*2.5. Scatter Plot Analysis of Tau vs. A*β *PET*

Scatter plot analysis of the two tracers revealed that the FAD (Osaka) patient exhibited a distinguishing pattern with a high tau burden and subtle Aβ accumulation in all parts of the AD signature cortices (Figure 5). In contrast, the SAD patients exhibited high accumulation of both tau and Aβ, whereas both were negligible in the HCs.

**Figure 5.** Scatter plot analysis between tau (PBB3-SUVR) and Aβ (PiB-SUVR) accumulation in the frontal cortex ((**A**): FRC); lateral temporal cortex ((**B**): LTC); posterior cingulate gyrus ((**C**): PCG); precuneus ((**D**): PC); and parietal cortex ((**E**): PAR) in a patient with familial Alzheimer's disease with Osaka mutation (FAD (Osaka)), patients with early and advanced sporadic Alzheimer's disease (SAD) and healthy controls (HCs). The FAD (Osaka) patient showed a distinguishing pattern with highly elevated PBB3 uptake and subtle uptake of PiB in all regions. In contrast, the SAD patients exhibited highly elevated accumulation of both PBB3 and PiB, whereas both were negligible in HCs.

#### **3. Discussion**

This report is the first report of tau PET imaging in the proband of familial AD with Osaka mutation, which revealed heavy tau burden in the cerebral cortex and cerebellum with only negligible Aβ in the cerebral cortex. The findings indicate that Aβ may induce tau accumulation and neuronal degeneration without forming senile plaques as previously reported in basic experiments [10–12].

We previously reported that in SAD, tau accumulation spreads from the parahippocampal gyrus to the cerebral cortex with advancing phases of AD, whereas Aβ distribution is already advanced in the clinically earliest stage [15]. Similar reports supporting our data have been recently published by Jack et al. [16]. The increase in tau accumulation in a patient with advanced SAD compared with early SAD patients was also confirmed in the present study. The FAD (Osaka) patient also exhibited higher tau accumulation than the HCs in all the AD signature ROIs in the cerebral cortex. It is noteworthy that the accumulation was even higher than early SAD by more than 2 SD in the lateral temporal cortex and parietal cortex. Although the difference between FAD (Osaka) and advanced SAD was also noticeable in these ROIs, whether this distribution pattern is specific to FAD (Osaka) or just reflecting the advanced stage of AD in general, requires further validation with more advanced SAD cases.

As is widely known, Aβ accumulates heavily and diffusely in the cerebral cortex even in the early stages of SAD and remains high in the advanced stage as in the present study. In contrast, Aβ accumulation was negligibly low in earlier cases of FAD (Osaka) previously reported [1] and remained low in the advanced stage in the present study. These findings strongly support our hypothesis that accumulated Aβ that is usually found with senile plaques is not a prerequisite for the disease progression that includes tau accumulation and neurodegeneration in any phase of FAD (Osaka). Instead, small oligomeric aggregates of Aβ, not detectable with PiB, may play a key role in the onset and progression of the disease. These mechanisms were experimentally confirmed by our previous studies revealing that the Osaka mutation promotes Aβ oligomerization in the brain without forming senile plaques [10–12]. In addition to Aβ, more recent evidences have pointed to the pertinent role of soluble oligomeric tau in AD onset and progression. These oligomers may share a common mechanism of toxicity [17]. As the present case of FAD (Osaka) exhibited heavy tau burden on PBB3 PET, any other FAD mutation without tau accumulation, if found, might prove the toxicity of tau oligomer.

The possible non-harmful profile of accumulated Aβ is critical for the development of AD treatment. Previous clinical trials targeting accumulated Aβ were mostly successful in reducing Aβ but failed to improve the clinical outcomes and even worsened the outcomes in some studies [18]. In contrast, in the present and previous studies, we demonstrated that tau accumulates more widely as the disease progresses in SAD [15]. The burden of tau was also heavy in FAD (Osaka) without amyloid accumulation. These findings strongly warrant further development of novel treatments to reduce soluble Aβ oligomer or target tau, including aducanumab, which is a human monoclonal antibody that selectively binds to Aβ fibrils and soluble oligomers [19].

Cerebellar lesions with senile plaques and/or neurofibrillary tangles as well as Purkinje cell loss were pathologically reported in some FAD patients [20–23], whereas these pathological changes were usually not found until the end stage in SAD [24]. In the present study, Aβ and tau accumulation in the cerebellum were only found in FAD (Osaka). The present patient and other family members with dementia symptoms demonstrated cerebellar ataxia even in the early phase, which is quite rare in most patients with SAD. Cerebellar accumulation of Aβ or tau may be helpful in distinguishing FAD from SAD if familial history cannot be obtained.

In the advanced stage of AD, neuronal degeneration results in cerebral atrophy. The process may decompose and reduce the actual accumulation of Aβ and tau. In addition, atrophy reduces apparent tracer accumulation. The accumulation of PBB3 was even higher in the lateral temporal and parietal cortex in the FAD (Osaka) patient than in the advanced SAD patients with similar cerebral atrophy, indicating substantial tau accumulation in these regions. In contrast, PiB accumulation seemed much lower in the patient with FAD (Osaka) than in the patient with advanced SAD, suggesting much lower Aβ accumulation in FAD (Osaka), but the small amount of Aβ in the FAD (Osaka) patient compared to that in the HCs cannot be ruled out completely.

#### **4. Materials and Methods**

#### *4.1. Ethical Approval*

All procedures performed in studies involving human participants were approved by the Institutional Research Ethics Committee of Osaka City University Graduate School of Medicine (IRB# 3009 approved on 25 December 2014) and were conducted in accordance with the 1964 Helsinki Declaration and its later amendments.

#### *4.2. Informed Consent*

Written informed consent was obtained from all participants or from close family members when the participants were cognitively impaired.

### *4.3. Subjects*

#### 4.3.1. Familial Alzheimer's Disease with APP Mutation

Details of the FAD (Osaka) pedigree were described elsewhere [10]. We previously reported PET imaging of Aβ accumulation in two sister patients of the pedigree [13]. In the present study, the elder sister, who was the proband, was examined. She was in the advanced stages of dementia, bed-ridden and did not speak.

#### 4.3.2. Sporadic AD

We recruited SAD patients from patients attending the memory clinic of Osaka City University Hospital. We evaluated medical history, neurological findings and general blood tests. Two qualified clinical psychologists (M.A. and N.K.) scored the MMSE. An MRI of the brain including coronal sections for hippocampal evaluation was taken.

AD was diagnosed based on the IWG-2 criteria for typical AD with amyloid PET as in vivo evidence of AD pathology [4]. Apparent familial AD was not included in the SAD diagnosis. The same exclusion criteria applied to the HCs were applied to the AD patients to exclude confounding diseases, such as diabetes, dyslipidemia and hypertension.

#### 4.3.3. Healthy Controls

HCs without a history of brain disorders or subjective abnormalities were openly recruited as candidates. All candidates took physical examinations covering vital and cardiopulmonary systems. Tests that were administered to the AD patients were applied to the HCs.

The exclusion criteria were (1) a history of any brain diseases, brain surgery, or head trauma that requires hospitalization; (2) high risk for cerebrovascular diseases, including poorly controlled diabetes, severe dyslipidemia, and hypertension above the recommended level of standard guidelines; (3) any neurological findings suggesting brain diseases; (4) low cognitive test scores [MMSE, and Rivermead Behavioral Memory Test (RBMT)]; (5) significant MRI lesions including asymptomatic lacunas (less than 15 mm in diameter, high in T2, FLAIR and low in T1), severe white matter lesions (more than 1 grade in Fazekas score [25]), more than 4 cerebral microbleeds, and atrophy beyond average for their age by visual inspection; and (6) positive amyloid PET imaging. No other biomarkers were evaluated in the HC group.

#### *4.4. PET Data Acquisition*

11C-PBB3 and 11C-Pittsburgh compound-B (2-[4-(11C-methylamino) phenyl]-1,3-benzothiazol-6-ol, 11C-PiB) were produced following the methods previously reported [2,26,27]. 11C-PBB3-and 11C-PiB-PET images were obtained with a Siemens Biograph16 scanner (Siemens/CTI, Knoxville, TN, USA) and with an Eminence-B PET scanner (Shimadzu Co., Kyoto, Japan), respectively. 11C-PBB3, a tau tracer, was intravenously injected in the range of 370 MBq (body weight ≤ 50 kg) to 555 MBq (body weight ≥ 70 kg) in a dimly lit room to avoid photoracemization. A 60-min PET scan was performed in list mode. The acquired data were sorted into dynamic data with 6 × 10 s, 3 × 20 s, 6 × 60 s, 4 × 180 s, and 8 × 300 s frames. To evaluate Aβ accumulation, each subject received 400 to 500 MBq of 11C-PiB intravenously over 1 min. After the injection, a static scan image acquisition was performed for 50 to 70 min. Reconstruction of PET images for 11C-PBB3 and 11C-PiB was performed by filtered back projection using a 4-mm full width at half maximum (FWHM) Hanning filter and a 5-mm FWHM Gaussian filter with attenuation and scatter correction, respectively.

#### *4.5. Criteria for A*β *Accumulation in SAD*

Aβ accumulation was determined visually based on the Japanese Alzheimer's Disease Neuroimaging Initiative (J-ADNI) Visual Criteria for 11C-PiB-PET (J-ADNI\_PETQC\_Ver1.1) modified from the ADNI PET core criteria [28]. The four regions selected for the assessment were the frontal lobe, lateral temporal lobe, lateral parietal lobe and the combined area of precuneus and posterior cingulate gyrus. The patient was considered positive when the accumulation in one of the four cerebral cortices was higher than that in the white matter just below the cortex and negative when none of the four cortices had higher accumulation than the white matter.
