Graphical abstract graphic file with name fx1.jpg [57]Open in a new tab Highlights * • Aβ induces olfactory memory impairment by piriform cortex neurons in 5xFAD * • Aβ accumulation mainly affects glial cells and synapse abnormal in 5xFAD * • Astrocyte-microglia interaction declines appear in 4-month-old 5xFAD and MCI patient __________________________________________________________________ Classification Description: Health sciences; Disease; Biological sciences Introduction Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases that severely affects the quality of life of the elderly, placing a significant burden on individuals, families, societies, and the global economy. The pathological symptoms, such as Aβ plaques, appeared before the clinical symptoms in AD.[58]^1 But the relationship between pathological changes and clinical features is still unknown. Thus, it is important to explore the molecular alterations in Aβ induced memory impairment.[59]^1^,[60]^2 APP and PS1 mutations are the most potential cause in most cases of familial AD (FAD) based on genetic analysis, and it is essential to elucidating Aβ-induced cognitive dysfunction in AD. The Aβ burden differs in various brain regions and can spread to unaffected brain regions, leading to neurodegeneration.[61]^3 And cognitive function can also be divided into multiple aspects: attention, memory, perception, and so on. Therefore, confirming a brain region that is related to one of the earliest cognitive impairments and suffers Aβ burden at the same time shows great importance in AD. Olfactory dysfunction has been reported as a common clinical symptom in early AD before other cognitive impairments.[62]^4^,[63]^5^,[64]^6^,[65]^7^,[66]^8 In patients with mild cognitive impairment (MCI) or AD, olfaction scores highly correlate with brain Aβ levels. Specific brain regions, such as the olfactory bulb and the piriform cortex (PCx), execute olfactory functions. The PCx is related to the olfactory and memory.[67]^9^,[68]^10^,[69]^11^,[70]^12 In human, different odorants have been reported to evoke different ensemble activity patterns in the PCx, which could discriminate between odor categories.[71]^13^,[72]^14^,[73]^15 In addition, several studies have also shown that the PCx may be impaired in the AD olfactory system.[74]^16^,[75]^17^,[76]^18 In clinic, patients at the early onset of AD usually show perceived deficits in olfaction prior to significant memory loss, which may be associated with pathological lesions in the PCx.[77]^16^,[78]^17^,[79]^18 Therefore, exploring the molecular changes in the PCx may provide clues to investigate the mechanism of AD pathogenesis involved with olfactory function. In this study, we first confirmed that Aβ induced olfactory memory dysfunction, which starts at 4 months old, is earlier than sound and working memory dysfunction in the 5xFAD mouse model. Abnormal PCx neural activity appears in the olfactory-associated memory recall process, accompanied by Aβ accumulation. Then, a consensus protein co-expression network from 4 months old to later, which related to astrocytes and microglia, was identified by 5xFAD mice PCx proteomics analysis. The astrocyte-microglia interaction in the network showed significant declines only in MCI n multiple human datasets. It implies that glial cells interaction may play a pivotal role in the early progress of AD. Result Olfactory memory impairment accompanies by abnormal piriform cortex neuronal activity in 4-month-old 5xFAD mice To determine if Aβ can induce olfactory dysfunction in the 5xFAD model, which was reported to be observed in the early stages of patients with AD, we designed three behavioral paradigms to detect different memory impairments in 4 months old. In the odor-associated fear memory test ([80]Figure 1A), mice were delivered foot shocks (3 times) paired with an odor cue in context A during the encoding stage (day 1), and there was no significant difference in the freezing ratio between WT and 5xFAD ([81]Figure 1B). However, during the recall stage (day 2), mice were exposed to one time of the same odor cue at day 1 in the context B, and showed a significantly lower freezing growth score in the 5xFAD group compared with WT (delta score = −0.94, p = 0.029, [82]Figure 1C). It indicated that Aβ can induce olfactory memory retrieval impairment in 4-month-old mice. In contrast, both the encoding and recall stages of the sound-associated fear memory test showed no significant difference between 5xFAD and WT mice ([83]Figures 1D–1F), indicating no auditory memory impairment in 4-month-old 5xFAD mice. These results showed that Aβ can induce olfactory memory loss but no auditory memory impairments in a 4-month-old 5xFAD mouse model. Figure 1. [84]Figure 1 [85]Open in a new tab 4-month-old 5xFAD mice exhibit olfactory memory retrieval impairment (A) Behavioral design for olfactory memory. (B and C) Behavioral performances on day 1 and day 2 (WT, n = 10; 5xFAD, n = 10). (D) Behavioral design for auditory memory. (E and F) Behavioral performances on day 1 and day 2 (WT, n = 8; 5xFAD, n = 8). (G) Up, schematic of virus injection; down, Behavioral design of olfactory memory; insert, the representative confocal images of Gcamp6s expression in the PCx. Scale bar: 200μm. (H and I) main, the mean and SEM of the corresponding Ca^2+ signal of 4-month-old mice PCx neurons during day 1 and day 2; insert, quantification of the average amplitude during 2-4s (colored area) after odor stimulation on day1 and day2 between WT and 5xFAD mice (WT, n = 6; 5xFAD, n = 6, day 1 had 3 trial for each mouse, thus have 18 dots each group). (J and K) Quantification of Aβ plaque numbers between 4-month-old WT and 5xFAD mice in PCx (WT, n = 3; 5xFAD, n = 3, 3 slices each mouse, thus 9 dots each group). Scale bar: 50μm. The numbers counting was done in the 150 μm × 150 μm area. (L) Behavioral design for working memory. (M) Behavioral performance (WT, n = 6; 5xFAD, n = 6).All statistical analyzes are used by t-test, ns, not significant; ∗p < 0.05, ∗∗∗p < 0.001. Data are presented as mean ± SEM. Since PCx is a crucial region related to olfactory and memory, PCx neuronal activity was further recorded using fiber photometry in the odor fear memory test, and the fluorescence imaging showed that the virus carrying Gcamp6s was expressed in PCx neurons ([86]Figure 1G). During the encoding stage, there was no change in PCx neuronal activity ([87]Figure 1H, data among 2-4s was used in statistical analysis). However, during the recall stage, there was a significant decline in PCx neuronal activity (0.21-fold, p < 0.001, [88]Figure 2I, data among 2-4s was used in statistical analysis). The decline in PCx neuronal activity was also accompanied by Aβ deposit accumulation, as shown by the immunohistochemistry (p < 0.001, [89]Figures 1J–1K). These results indicated that abnormal PCx neuronal activity in the recall stage is involved in Aβ-induced olfactory memory impairment. Figure 2. [90]Figure 2 [91]Open in a new tab The alteration of PCx proteome between WT vs. 5xFAD in different stages (A) Workflow summarizing the process of mass-spectrum based proteomics. (B) PCA of proteome data showed main difference in age (3-month-old: WT = 4, 5xFAD = 5; 4-month-old: WT = 4, 5xFAD = 4; 6-month-old: WT = 4, 5xFAD = 5; 9-month-old: WT = 3, 5xFAD = 7; 11-month-old: WT = 7, 5xFAD = 4). (C) Diagram of DEPs in different month-old mice, degree of red color indicated their p values, nodes size indicates their log2FC, bar in nodes indicate their overlapping status among different month-old. (D) GO pathway enrichment of DEPs of different month-old mice. The key pathways were colored in green, blue, red fonts which show specific enrichment in different month-old and purple box showed synapse related functions. (E) GSEA enrichment of DEPs in different month-old mice. Results from left to right were sorted in the order of 3-month to 11-month. (F) Classic synaptic proteins expression showed synapse impairment in 4-month-old 5xFAD. Left, Western blot images; right, quantification of western blots, t-test, ns, not significant; ∗p < 0.05, ∗∗∗p < 0.001. Data are presented as mean ± SEM. To further investigate the different memory declines in 5xFAD mice, we assessed one more behavior test, the T-maze test, in 4-month-old mice, olfactory and auditory memory test in 6-month-old mice, olfactory memory and novel odor recognition test in 3-month-old mice. The T-maze test in 4-month-old mice showed no difference in working memory between 5xFAD and WT mice ([92]Figures 1L and 1M). Olfactory and auditory memory tests in 6-month-old mice showed that olfactory and auditory fear memory were both impaired in 6-month-old 5xFAD mice ([93]Figures S1A–S1D). In 3-month-old mice, olfactory fear memory showed no decline ([94]Figures S2A and S2B) but olfactory recognition function is decline by novel odor recognition test ([95]Figures S2C and S2D, delta index = −0.24, p = 0.029). These results imply that Aβ damage olfactory related function earlier than auditory and working memory. Therefore, investigating the molecular alterations in PCx at different ages, especially in 4-month-old mice, may help elucidate the molecular mechanism of Aβ induced memory progress. Proteomics revealed various function changes of piriform cortex in different ages of 5xFAD mice To discover the Aβ accumulated effect in PCx, age- and sex-matched WT and 5xFAD mice were chosen to conduct PCx proteomics experiments across 5 ages (3-month-old: WT = 4, 5xFAD = 5; 4-month-old: WT = 4, 5xFAD = 4; 6-month-old: WT = 4, 5xFAD = 5; 9-month-old: WT = 3, 5xFAD = 7; 11-month-old: WT = 7, 5xFAD = 4). The PCx was isolated based on a mouse brain atlas, and a total of 47 samples from 5 different ages were subjected to LC-MS/MS for proteomic analysis, as shown in [96]Figure 2A. Outlier samples were removed, and proteins with high missing rates were excluded, resulting in the quantification and analysis of 3913 out of 5359 proteins and 45 out of 47 samples ([97]Tables S1 and [98]S2). Principal component analysis (PCA) indicated that proteomic patterns mainly differed among ages in the PC1 dimension (younger group prefers to cluster in the left, older in the right), differed between genotypes in the PC2 dimension (WT prefers to cluster in the upper area, 5xFAD in the lower area) ([99]Figure 2B). Then, the differentially expressed proteins (DEPs) between WT and 5xFAD in these 5 ages were calculated based on FDR <0.05 and absolute log2FC > 1.2. The numbers of DEPs increased with age, except for the 3-month-old stage (3-month-old = 120; 4-month-old = 85; 6-month-old = 91; 9-month-old = 171; 11-month-old = 217, [100]Table S3). Interestingly, the DEPs in these ages had few overlapping proteins (Chi-Squared Test, P^3vs.4 = 0.74, P^4vs.6 = 0.90, P^6vs.9 = 0.15, P^9vs.11 = 0.013). And the overlapping DEPs number between 3 and 4, 4–6, 6–9, 9–11 increased with age (2, 3, 8, 19 respectively, [101]Figure 2C). Only one protein (APP) was shared among all ages ([102]Figure 2C). The protein expression changes between WT and 5xFAD in the proteomics levels also showed a lower conservation property in each age, except for the relatively high conservation property seen in 6 and 9 months old ([103]Figure S3). These results suggest that the PCx proteomics alteration compared to WT vs. 5xFAD have distinct features in each age, but showed relatively high similarity among 6/9/11-month-old. GO enrichment analysis of DEPs further confirmed the observed changes in protein functions at different stages ([104]Figure 2D). The 3-month-old DEPs were found to be enriched in ensheathment-related functions ([105]Figure 2D, colored in green), while the 4-month-old and 6/9/11-month-old DEPs were enriched in transporter activity- and Aβ clearance-related functions ([106]Figure 2D, colored in blue and red), respectively. This finding suggests that there is a shift in the protein functions involved in the progression of olfactory memory impairment. Moreover, the 4-month-old stage is a critical turning point in the dysfunction process, as the enriched pathways are strongly different from those observed in the 3-month-old stage and start to be involved in synapse-related pathways ([107]Figure 2D, boxed in purple). This finding provides supportive evidence for the behavioral changes observed in olfactory memory dysfunction starting in the 4-month-old 5xFAD mice. In addition, gene set enrichment analysis (GSEA) provided an overall view of functional changes at the proteomic level in different ages ([108]Figure 2E). The significant functional changes observed in the 3-month-old mainly involved morphogenetic, tube-related, and development-related pathways, while those observed in the 4-month-old were related to axonal transport, secretion, and extension, which may all potentially contribute to the pathological changes observed in early AD. The significant function terms of the 6, 9, and 11-month-olds were not as prominent in the GSEA results, but were mainly related to phos/dephosphorylation, Aβ-related, ion transport, and autophagy-related functions. These results suggest that the altered proteins and functions are dynamic and shift along with Aβ accumulation, especially showing a turning point at the 4-month-old stage and starting to be more involved in AD progress. Since the results of different analyses all showed several synapse-related functions and pathways, especially in 4-month-olds ([109]Figure 2D purple box; [110]Figure 2E), Western blot (WB) was performed. Classic synaptic biomarkers were used to investigated the changes in the synapse. The result of WB confirmed Aβ can induced synapse impairment in 4-month-old 5xFAD, with the up regulation of pre-synaptic protein synaptophysin (Syp, p = 0.047), and down regulation of glutamate receptors glutamate receptor 1, 2 (Gria1, p = 0.030, Gria2, p = 3.0e-4) and vesicular glutamate transporter1 (Slc17a7, p = 0.043) ([111]Figures 2F and [112]S7A). Differentially expressed proteins in different stages shared the conserved network connection associated with Alzheimer’s disease and glial cells The significant variation in protein changes between WT and 5xFAD in different stages begs the question of how these changes are reflected in the co-expression network. To address this, PCx proteomics data from all stages (45 samples x 3913 proteins) were used to construct a co-expression network using weighted correlation network analysis (WGCNA) ([113]Figure 3A). The analysis generated 14 co-expression modules (M1-M14). We used module eigenvalue (ME) to calculate the correlation of the module and different traits (group, month-old, sex and genotype (WT or 5xFAD)). The results showed that one module was negatively correlated to group (M6), eight modules were correlated to month-old (M1, M4, M5, M7 were negatively correlated, M3, M6, M8, and M13 were positively correlated), five modules were correlated to genotype (M3, M4 were negatively correlated, M2, M6, and M13 were positively correlated), and four modules were correlated to both month-old and genotype (M3, M4, M6, and M13) ([114]Figures 3B and [115]S4). Figure 3. [116]Figure 3 [117]Open in a new tab Proteome in different stages shared the conserved co-expression network (A) Dendro-tree plot of WGCNA. All protein were used to build up WGCNA module. (B) Heatmap of module-trait relationship. Diagnose, age, sex and disease were all included to see their pearson correlation with different module from WGCNA (Module eigenvalue (ME) were used to calculate pearson coefficient, number in the upper cell block is Pearson coefficient and in lower cell block is p value). (C) Heatmap of DEPs and Human postmortem brain data enrichment in WGCNA module. Enriched number and p value were showed in the matched cell block (Enriched fold in upper cell block and p value in lower cell block). (D) Histogram of DEPs and DAPs enrichment in different type of cell. Enrichfold was taken –log10 and gray line indicate p < 0.05. (E) KEGG pathway enrichment of proteins in M3. (F) KEGG pathway enrichment of DAPs core (overlapping DAPs in M3). (G) Network of all DEPs and DAPs. Size of the nodes indicate their fold change. Green nodes indicate they are down-regulated, red nodes indicate they are up-regulated, both color shade along with age. The size of hub protein nodes are 5 times enlarged and are in the middle of up-regulated proteins. The DEPs at different ages were then subjected to an enrichment analysis with the co-expression modules. Surprisingly, almost all DEPs were enriched in M3, with the exception of DEPs in 3-month-old (DEPs 3M), and the extent of enrichment increased with age (4-month-old: enrichfold = 2.54, p = 6.0e-4; 6-month-old: enrichfold = 2.47, p = 4.1e-4; 9-month-old: enrichfold = 4.84, p = 1.8e-19; 11-month-old: enrichfold = 3.36, p = 3.0e-10; [118]Figure 3C). This finding indicated that DEPs across different ages shared a conserved protein network dysfunction that began with 4-month-old mice, despite high DEP heterogeneity. Furthermore, the module enrichment analysis of AD DEPs in human postmortem prefrontal cortex (ROSMAP dataset)[119]^19 also enriched in M3 (enrichfold = 1.55, p = 1.5e-3, [120]Figure 3C) indicated that M3 is highly correlation with AD. Summarizing the findings with the previous behavioral results suggests that PCx proteome changes accompany with olfactory memory impairment are associated with AD progress. Additionally, KEGG enrichment revealed that proteins in M3 participate in immune-, synaptic-, glia- and even Aβ-related pathways ([121]Figure 3E), all of which are the main pathological impairments of AD.[122]^20^,[123]^21^,[124]^22 In addition, we utilized the DDPNA method to obtain DEPs-associated proteins (DAPs) in order to gain a better understanding of their underlying mechanisms and relations. DAPs are sets of proteins that are significantly correlated with DEPs in M3 and are considered to be involved in a common function. DAPs from various stages were combined and constructed through a co-expression protein network based on the Pearson’s correlation coefficient ([125]Figure 3G), where the up-regulated and down-regulated DAPs are presented in the middle circle. The network showed highly connected and a total of nine hub genes were identified, all of which were up-regulated. Most of these hub genes, such as Apoe, Clu, Itm2b, Ctsd, and C1qc, have been reported to be associated with AD.[126]^23^,[127]^24^,[128]^25^,[129]^26^,[130]^27 Many of these genes are primarily expressed in microglia and astrocytes, such as Hexb, Apoe, and C1qc in microglia and Gfap in astrocytes. This indicates that these DEPs or DAPs may have a cell preference. To answer wheather DEPs and DAPs present cell preferences, we performed