk factors for multiple proteinopathies (TDP-43 proteinopathy, tauopathy or α-synucleinopathy).
Immunohistochemical analyses of phosphorylated TDP-43 revealed a considerable number of neuronal cytoplasmic inclusions and dystrophic neurites in all GRN mutation cases (Fig. 1, Tables 2 and 3). In FTLD-TDP, TDP-43 pathology falls within four histological subtypes (types A-D) based on the predominant type of TDP-43-positive structures exhibited 32 . Type A is characterized by numerous short dystrophic neurites and crescentic or oval neuronal cytoplasmic inclusions. Cases of FTLD-TDP with a GRN mutation invariably display type A pathology [34][35][36] , and present observations were in accordance with this.
The very high sensitivity staining method that we performed for Study A (cases 1-7) revealed Case 2 to show atypical tauopathy with massive tau deposition in neuron, astrocytes and oligodendrocytes without Aβ deposition. Case 4 was atypical synucleinopathy with diffuse α-synuclein positive structures that were observed mainly in the neocortex. Case 3 exhibited massive Aβ deposition corresponding to Braak Stage C and tau deposition corresponding to Braak stage V in AD pathology. Case 1 exhibited tau deposition that corresponded to Braak stage IV. It is possible that case 3 might be an incidental complication of AD because the age was late 70 s' . The pathology in the other two cases (case 2 and 4), however, is very rarely observed in the normal aging brain at mid-50 years of age. The control cases in Study A also had levels of tau deposition that corresponded to Braak stage I to IV (Table 2), but the average age was higher than that of the GRN mutation cases. We compared abnormal tau deposition using the paraffin-embedded tissues of GRN mutation and control cases, and there were significantly differences (Study B, Table 3). Though Study B was less obvious differences than Study A. Braak et al. reported that for Braak NFT stage III-IV, the ratio was less than 10% at ages 50 s' to 60 s' 37 , so that our GRN mutation cases in Study A showed tau accumulation atypical for normal aging.
It has been widely accepted for the past decade that there is no tau deposition in GRN mutation brains. However, using high-sensitivity immunohistochemical staining, we have found that hyper-accumulated tau and α-synuclein can occur in younger GRN mutation cases. Part B of the present study, using paraffin-embedded tissues, however, showed only mild tau deposition, as has been previously reported 8,9 . Hence, GRN mutation may accelerate deposition of tau and α-synuclein but the level of abnormal protein deposition seen in routine paraffin-embedded sections from GRN cases might not be as strong as that seen than in free-floating sections and therefore go unrecognized. Re-analysis might be necessary in other GRN mutation cases using this high-sensitivity immunohistochemical staining method, or immunoblot analyses on frozen brain tissue, in order to gain a fuller appreciation of the level of tau pathology present in such cases.
Our previous report made mention of the fact that a GRN mutation in P301L tau transgenic mice affected phosphorylated tau deposition 28 . The results of the present study support our previous observations in mice. It has been reported that PGRN deficiency causes lysosomal dysfunction 38 . We hypothesized that lysosomal dysfunction might reduce protein degradation in brain cells allowing aggregation-prone neurodegenerative disease-related proteins to deposit more easily.
The features of tau pathology in GRN mutation cases in this study are of predominantly neuronal pretangles, abundant fine granules in the neuropil, and astrocytic and oligodendroglial pathology. It is interesting that fine tau-positive granules were reported in the striatum of a brain with a GRN c.709-2A > G mutation 27 . Among tauopathies, the tau pathology most similar to our cases might be found in AGD. However, the size of the fine granules in our cases seemed smaller than that of the grains in AGD and they were negative for Gallyas-silver staining (data not shown). Although the form of tau-positive astrocytes in our cases was similar to the "bush-like" astrocytes in AGD, their Gallyas-positive status in contrast to the Gallyas-negative status of the "bush-like" AGD astrocytes 33 . No FUS accumulation was found in any GRN mutation cases in Study A, thus there might be no or little relationship between the GRN mutation and FUS deposition (data not shown).
Immunoblot analysis of our cases using C-terminal tau antibody revealed that the banding patterns of the full-length tau in the sarkosyl-insoluble fraction appeared to be essentially the same as that seen in AD (Fig. 5). The staining using three and four repeat tau specific antibodies revealed that tau in the sarkosyl-insoluble fraction consists of both forms of tau (Supplementary Figure 2). These results suggest that accumulated tau in cases of GRN mutation cases contains six tau isoforms just as in AD. However, the distribution of fine granular tau and the lack of any or only light Aβ accumulation (Fig. 4) is different from AD pathology. Tau isoforms in GRN mutation cases were biochemically different from those in CBD and PSP (Fig. 5). Cases of GRN mutation may therefore represent a different tauopathy from that of AD, CBD, PSP and AGD.
In addition to neuronal and glial tau accumulation, the present study also revealed α-synuclein-positive structures, including small round, dot-like or thread-like structures in the cortex and oligodendroglial coiled body-like  structures in the white matter in the GRN mutation cases in Study A (cases 1-4, Fig. 3). Case number 4 showed particularly striking phosphorylated α-synuclein pathology. However, the nine paraffinized GRN mutation cases showed no α-synuclein-positive structures. This discrepancy might be caused by fixation or preservation methods. Leverenz and colleagues reported that α-synuclein pathology was observed in two of seven brains with a familial GRN mutation. One case showed brainstem α-synuclein pathology while the other was cortical 27 .
Accumulations of phosphorylated-tau, α-synuclein and TDP-43 were reported in the brains of Guam/Kii-amyotrophic lateral sclerosis (ALS)-parkinsonism-dementia complex (PDC) patients 39 . The triplet tau band patterns (68, 64, and 60 kDa) of immunoblot analysis in the sarkosyl-insoluble fraction of GRN mutation cases (Fig. 5) appeared to be essentially the same among cases with the GRN mutation, AD and Guam/Kii ALS-PDC. Fine tau-positive granules were also reported in the cerebral white matter of Guam-PDC cases, in which the morphology seemed to resemble that of our cases. Hazy astrocytes were observed in Guam-PDC cases, but their morphology seemed to differ from that of our cases. To date, no GRN mutations in Guam/Kii ALS-PDC cases have been reported. Very recently we reported that the GRN mutation leads lysosomal dysfunction 40 . We speculated that there could be common pathway(s) in lysosomal function or that these diseases have something common features of protein aggregation because of the similarities (TDP-43, tau, α-synuclein deposition and 3R/4R tau isoform aggregation).
In conclusion, when using a highly sensitive free-floating immunohistochemical technique combined with western blotting, we have shown widespread pathological tau and α-synuclein deposition in neurons and glial cells in familial GRN mutation cases that are not apparent when using standard immunohistochemical methods based on routine paraffin embedded sections. Although, the number of samples for this study was small and we recognize the limits of this study, our findings suggest that the pathologies seen in GRN mutation cases may possibly be renamed "neuronoglial multiple proteinopathies".