The molecular spectrum of amyloid‐beta (Aβ) in neurodegenerative diseases beyond Alzheimer's disease

Abstract This study investigated the molecular spectrum of amyloid‐beta (Aβ) in neurodegenerative diseases beyond Alzheimer's disease (AD). We analyzed Aβ deposition in the temporal cortex and striatum in 116 autopsies, including Lewy body disease (LBD; N = 51), multiple system atrophy (MSA; N = 10), frontotemporal lobar degeneration‐TDP‐43 (FTLD‐TDP; N = 16), and progressive supranuclear palsy (PSP; N = 39). The LBD group exhibited the most Aβ deposition in the temporal cortex and striatum (90/76%, respectively), followed by PSP (69/28%), FTLD‐TDP (50/25%), and the MSA group (50/10%). We conducted immunohistochemical analysis using antibodies targeting eight Aβ epitopes in the LBD and PSP groups. Immunohistochemical findings were evaluated semi‐quantitatively and quantitatively using digital pathology. Females with LBD exhibited significantly more severe Aβ deposition, particularly Aβ42 and Aβ43, along with significantly more severe tau pathology. Furthermore, a quantitative analysis of all Aβ peptides in the LBD group revealed an association with the APOE‐ε4 genotypes. No significant differences were observed between males and females in the PSP group. Finally, we compared striatal Aβ deposition in cases with LBD (N = 15), AD without α‐synuclein pathology (N = 6), and PSP (N = 5). There were no differences in the pan‐Aβ antibody (6F/3D)‐immunolabeled deposition burden among the three groups, but the deposition burden of peptides with high aggregation capacity, especially Aβ43, was significantly higher in the AD and LBD groups than in the PSP group. Furthermore, considerable heterogeneity was observed in the composition of Aβ peptides on a case‐by‐case basis in the AD and LBD groups, whereas it was relatively uniform in the PSP group. Cluster analysis further supported these findings. Our data suggest that the type of concomitant proteinopathies influences the spectrum of Aβ deposition, impacted also by sex and APOE genotypes.

While age is the most important risk factors for NDDs, sex and certain genotypes, especially apolipoprotein E (APOE) ε4, are considered significant risk factors for developing late-onset AD [9][10][11][12].We recently reported that females with Lewy body disease (LBD) have significantly more severe Aβ deposition, labeled by a pan-Aβ antibody (clone 6F/3D; epitope lies within 10-15 amino acids) than males [13].Several studies have also reported sex-based differences in individuals with LBD, including the concentration of Aβ in cerebrospinal fluid and the severity of AD pathology [14][15][16][17][18][19][20].Furthermore, APOE ε4 status has been reported as a risk factor for the presence of copathologies independent of NDD [3].Thus, these factors are also speculated to influence the spectrum of Aβ pathology in non-AD NDDs.
Striatal Aβ deposition is an important pathological finding as it is closely associated with the development of dementia in individuals with LBD [21][22][23].Kalaitzakis et al. reported that the striatal Aβ deposition burden was higher both in Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) compared to that in PD, multiple system atrophy (MSA), and progressive supranuclear palsy (PSP) [23].Furthermore, they also reported that striatal cored plaques were only observed in patients with DLB [23].As the composition of Aβ peptides varies depending on the morphology of the senile plaques [24,25], it is possible that the composition of striatal Aβ peptides differs in each NDD.
Amyloid-centric therapeutic strategies have been rapidly evolving [26][27][28][29].Consequently, it is crucial to comprehend the molecular characteristics of pathological proteins deposited in each non-AD NDD to enhance therapeutic efficacy.However, there is a lack of knowledge regarding the spectrum of Aβ deposition and its association with some factors such as sex and APOE genotype, particularly in non-AD NDDs.To elucidate this, we conducted a comprehensive immunohistochemical study of non-AD NDDs, including LBD, MSA, frontotemporal lobar degeneration-TDP43 (FTLD-TDP), and PSP.Initially, we performed a semi-quantitative assessment of Aβ burden in the neocortex and striatum using anti-pan-Aβ antibodies (6F/3D) to explore potential differences in Aβ deposition patterns among these diseases.We also assessed the effects of age, sex, and APOE genotypes on Aβ deposition in those diseases.Subsequently, we evaluated the spectrum of deposited Aβ peptides using antibodies targeting eight different epitopes of Aβ focusing on LBD and PSP, as these two groups had the most cases in our study.Lastly, we investigated whether the spectrum of deposited Aβ peptides in the striatum differs between LBD, PSP, and AD showing similar levels of 6F/3Drecognized amyloid deposition by semiquantitative analysis or Thal phase classification [30].

| Subjects
We reviewed the archives of neuropathological autopsies from the University Health Network Neurodegenerative Brain Collection (UHN-NBC) and selected 120 consecutive brain donation cases, where, based on clinical and pathological interpretation LBD, MSA, FTLD-TDP, and PSP were diagnosed.Patients with a clear history of repetitive head injury were excluded from this study Finally, we included 116 cases, including 51 cases of LBD, 10 cases of MSA, 16 cases of FTLD-TDP, and 39 cases of PSP (Table 1).Regarding the LBD group, we assessed the severity of Lewy-related pathology using α-synuclein immunohistochemistry (antibody 5G4) based on the Lewy pathology consensus criteria (LPC) [31,32].We included only cases with neocortical and limbic Lewy-related pathology, as previously described [14].For comparison, we included 14 AD cases without LBD-related pathology.All brains were obtained at autopsy through appropriate consenting procedures with Local Ethical Committee approval.This study was approved by the UHN Research Ethics Board (No.  and University of Toronto (No. 39459) and was performed per the ethical standards established in the 1964 Declaration of Helsinki, updated in 2008.

| Neuropathological case selection
Following Aβ and tau immunostaining, we classified all cases according to the National Institute on Aging-Alzheimer's Association (NIA-AA) guidelines, dividing the level of AD neuropathologic change (ADNC) into four categories (not, low, intermediate, and high) [33].Additionally, we evaluated the type of cerebral amyloid angiopathy (CAA) according to the Thal classification [34].For detailed pathological analysis in the TL, we selected cases with an A score of 2 or higher [30,33] and with mild (G2) or higher Aβ deposition on the 6F/3D-immunostained specimen (see below).For Aβ analysis in the striatum, we selected cases with moderate (G3) or higher striatal Aβ deposition on the 6F/3Dimmunostained specimen (see below). Figure 1 shows the flow chart of case selection in the TL (Figure 1A) and striatum (Figure 1B).

| Semiquantitative grading system for Aβ and tau pathology
In addition to the staging systems described above, we semiquantitatively graded the severity of immunohistochemical findings of each Aβ peptide and tau (AT8).The severity of Aβ pathology within brain parenchyma of the TL and striatum (as amyloid plaques) and cerebral vessels (as CAA) was evaluated using a five-point scoring system [35,36] as follows: Plaque Grade 0, no Aβ plaques in parenchyma/layer; Grade 1, a few Aβ plaques in parenchyma/layer occupying each low power (Â10 microscope objective) field; Grade 2, a moderate number of Aβ plaques in parenchyma/layer occupying each low power field; Grade 3, many dispersed Aβ plaques in parenchyma/layer occupying each low power field; Grade 4, abundant densely packed Aβ plaques in parenchyma/layer occupying each low power field.CAA Grade 0, no CAA in blood vessel walls in leptomeninges or brain parenchyma; Grade 1, occasional blood vessels with CAA in leptomeninges and/or within brain parenchyma, usually not occupying the full thickness of the wall; Grade 2, a moderate number of blood vessels with CAA in leptomeninges or brain parenchyma in leptomeninges or within brain parenchyma, some occupying the full thickness of the wall; Grade 3, many blood vessels with CAA in leptomeninges or brain parenchyma, most occupying the full thickness of the wall; Grade 4, most or all blood vessels with severe CAA in leptomeninges or within brain parenchyma, occupying the full thickness of the wall.
The staging system we used for CAA [36] follows the concept of the grading proposed by Love et al. [37] but includes five grades (0-4) instead of four (0-3) and better comparable to the grading we used to evaluate parenchymal deposits.Representative images are shown in Figure S1.Additionally, we evaluated the severity of coarse-grained plaque (CGP) pathology [24] using the same grading system (representative microphotographs of CGPs are shown in Figure 2A,B).The severity of overall tau (AT8) pathology, including pre-tangles, NFTs, and neuropil threads, was evaluated using a five-point scoring system [35,36] as follows: Grade 0, no tau pathology present; Grade 1, very few and scattered neuronal cytoplasmic tau immunoreactivity and neuropil threads in each low power (Â10 microscope objective) field; Grade 2, a mild number of neurofibrillary tangles and neuropil threads in each low power field; Grade 3, a moderate number of neurofibrillary tangles and neuropil threads in each low power field; Grade 4, many densely packed neurofibrillary tangles and

| Quantitative image analysis
Regarding the TL, the region of interest was defined as the cortical gray matter of an entire involved sulcus and at least one side of the adjacent gyral crest where Aβ deposition was present because the distribution of senile plaques can vary within the cortex [13,24,38].Immunostained sections were scanned at a magnification of Â40 over the entire region with a TissueScope LE120 slide scanner (Huron Digital Pathology, Ontario, Canada).All scanned slides were separately imported to Photoshop (version 24.0; Adobe Inc., CA, USA) and one region of interest was cropped from each image and saved as a new image.The new images were pre-processed by adjusting the contrast to improve the recognition of small diffuse plaques and reduce the background noise of non-specific immunoreactivity.
For the striatum, the region of interest was delineated based on anatomical definitions.According to a previous study [22], 10 image squares were taken within the regions of interest as evenly as possible without bias over the entire area (area per image: 0.7 Â 0.5 mm) using a bright field microscope (Nikon Eclipse Ci microscope, Nikon Instruments Inc, Tokyo, Japan) equipped with a DS-Fi3 microscope camera and NIS-Elements imaging software.All microphotographs were separately imported to Photoshop and pre-processed by adjusting the contrast to improve the recognition of small diffuse plaques and reduce the background noise of non-specific immunoreactivity.
The burden of Aβ deposition in each image was quantified using ImageJ/Fiji (ImageJ Version 1.53q) [39] as described previously [13].This method allows avoiding potential technical bias due to different fixation and enzymatic diaminobenzidine reactions (i.e., the labeling intensity) [13,40].
Disease category grouping of the cases was performed by one neuropathologist (G.G.K.) based on clinical information and current immunohistochemistry-based neuropathological criteria.The genotypes for APOE and MAPT were obtained as described previously [41].The semiquantitative case selection and quantitative Aβ burden analysis were performed separately by another neuropathologist (S.I.).For the Aβ burden analysis processes, all assessments were performed blinded to clinical and neuropathological diagnoses.

| Statistical analysis
Data were analyzed using IBM SPSS Statistics (version 28.0.0.0;SPSS Inc, Chicago, IL, USA), with a significance level set at p < 0.05.Fisher's exact test was used for categorical variables (sex and pathological findings).Mann-Whitney U test was used for comparing continuous variables (age at death and Aβ deposition burden) between two groups, while Kruskal-Wallis test with post hoc Bonferroni correction was used for comparing three or more groups.Spearman rank correlation coefficient test was used to analyze the relationship between the CGP pathological grading and the number of APOE ε4 alleles (APOE ε4 score; n = 0-2) or the CAA pathological grading.An ordinal regression analysis (ORA) was used to adjust for the effect of age and the APOE ε4 score on the Aβ pathological grading for semiquantitative analysis results.For quantitative analysis results, analysis of covariance (ANCOVA) was used to adjust for the effect of age and the APOE ε4 score on the Aβ pathological grading.Furthermore, using JMP Pro (version 11.2.0;SAS Institute Inc., Cary, NC, USA), we conducted Ward's hierarchical cluster analysis based on the quantitative results of each Aβ peptide deposition burden.significantly lower than that in the LBD group ( p < 0.01).Additionally, females were older than males in all disease groups, but the differences were not statistically significant ( p = 0.08/0.48/0.09/0.96 in the LBD/MSA/FTLD-TDP/PSP groups, respectively).APOE ε4 allele frequency was significantly higher in the LBD group than in the FTLD-TDP and PSP groups ( p = 0.03, <0.01, respectively), while MAPT H1 allele frequency was significantly higher in the PSP group than in the LBD group ( p < 0.01).In the LBD group, 33 out of 51 cases (67%) showed intermediate or higher ADNC levels, which was significantly higher than in the other groups (vs.all groups; p < 0.01).In the LBD group, females had significantly more ADNC-high cases than males ( p = 0.01).After adjusting for age, this difference retained its significance (p < 0.01; binomial logistic RA).Although Aβ deposition was observed in more than half of the patients in all groups, the positivity was significantly higher in the LBD group than in the other groups (vs. the MSA/FTLD-TDP/PSP groups; p < 0.01/<0.01/0.01).Moreover, the positivity of striatal Aβ deposition was significantly higher in the LBD group than in the other groups (vs.all groups; p < 0.01).After selecting cases based on the neuropathological evaluation described above, we excluded the MSA and FTLD-TDP groups because only one and four cases, respectively, were suitable for further detailed histopathological evaluation.

| NDD group characteristics
A summary of the cases that underwent detailed histopathological evaluation, including the evaluation of various Aβ peptides, is shown in Table 3.In the LBD group, the dementia status was available for 31 of 36 patients, and all of them had a clinical diagnosis of dementia.Furthermore, 32 out of 36 cases (89%) exhibited moderate or higher levels of AD co-pathology.APOE genotypes were available for 34 cases in this group, and 29 cases (85%) had one or more APOE ε4 allele(s) (7 cases, 21%, were homozygous).Additionally, all cases that showed CGP formation in the TL had at least one APOE ε4 allele, and the severity of CGP pathology was significantly correlated with the APOE ε4 score (R = 0.59, p < 0.01; Figure 2C).The effect of the APOE ε4 score on the CGP severity remained significant after adjusting for age and sex (p < 0.01; ANCOVA).Furthermore, the severity of CGP pathology correlated with the severity of CAA pathology, evaluated using immunostaining for 6F/3D (R = 0.76, p < 0.01).In the PSP group, seven out of 10 cases (70%) exhibited moderate or higher levels of AD co-pathology.APOE allele status was available for nine cases, seven of which (78%) had one APOE ε4 allele.Notably, none of the cases were homozygous for APOE ε4 allele in this group, and only one case (10%) showed CGP formation.Seven cases in the LBD group and two cases in the PSP group exhibited Type 1 CAA lesions.Among these, seven out of eight cases (88%) genotyped for APOE had one or more APOE ε4 allele(s).

| Evaluation of the neocortical Aβ and tau deposition severity in the LBD and PSP groups
The representative histological photographs of Aβ plaques and the analysis region are presented in Figure 3, and the representative histological microphotographs of CAA lesions, including Type 1 and 2 lesions, are displayed in Figure 4.A summary of the semi-quantitative and quantitative analyses results is presented in Table 4, and all analysis results are shown in Table S1.Based on subjective histopathological observations in both disease groups, the amount of neocortical Aβ deposits immunolabeled by Aβ 42 was the highest (Figure 3E), and the parenchymal deposition of shorter peptides (Aβ 38 and Aβ 39 ) and Aβ pSer8 was relatively mild compared to others (Figure 3A-H).Type 2 CAA lesions showed the most robust immunoreactivity for Aβ 40 and relatively weak immunoreactivity for longer Aβ peptides (Aβ 42 and Aβ 43 ) (Figure 4A-H, upper panels).In contrast, in Type 1 lesions, the immunoreactivity was stronger for these longer peptides than for the shorter peptides (Aβ 38 and Aβ 39 ) (Figure 4A-H, lower panels).The immunoreactivity of Aβ Np3E and Aβ pSer8 was strong and intermediate in both types of CAA lesions, respectively (Figure 4G,H).Regarding the semiquantitative severity of CAA, there were no significant differences between the two groups in all Aβ peptides investigated.Considering the extent of deposition and background non-specific immunoreactivity, we conducted a quantitative analysis on sections immunostained for 6F/3D, Aβ 40 , Aβ 42 , Aβ 43 , and Aβ Np3E .
In the LBD group, females showed more severe Aβ plaque pathology than males in most of Aβ-immunostainings investigated.The statistical significance was reached for semiquantitative grading of Aβ 42 and Aβ 43 , as well as for the quantitative burden of pan-Aβ (6F/3D) and Aβ 43 .Notably, only males displayed mild Aβ 43 deposition (G2), while severe Aβ 43 deposition (G4) was observed exclusively in females (Table S1).After adjusting for age and the APOE ε4 score, the differences remained statistically significant for semiquantitative grading of Aβ 42 (p = 0.02; ORA) and quantitative burden of 6F/3D (p = 0.04; ANCOVA).Additionally, although the observed difference did not reach statistical significance, there was a tendency for females to exhibit a higher quantitative Aβ 43 deposition burden compared to males, following adjustment (p = 0.054; ANCOVA).Similar levels of Aβ 38 , Aβ 39 , and Aβ pSer8 deposition were observed between sexes.Furthermore, no significant differences were found in the severity of CAA pathological grading between males and females.Regarding p-tau pathology, females showed significantly more severe tau deposition than males.After adjusting for age and the APOE ε4 score, the difference remained statistically significant (p = 0.03; ORA).We additionally conducted an analysis to explore the impact of the APOE ε4 score on the quantitative burden of Aβ deposition.The findings are summarized in Table 5.Out of the 30 cases included in the quantitative analysis, four cases were classified as Group 0 (score of 0), 19 cases as Group 1 (score of 1), and seven cases as Group 2 (score of 2).Group 2 exhibited higher quantitative Aβ deposition burdens across all peptides investigated compared to the other groups, with significant differences observed for Aβ 42 .Moreover, there was a positive correlation between the APOE ε4 score and the quantitative deposition burden of all investigated Aβ peptides in this cohort, with significant correlations found for Aβ 40 , Aβ 42 , and Aβ Np3E .
In the PSP group, the mean values of all Aβ peptides subjected to quantitative analysis were lower compared to the LBD group, with significant differences observed Unlike the LBD group, there were no notable differences in Aβ pathology between females and males.In terms of p-tau pathology, there was no significant difference observed between the sexes.6 and all the analysis findings in Table S2.Based on the criteria detailed above only 15 LBD, 6 AD, and 5 PSP cases were selected for detailed analysis (i.e., those showing G3 or above severity of striatal Aβ deposition; see Figure 1 for case selection details).Most Aβ plaques displayed diffuse-type morphology (Figure 5A-O).There were no significant differences in the severity of Aβ deposition labeled by antibody 6F3D among the three groups in the semiquantitative analysis.Although not statistically significant ( p = 0.07, see Table 6), cored plaque formation tended to be greater in the AD and PSP groups and less in the LBD group (   significantly higher Aβ 40 deposition in the CN and putamen than the other groups (Figure 5Q,V), the amount of deposition was low in all groups.In contrast, the AD group showed significantly higher Aβ 42 (Figure 5R,W), Aβ 43 (Figure 5S,X), and Aβ Np3E (Figure 5T,Y) deposition in the CN and putamen than the other groups.To rigorously investigate these differences, we further compared the burden of Aβ deposition among only individuals with ADNC-A score of 3 in the three groups.The data are provided in Table S3 and Figure S3.In comparing the AD and LBD groups, only the deposition burden of Aβ 42 in the putamen was significantly more severe in the AD group ( p = 0.02).In contrast, when comparing the AD and PSP groups, 6F/3D deposition was significantly more severe in the PSP group in both the CN and putamen ( p < 0.01, 0.04, respectively), whereas the deposition burden of Aβ 42 , Aβ 43 , and Aβ Np3E were significantly more severe in the AD group in both the CN and putamen (Aβ 42 , p < 0.01 in both; Aβ 43 , p = 0.03, < 0.01; Aβ Np3E , p < 0.01 in both, respectively).Due to the unavailability of APOE genotypes for all AD cases in this cohort, a comparative evaluation of the impact of APOE score among the three groups could not be performed.However, no significant difference was observed between the LBD and PSP groups ( p = 1.00;Mann-Whitney U test).Furthermore, we compared the burden of Aβ deposition among only individuals with ADNC-A score of 2. In this cohort, only cases in the LBD and PSP groups were included.The data are provided in Table S4 and Figure S4.Although there were no significant differences in sex ratio or the APOE score between the two groups (p = 1.00, 0.79, respectively), the deposition burden of 6F/3D and Aβ 43 was significantly more severe in the LBD group in both the CN and putamen (6F/3D, p = 0.02, < 0.01; Aβ 43 , p < 0.01 in both, respectively).

| Subgroups classified with cluster analysis
We conducted a cluster analysis based on the quantitative deposition burden of each Aβ peptide in the TL and striatum (Figure 6).Clinical and pathological information for each group can be found in Tables S5 and S6.
In the TL, the cluster analysis categorized the cases into two groups (Figure 6A).Table S5 provides details for each group.Group 1 (consisting of 28 cases) exhibited lower levels of Aβ deposition compared to Group 2 (consisting of 8 cases).Therefore, Group 1 was designated as the "low Aβ deposition group."Notably, all PSP cases were classified within this group, supporting the finding of less Aβ deposition compared to LBD cases.Group 2 displayed significantly higher levels of Aβ deposition compared to Group 1 across all investigated Aβ peptides (p < 0.01).Hence, Group 2 was identified as the "high Aβ deposition group."Moreover, Group 2 demonstrated a significantly greater proportion of cases with ADNC-high in comparison to Group 1 (75% vs. 29%; p = 0.04), providing support for the association between tau pathology and the extent of Aβ deposition in the cerebral cortex.Among the eight cases in this group, seven were females.The frequency of APOE ε4 allele was significantly higher in Group 2 compared to Group 1 (p = 0.046).
In the striatum, the cluster analysis divided the cases into three groups (Figure 6B).Table S6 provides additional information for each group.This study aimed to investigate the spectrum of AD-related pathology in four non-AD NDDs using immunohistochemistry.Our findings revealed several significant observations: (1) there are notable variations in the severity of AD-related pathology among non-AD NDDs; (2) the frequency of the APOE ε4 allele is higher   6) the deposition burden of peptides with high aggregation capacity in the striatum in the AD and LBD groups, especially Aβ 43 , was significantly higher than in the PSP group; (7) the composition of Aβ peptides deposited in the striatum varies considerably from case to case in the AD and LBD groups, but is relatively consistent in the PSP group; and (8) cluster analysis corroborated these observations.We have demonstrated that the LBD group exhibited the most severe AD-related pathologies among the four non-AD NDDs investigated, which aligns with previous neuropathological studies [3,5].In contrast, the MSA group, also categorized as an α-synucleinopathy [42], displayed the mildest AD pathology.One possible reason for this disparity is that the mean age at death in the MSA group was significantly younger than that in the LBD group in this study.However, previous studies have indicated that the incidence of concomitant ADNC in MSA is generally lower than in LBD [1,16,43,44], suggesting that factors beyond age contribute to these differences.There is a growing understanding that the pathological aggregation of one protein can synergistically initiate or promote the aggregation of different species [45].Synergistic interactions between Aβ and α-synuclein, tau and α-synuclein, as well as these three proteins together, have been reported [46][47][48][49][50].Given that the strain of α-synuclein differs in LBD and MSA [51][52][53], we can speculate that the interaction between α-synuclein and AD-type pathological proteins is weaker in MSA compared to LBD.
Furthermore, it is noteworthy that the severity of Aβ pathology was significantly lower in the PSP group compared to the LBD group, despite no significant difference in mean age at death between the two groups.It is important to mention that the APOE ε4 and MAPT H1 allele frequencies differ between the two disease groups in our cohort.In particular, there were no cases homozygous for APOE ε4 in the PSP group.However, recent studies on large PSP cohorts have consistently demonstrated low levels of ADNC among individuals with PSP [54,55].Furthermore, Robinson et al. found that the overall burden of FTLD-tau, which includes neuronal, astrocytic, and oligodendrocytic tau, did not correlate with Thal phase and CERAD neuritic plaque scores [56].The properties of deposited tau are known to be different between PSP and AD [57][58][59], suggesting that the synergistic interaction between tau and Aβ may be weaker in PSP compared to AD.
Semi-quantitative and quantitative analyses revealed that females in the LBD group had higher AD-related pathologies than males.These findings support previous results reported by Bayram et al., who evaluated a similar cohort [14].Specifically, females in this group exhibited statistically significant higher Aβ deposition, labeled by immunostaining for 6F/3D, Aβ 42 , and Aβ 43 .The 6F/3D antibody is predicted to react primarily with Aβ generated through β-secretase and γ-secretase, known as the amyloidogenic pathway [60].Additionally, both Aβ 42 and Aβ 43 are hydrophobic and prone to form toxic aggregates [7,61,62].Hence, it is suggested that females have more neurotoxic Aβ peptides deposited than males in the LBD group.It is well known that approximately twothirds of individuals with AD are females, and the female sex is considered a major risk factor for developing AD [10,17,[63][64][65].Sex hormones have been proposed as a potential explanation for this difference, and several studies have indicated that sex hormones have protective effects against Aβ, such as regulating the production, transport, and removal of Aβ [66][67][68], and preventing Aβ toxicity to mitochondria and reducing neuronal vulnerability to apoptosis [69,70].Therefore, we can speculate that females with LBD tend to be more predisposed and vulnerable to greater Aβ deposition, and associated tau deposition [71][72][73], resulting in differences in the severity of AD pathology between the sexes.APOE ε4 is a significant genetic risk factor for lateonset AD dementia [64].As the apoE4 protein is less efficient at clearing Aβ [74], the abnormal accumulation of Aβ protein is considered one of the main causes of increased risk for AD [75].Interestingly, Dickson et al. reported that APOE ε4 is associated with an increased risk of transitional and diffuse LBD in cases with moderate or high AD pathology [76].In our cohort, approximately 90% of LBD cases with moderate or higher AD levels had at least one APOE ε4 allele, corroborating their result.Therefore, it is suggested that APOE ε4 plays a crucial role in the pathophysiology of not only AD but also LBD.We can speculate that the deposited Aβ facilitates α-synuclein deposition analogously to that proposed for neurofibrillary pathology in AD [71][72][73]76].
The CGP is a recently described type of Aβ plaque that mainly consists of Aβ 40 [24].In this study, approximately half of the individuals with LBD showed CGPs.It is more common in individuals with early-onset AD and is associated with an APOE ε4 homozygous status and CAA [24].Consistent with this study [24], we showed that the APOE ε4 score and the severity of CAA pathology were significantly correlated with the CGP grading.Vidal et al. have reported two cases of neocortical LBD homozygous for the APOE ε4 alleles showing Aβ 40 -predominant plaques consistent with the CGP [77].Thus, it is speculated that the formation of CGP in LBD occurs based on a similar pathophysiology as in AD, especially associated with APOE ε4.Furthermore, CGPs were only observed in AD cases with clinical dementia [24].Whether the presence of CGPs contributes independently to the cognitive decline in LBD merits further studies.
We found that Type 2 CAA lesions contain more Aβ peptides with shorter C-termini (Aβ 38 , Aβ 39 , Aβ 40 ), while Type 1 CAA lesions contain more Aβ peptides with longer C-termini (Aβ 42 , Aβ 43 ).These findings are consistent with the results of some previous neuropathological studies that found Aβ 40 predominated over Aβ 42 in Type 2 CAA lesions, whereas Aβ 42 predominated over Aβ 40 in Type 1 CAA lesion [78,79].It is reasonable to hypothesize that Aβ peptides with longer C-termini are more prone to aggregate and therefore show deposition on more peripherally-located vessels (capillaries).To the best of our knowledge, this is the first report to comprehensively examine the immunohistochemical composition of deposited Aβ peptides in Type 1 and Type 2 CAA lesions.
The discrepancy in the severity of quantitative Aβ deposition burden in the striatum, as indicated by 6F/3D and other antibodies (especially anti-Aβ 42 , Aβ 43 , and Aβ Np3E antibodies), was significant in the AD and LBD groups, but comparatively smaller in the PSP group.Additionally, cluster analysis revealed a relatively homogeneous spectrum of deposited Aβ peptides among cases in the PSP group, whereas notable heterogeneity was observed among cases in the AD and LBD groups.These findings suggest a similarity in the progression of Aβ pathology between LBD and AD, while PSP exhibits distinct characteristics.Importantly, the significant presence of highly neurotoxic Aβ variants, such as Aβ 42 , Aβ 43 , and Aβ Np3E , in the plaques of LBD patients suggests their potential as promising targets for treatment with anti-Aβ antibodies.Importantly, the striatum is affected by astrocytic tau pathology in PSP, and thus it is tempting to speculate that the altered microenvironment associated with this contributes to distinct processing of Aβ.
It should be noted that LBD cases tended ( p = 0.07) to have less cored plaque formation than AD and PSP cases, despite the presence of Aβ deposition similar to that of AD.The reason for this is unknown and is a subject for further study.Ikonomovic et al. have reported that the intensity of [ 18 F]flutemetamol and [ 11 C]Pittsburgh Compound-B positron emission tomography signal is influenced by both diffuse plaques and cored plaques, but total fluorescence output from a cored plaque is approximately three times that from a diffuse plaque of the same volume [80].Thus, it is suggested that amyloid deposition in the striatum may be underestimated in individuals with LBD.
In conclusion, our data imply that the type and strain of concomitant proteinopathy may affect the spectrum of deposited Aβ in non-AD NDDs.Interactions between abnormal proteins are speculated to be one of the causes of this phenomenon.The female sex and APOE ε4 promote Aβ deposition and may accentuate this difference.Aβ deposition is strongly linked to more rapid cognitive deterioration and earlier mortality in individuals with LBD [81,82].Thus, we suggest that individuals with LBD, particularly females with one or more APOE ε4 alleles, could be considered candidates for treatment with amyloidocentric drugs, should they show efficacy in slowing cognitive decline in patients with AD.By reducing Aβ deposition, the progression of α-synuclein pathology may also be inhibited by suppressing the synergistic interaction between the two pathological proteins.Additionally, combining tau-targeting drugs may further augment their therapeutic effectiveness.Furthermore, we found that the spectrum of Aβ peptides deposited in the striatum differs from that in the neocortex.This might also be influenced by the type of the non-Aβ protein cytopathology.Therefore, a comprehensive analysis of each anatomical site is considered necessary to enhance our understanding of the effects of Aβ, especially for improving diagnostic accuracy in imaging analysis.Establishing therapeutic strategies that integrate these factors may lead to improved treatment outcomes using drugs.

F
I G U R E 1 Flow chart of patient and sample selection.Aβ, amyloid-beta; AD, Alzheimer's disease; FTLD, frontotemporal lobar degeneration; LBD, Lewy body disease; MSA, multiple system atrophy; PSP, progressive supranuclear palsy; TDP, TAR DNA-binding protein 43.neuropil threads in each low power field.Representative images are shown in Figure S1.

F
I G U R E 2 Representative microphotographs of the coarse-grained plaques (CGPs) and the relationship between the CGP pathological severity and the genotype of APOE in individuals with Lewy body disease.(A, B) Immunohistochemistry for 6F/3D.(A) The CGPs are observed as clusters (arrows).(B) The plaque shows multiple cores, Aβ-devoid pores, a vague rim and tubular-like and trabecular structures (arrowheads).(C) No CGP formation is observed in non-APOE ε4 carriers.Statistical analysis was performed using Kruskal-Wallis test with post hoc Bonferroni correction.Scale bar = 200 (A), 20 μm (B).

Figure 5
Figure 5 exhibits representative histological microphotographs and analysis results of the three disease groups.The representative histological microphotographs of Aβ plaques immunolabeled by Aβ pSer8 are presented in FigureS2, a summary of the clinical information and the results from both the semiquantitative and quantitative analyses in Table6and all the analysis findings in TableS2.Based on the criteria detailed above only 15 LBD, 6 AD, and 5 PSP cases were selected for detailed analysis (i.e., those showing G3 or above severity of striatal Aβ deposition; see Figure1for case selection details).Most Aβ plaques displayed diffuse-type morphology (Figure5A-O).There were no significant differences in the severity of Aβ deposition labeled by antibody 6F3D among the three groups in the semiquantitative analysis.Although not statistically significant ( p = 0.07, see Table6), cored plaque formation tended to be greater in the AD and PSP groups and less in the LBD group (Figure5A,F,K).Similar to the TL, striatal Aβ deposits mainly consisted of Aβ 42 (Figure5C,H,M), and Aβ 38 and Aβ 39 deposition was minimal.In contrast, there was less Abbreviations: IR, immunoreactivity; NE, not evaluated; SP, senile plaque pathology grading; VP, vascular pathology grading.a Comparison of the SQ-SP/SQ-VP/Q values between females and males (Mann-Whitney U test).

b
Kruskal-Wallis test with post hoc Bonferroni correction.c Correlation between the APOE ε4 score and the quantitative Aβ deposition burden in the temporal lobe (Spearman rank correlation coefficient test).THE SPECTRUM OF AΒ IN NON-AD DISEASES Group 1 (consisting of 12 cases) exhibited the lowest degree of Aβ deposition among the three groups, particularly with respect to Aβ 42 , Aβ 43 , and Aβ Np3E .Consequently, Group 1 was termed the "low Aβ deposition group."Four out of five PSP cases (all with intermediate ADNC level) were included in this group.Group 2 (consisting of eight cases) displayed significantly higher levels of Aβ deposition compared to Group 1, except for Aβ 40 ( p < 0.01).Thus, Group 2 was designated the "Intermediate Aβ deposition group."This group included one PSP case with a high ADNC level.Group 3 (consisting of six cases) exhibited the most severe Aβ deposition among the three groups, leading to its identification as the "high Aβ deposition group."No PSP cases were included in this group.Interestingly, cases in the LBD and AD groups were distributed relatively evenly across the three groups, indicating a significant level of heterogeneity among cases within these groups.

a
Evaluated using the Kruskal-Wallis test with post hoc Bonferroni correction.b The CERAD score A (sparse) corresponds to score of 1, B (moderate) corresponds to score of 2, and C (frequent) corresponds to score of 3. THE SPECTRUM OF AΒ IN NON-AD DISEASES in the LBD group compared to the PSP group, and the APOE ε4 score is associated with the severity of coarsegrained plaque (CGP), a recently described type of Aβ plaque primarily composed of Aβ 40 [24]; (3) females in the LBD group exhibit a significantly higher burden of Aβ (6F/3D, Aβ 42 , and Aβ 43 ) and p-tau pathology compared to males; (4) in the LBD group, APOE ε4 status is positively correlated with the deposition burden of all investigated Aβ peptides; (5) differences exist in the composition of deposited Aβ peptides between Type 1 and Type 2 CAA lesions; (

F I G U R E 6
Wards hierarchical cluster analysis using the quantitative Aβ deposition burden.(A) Temporal cortex.(B) Striatum.(A) Individuals in the Lewy body disease (LBD) group (1-29) and progressive supranuclear palsy (PSP) group (30-36).(B) Individuals in the LBD group (1-15), Alzheimer's disease (AD) group (16-21), and PSP group (22-26).Dendrogram of cluster analysis (left panel) and the severity of deposition burden in each Aβ peptide (right panels).Regular typeface: LDB cases; bold typeface: AD cases; underlined typeface: PSP cases.Cases highlighted in blue indicate low Alzheimer's disease neuropathologic change (ADNC) levels, cases highlighted in green indicate intermediate ADNC levels, and cases highlighted in red indicate high ADNC levels.
Summary of the cases for the temporal lobe evaluation.

Table 1
presents a summary of the cases in the four groups.The mean age at death in the MSA group was T A B L E 3 Comparison of general pathological findings of suitable cases for detailed histopathological evaluation.The type of CAA and presence or absence of the CGP in the temporal lobe were evaluated using specimens immunostained for Aβ (6F/3D).
T A B L E 5 Summary of clinical information and analysis results for each APOE ε4 score group within the LBD cohort.a a Group 0 comprises individuals who are not carriers of APOE ε4 allele (APOE score 0), Group 1 includes cases of heterozygosity for APOE ε4 allele (APOE score 1), and Group 2 includes cases of homozygosity for APOE ε4 allele (APOE score 2).
Summary of the clinical, pathological, semiquantitative (SQ), and quantitative (Q) results of striatal Aβ analysis.
T A B L E 6