BACE1 regulates expression of Clusterin in astrocytes for enhancing clearance of β-amyloid peptides

Deletion of Bace1 in astrocytes alters transcriptomic profiles of reactive population

To understand the role of BACE1 in astrocytes, we enriched astrocytic cell population from 3 brains of wild-type (WT) and Bace1 KO mice at 2-months of age using astrocyte-cell specific antigen 2 (ACSA2) immunomagnetic beads. Purified astrocytes were subjected to 10 × Genomic single cell RNA sequencing and initial read count analysis by Cell Ranger (10 × Genomics). Overall gene number, read counts, and mitochondrial RNA appeared to be similar between all samples (Supplemental Figure S1A). Approximately 17,000 cells were identified for each genotype. Using Seurat R package (Version 4, Satija lab), we analyzed the sequencing results and clustered these cells into UMAP-defined clusters (Fig. 1A). Different cell clusters were arranged based on the unique gene signature as previously described [23, 24]: 1) major populations of non-reactive astrocytes [indicated by high Atp1b2 expression and low Vimentin (Vim) expression], 2) reactive astrocytes (high Atp1b2 and high Vim), 3) microglia [Cluster of differentiation 68 (CD68)], 4) oligodendrocytes [Myelin and lymphocyte protein (Mal)], 5) oligodendrocyte precursor cells (OPC) [Platelet-derived growth factor receptor A 2 (Pdgfr2) or Chondroitin sulfate proteoglycan 4 (CSPG4)], and 6) endothelial cells [Platelet endothelial cell adhesion molecule (PECAM1)] (Fig. 1B). From UMAP-defined clusters, it appeared that Cluster 0, 2, and 6 were non-reactive astrocytes while Clusters 3, 4, 8, and 10 were reactive astrocytes (Fig. 1B and Supplemental Figure S1B). As expected, astrocytes represented the majority of cells, and approximately 20% of remaining cells were identified as oligodendrocytes (ochre; Clusters 1 and 5), and OPCs (teal; Cluster 7 and 12). Although in low abundance, microglia (in cluster 9 in blue) and endothelial cells (cluster 7 in fuchsia) were present (Fig. S1B).

Fig. 1figure 1

BACE1 deficiency enhances reactive astrocyte population. A Total Uniform manifold approximation and projection (UMAP) clustering of ACSA2 + positive cells from derived from 2-month old Bace1-null and WT mice via single-cell RNA-seq, N = 3 samples, ~ 17,000 cells per genotype. Clusters are labeled by each cell type. B Violin plot representation of log2 fold change gene expression of known cell type gene markers and correlated with labeled clusters. C Proportion of indicated cell types. Comparison of average proportion of each cell types from each samples genotype (* p-value < 0.05, ** p-value < 0.01). Two-way ANOVA with Sidak multiple comparison post-test. D UMAP of only astrocyte clusters after removing other cell types. R Astrocytes refer reactive astrocytes while Non-R Astrocytes refer to non-reactive astrocytes. E Western blot of primary astrocytes cultures lysates derived from Bace1-null and WT mice to confirm increase in reactive astrocytes from Bace1-null mice. GFAP antibody was used for detecting GFAP levels, while actin was for loading controls. F GFAP levels were quantified based on Western blot band intensity normalized to actin (N = 3, ** p-value < 0.01, Student t-test)

After using known brain cell type markers to filter out non-astrocyte cells [23, 25], we re-clustered and compared gene profiles derived from Bace1-null and WT astrocytes. Quantification showed that reactive astrocytes comprised approximately 20% of total cells in Bace1−/− brains compared to only ~ 10% reactive astrocytes in Bace1+/+ controls (Fig. 1C). Correspondingly, the proportion of non-reactive astrocytes in WT controls was greater, while other cell populations were not significantly altered under this immune-panning condition (Fig. 1C). Furthermore, BACE1 deficiency visibly increased numbers of reactive astrocytes (labeled as R Astrocytes in teal, Fig. 1D).

Consistent with this unbiased result, we found that GFAP protein levels in lysates from BACE1 deficient primary astrocyte cultures were significantly higher compared to WT controls (Fig. 1E and F), further indicating that BACE1 deficiency promotes astrocytes in the reactive states.

Comparing the transcriptomes of pan reactive astrocytes from Bace1 KO and WT mice using Seurat V4, we identified 37,658 unique gene signatures, with about 610 significantly differentially expressed genes (DEGs) (adjusted p-value < 0.05, log2FC > 0.2; Supplemental Table 1). A volcano plot revealed many significant DEGs (Fig. 2A). Among these genes, we found several genes known to have roles in synaptic maintenance, clearance of β-amyloid peptides (Aβ), glutamate homeostasis, metabolism, hippocampal synaptogenesis, insulin and insulin growth factor signaling and AP-1 transcription factor family (Table 1 and Supplemental Table 1). Expectedly, Bace1 from Bace1 KO reactive astrocytes was also significantly reduced.

Fig. 2figure 2

Germline BACE1 deficiency results in unique reactive astrocyte transcriptomes. A Top differentially expressed genes comparing Bace1-null to WT reactive astrocyte transcriptomes expressed as a volcano plot of log2 fold change value gene expression and –log10(p-value), Wilcoxon ranked sum test was used to calculate p-values. Dotted lines indicate DEG cut-offs for |log2(fold change)|> 0.2 and -log10 (p-value) of 1.3, corresponding to p-value < 0.05. Green triangle highlights elevated expression of Clu and Cxcl14. Blue dots highlight genes that are members of the AP-1 transcription machinery (B) Violin plot of genes of interest, Clu, and Cxcl14, expression comparing Bace1-null and WT reactive astrocyte clusters (* p-value < 0.05, *** p-value < 0.001; Wilcoxon ranked sum test was used to calculate p-value). C Distribution of cells highly expression Clu and Cxcl14. Scale indicates gene expression levels. D Western blotting of genes of interest – Clu and Cxcl14 – in primary astrocyte culture lysates with or without 2 μM of aggregated Aβ42 treatment. E Quantification of Western blot band intensity normalized to actin with or without Aβ42 treatment (N = 3, * p-value < 0.05, ** p-value < 0. *** p-value < 0.001; One-way ANOVA with Sidak post-test)

Genes thought to regulate clearance of Aβ, marked in green triangles, received our particular attention. We showed that Clu and C-X-C Motif Chemokine Ligand 14 (Cxcl14) expression levels were significantly elevated in Bace1-null reactive astrocyte gene clusters (Fig. 2B and C). These changes were then further validated on the protein level. Western blot analysis showed that both CLU and CXCL14 protein levels were increased in Bace1-null primary astrocytes, both basally and when stimulated with Aβ, compared to WT astrocytes (Fig. 2D-E).

Deletion of Bace1 in 5xFAD mice enhances expression of Clu and Cxcl14

As previously mentioned, conditional knockout of BACE1 in the adult reverses previously formed amyloid plaques in adult 5xFAD;Bace1fl/fl; UBC-creER compared to non-BACE1 deleted 5xFAD;Bace1fl/fl adult mice [15]. To understand the role that adult BACE1 deficiency might play on astrocyte transcriptomes in adult mice with the 5xFAD background, we compared scRNA-Seq of astrocytes from 14-month old 5xFAD;Bace1fl/fl; UBC-creER and 5xFAD;Bace1fl/fl mice. Overall gene number, read counts, and mitochondrial RNA appeared to be similar between all samples (See Supplemental Figure S2). About 7,000 astrocytes were recovered from these mice after magnetic activated cell sorting (MACS) by ACSA2 immunomagnetic beads and cell type filtration. When comparing reactive astrocyte population transcriptomes from 5xFAD;Bace1fl/fl; UBC-creER and 5xFAD;Bace1fl/fl mice, 1,873 DEGs (P < 0.05) were noted (Supplemental Table 2). Among these DEGs, insulin degrading enzyme (IDE), klotho (Kl) and lysosomal associated membrane protein 2 (LAMP2) were upregulated in the 5xFAD;Bace1fl/fl; UBC-creER compared to 5xFAD;Bace1fl/fl mice. Furthermore, we once again found significantly elevated Clu and Cxcl14 gene expression in the reactive astrocyte population (Fig. 3A), and an increase in the number of Cluhigh and Cxcl14high astrocytes in the case of 5xFAD;Bace1fl/fl; UBC-creER mice (Fig. 3B).

Fig. 3figure 3

Bace1 deficiency in adult mice increases Clu and Cxcl14 gene expression in reactive astrocytes. A Transcriptomic analysis was performed on single-cell RNA-seq of astrocytes sorted from 14-month-old 5xFAD;Bace1fl/fl and 5xFAD;Bace1fl/fl;UBC-creER mice (N = 3, per genotype). Violin plot of genes showed high expression of Clu and Cxcl14 (log2 fold change expression) in 5xFAD;Bace1fl/fl; UBC-creER compared 5xFAD;Bace1fl/fl reactive astrocyte clusters. B Distribution of cells highly expressing Clu and Cxcl14 with indicated log2 fold change scaling based on UMAP clustering of astrocytes

Our data suggest that both germline and adult BACE1 deficiency increases a group of astrocytes that commonly express more Cxcl14 and Clu. These Cluhigh and Cxcl14high reactive astrocytes are likely capable of clearing more β-amyloid peptides (Aβ) as higher levels of CLU are known to increase Aβ clearance [26, 27].

BACE1 deficiency increases uptake and degradation of Aβ by astrocytes

Since BACE1 deficiency altered astrocytic transcriptomic profiles, we then asked whether astrocytes with BACE1 deficiency would enhance Aβ clearance, which might contribute to the reversal of pre-formed amyloid plaques in 5xFAD mice with deletion of Bace1 in the adult as previously reported [15]. We cultured primary astrocytes from WT and Bace1-null mice, and then treated these cultured astrocytes with aggregated oligomeric human Aβ42 according to the published procedure [28]. We found that BACE1 deficient astrocytes had visibly increased uptake of HiLyte Fluor-555-labeled Aβ42 (Anaspec, Fremont, CA) after 12 hrs incubation compared to WT controls (Fig. 4A). High magnification images showed that the majority of the HiLyte Fluor-555-labeled Aβ42 signal was found within astrocyte cell bodies (Fig. 4A, inset). Quantification showed that enhanced uptake of aggregated Aβ42 began at around 6 hrs post incubation and plateaued at around 12 hrs, while it took about 36 hrs for WT astrocytes to uptake about the same amount of HiLyte Fluor-555-labeled Aβ42 (Fig. 4B).

Fig. 4figure 4

BACE1 deficiency enhances astrocytic clearance of Aβ in vitro. A Confocal imaging of astrocyte primary cultures from BACE1-null and WT perinatal mice pups and treated with 2 μM of Aβ42 tagged with fluorescent Hilyte-555 for indicated incubation times. Stained with phalloidin (green) to mark F-actin and ToPro3 (blue) to mark nuclei. Scale bars represents 10 μm. Inset shows magnified image of Aβ42 within phalloidin marked astrocyte boundaries. B Quantification of Aβ42 integrated fluorescence within phalloidin marked astrocyte boundaries and normalized to number of nuclei and astrocyte area (N = 6, * p-value < 0.05, ** p-value < 0.01; One way ANOVA, with Tukey post testing comparing between time points. C Western blot of astrocyte primary culture lysates from indicated genotypes and treated with 2 μM of aggregated Aβ42 for the indicated incubation times. Images indicate major bands for BACE1, Aβ42 (human, oligomeric), and actin. D Quantification of Western blot Aβ42 band intensity normalized to actin (N = 3, * p-value < 0.05, One-way ANOVA, with Tukey post testing comparing between samples)

We also examined Aβ levels by Western blot. Although the levels of oligomeric Aβ42 in Bace1-null astrocytes were higher than that in WT controls during initial incubation (12 to 36 h), significantly less Aβ42 was found at the time of 72 hrs (Fig. 4C). This was further confirmed by multiple replication experiments (Fig. 4D), indicating that BACE1 deletion likely also enhanced degradation of oligomeric Aβ42 after initial uptake. In line with previous reports [20, 21, 29], astrocytes treated with Aβ42 appeared to enhance expression of BACE1 (Fig. 4C), suggesting that elevated BACE1 in AD brains might have vicious inhibitory effect on astrocytic Aβ clearance.

Clusterin contributes to enhanced Aβ uptake in Bace1-null astrocytes

Because CLU is known to regulate Aβ clearance and degradation and elevation of the CLU protein level was evident, we chose to examine whether elevated CLU levels would contribute to enhanced Aβ uptake and degradation in Bace1-null astrocytes. To this end, we identified that one well-characterized siRNA (Santa Cruz Biotechnology) was able to reduce CLU protein levels in primary astrocytes in a dose dependent manner, with astrocytes treated with 80 pmol of Clu siRNA down-regulated about 48.6% expression of CLU (Supplemental Figure 3A and B).

We then pretreated Bace1-null and WT astrocytes with 80 pmol of Clu or control siRNA before treating astrocytes with aggregated Aβ42 and examined intracellular Aβ levels by either Western blot (Fig. 5A and B), or fluorescently tagged Aβ by confocal imaging (Fig. 5C). We showed that knocking down Clu in Bace1-null astrocytes reduced the uptake of Aβ42, when compared to Bace1-null astrocytes treated with control siRNA (Fig. 5).

Fig. 5figure 5

Clusterin underlies astrocyte endocytosis of Aβ in vitro. A Western blot of Bace1-null and WT-primary astrocytes pretreated with either 80 pmol of Clu siRNA or 80 pmol of control scrambled siRNA and incubated with 2 μM of Aβ42 for indicated times. Images indicate major bands for Aβ42 (human, oligomeric) and GAPDH. B Quantification of Western blot Aβ42 band intensity normalized to actin (N = 3, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001; One-way ANOVA with Tukey post testing comparing between samples. C Confocal imaging of astrocyte primary cultures from BACE1-null and WT and pretreated with either Clu or control scrambled siRNA then treated with 2 μM of Aβ42 tagged with fluorescent Hilyte-555 for indicated incubation times. Stained with phalloidin (green) to mark F-actin and ToPro3 (blue) to mark nuclei. Scale bars represents 10 μm

Further quantification showed a clear shift in the uptake pattern (Fig. 5B): slowed uptake of Aβ42 by astrocytes when CLU levels were reduced, and treatment with Clu siRNA reduced total levels of Aβ42 in both Bace1-null and WT astrocytes from 0.5 to 36 hrs compared to control siRNA treated astrocyte (Fig. 5B). Confocal images also showed delaying uptake of Aβ42 when comparing Bace1-null astrocytes with and without specific Clu siRNA treatment (Fig. 5C).

Altogether, these results add evidence that CLU indeed plays a direct role in astrocytic clearance of Aβ. Furthermore, it suggests that increased CLU levels underlie the increased uptake of Aβ in Bace1-null astrocytes.

BACE1 deficiency increases P38, ERK1/2 and cJun activity

In order to investigate signaling pathways responsible for the increased expression of Clu and other aforementioned DEGs in BACE1 deficient astrocytes, we performed a database search using BioMart to determine common transcription factors for these DEGs. Members of the activated protein 1 (AP-1) transcription family were the most commonly found elements to be related to DEGs of BACE1 deficient astrocytes, including Cxcl14 and Clu. Furthermore, some members of the AP-1 family themselves were DEGs upregulated in BACE1 deficient reactive astrocytes (Fig. 2B, Table 1, and Supplemental Table 1). AP-1 activation is known to be modulated by upstream molecules such as P38, JNK1/2, and ERK1/2 [30, 31]. We found that the levels of JNK were not significantly changed, but levels of phosphorylated P38, Jun, and ERK1/2 were significantly elevated in Bace1-null astrocyte cultures (Fig. 6A); total Jun and P38 levels were not obviously changed. Quantification of replicated results confirmed this in Fig. 6B.

Fig. 6figure 6

BACE1 deficiency enhances P38, ERK1/2, and cJun phosphorylation. A Western blot of BACE1-null and WT primary astrocytes lysates. Images indicate major bands for pJun, total Jun, pP38, total P38, pERK1/2, pJNK and Calnexin. B Quantification of pJun/total cJun, pP38/total p38, and pERK1/2 band intensity normalized to Calnexin (N = 3, * p-value < 0.05, One-way ANOVA with Tukey post testing comparing between samples)

Our results imply that BACE1 deficiency in astrocytes results in an increase of pP38 and pERK1/2 activation, which then phosphorylates and activates downstream Jun and AP-1 mediated transcription of its downstream molecules such as Clu, which is related to enhanced astrocytic clearance of Aβ.

BACE1 deficiency increases astrocytic insulin receptor signaling

We further explored how BACE1 deficiency would activate the aforementioned signaling molecules in astrocytes. It was previously reported that insulin receptor (IR) is a BACE1 cleavage substrate in the liver [32], and insulin signaling regulates both P38 and ERK1/2 MAPK activity. Furthermore, some DEGs of the insulin and insulin growth factor families were altered in Bace1-null astrocytes (Table 1 and Supplemental Table 1). IR is composed of a heterodimer dimer, α and β subunits, and the membrane anchored β subunit is identified as a BACE1 substrate. When Bace1 is deleted, the IRβ subunit is no longer cleaved and more preserved IRβ subunit might be available for enhancing P38 and ERK1/2 activity. We therefore investigated whether BACE1 deficiency might also enhance astrocytic insulin receptor availability.

Western blot analysis revealed that BACE1 deficiency significantly reduced levels of BACE1-cleaved IRβ C-terminal fragment (CTF) in Bace1-null astrocytes compared to their WT controls (Fig. 7A), consistent with prior results that BACE1 deficiency abrogated cleavage of IRβ in astrocytes. An increase of total mature IRα and IRβ levels was also noted, while as well as a much larger increase in the phosphorylated IRβ (pIRβ) subunit (Fig. 7A), which is required for downstream activation for pP38 and ERK1/2 MAPK. Quantification of replicated results confirmed changes of these proteins (Fig. 7B). Altogether, this suggests that astrocytic BACE1 regulated IR receptor availability by cleavage in a manner similar to liver BACE1 and IR.

Fig. 7figure 7

BACE1 deficiency preserves astrocytic IR bioavailability. A Western blot of Bace1-null and WT primary astrocytes culture lysates. Images indicate major bands for IRβ, IRβ CTF, pIRβ, IRα, and actin. B Quantification of IRβ, IRβ CTF, pIRβ, and IRα band intensity normalized to actin (N = 4, * p-value < 0.05, ** p-value < 0.01, One-way ANOVA with Tukey post testing comparing between samples. C Western blot of BACE1-null primary astrocytes culture lysates treated with 1 μM of BMS-754807 or control. Images indicate major bands for CLU, CXCL14, and actin. D Quantification of IRβ, IRβ CTF, pIRβ, and IRα band intensity normalized to actin (N = 4, * p-value < 0.05, One-way ANOVA with Tukey post testing comparing between samples)

We further tested whether insulin signaling might be responsible for the elevated CLU and CXCL14 levels in Bace1-null astrocytes. Since insulin is already present in the G-5 supplemented astrocyte media, we inhibited insulin signaling by using BMS-754807 (SelleckChem), a potent IR tyrosine kinase phosphorylation inhibitor (Fig. 7C and D). BMS-754807 treatment resulted in a potent reduction of both CLU and CXCL14 levels in Bace1-null astrocytes (Fig. 7C). Quantification of replicated results confirmed changes of these proteins (Fig. 7D). This suggests that elevated CLU and CXCL14 levels in Bace1-null astrocytes is dependent on IR signaling.

Altogether, this suggests that astrocytic BACE1 regulated IR availability by cleavage in a matter similar to liver BACE1 and IR and that IR signaling may play a role in reactive astrocyte amyloid clearance.

Targeted deletion of Bace1 in astrocytes increases astrocytic levels of IR, CLU and CXCL14

To investigate whether in vivo astrocytic BACE1 deficiency results in increased IR, CLU and CXCL14, we bred Bace1fl/fl mice with Gfap-cre mice [Tg-Gfap-cre 73.12Mvs, the Jackson lab] and examined Bace1fl/fl;Gfap-cre mice, which conditionally deleted  Bace1 mostly in astrocytes due to expression of Cre recombinase by the mouse Gfap promoter [33]. To avoid potential effects from other Gfap-expressing lineage cells, we isolated astrocytes from 2-month old Bace1fl/fl;Gfap-cre mice using ACSA2+ immunobeads and the purified ACSA2 + astrocytes were used for Western blot (Fig. 8).

Fig. 8figure 8

Targeted astrocytic deletion of Bace1 enhances IR bioavailability and downstream amyloid clearance proteins. A Western blot of ACSA2 + astrocyte enriched lysates from Bace1fl/fl;Gfap-cre and Bace1fl/fl mice. Images indicate major bands for IRβ, IRβ CTF, CLU, CXCL14, and actin. B Quantification of indicated protein intensity normalized to actin (N = 3, * p-value < 0.05, One-way ANOVA with Tukey post testing comparing between samples)

We found that astrocytes from Bace1fl/fl;Gfap-cre mice had an increased amount of mature IRβ and reduced IRβ CTF compared to Bace1fl/fl astrocytes in a manner similar to above in vitro conditions (comparing Fig. 7 and 8). We also found a significant increase in CLU and CXCL14 protein levels (Fig. 8). Altogether, this suggests that BACE1 deficiency increases astrocytic IR signaling and its downstream molecule CLU and CXCL14 in a mostly cell autonomous manner both in vivo and in vitro.

Bace1 deletion in astrocytes decreases amyloid plaque levels

To further understand the effect of astrocytic Bace1 deletion on amyloid pathology, we bred Bace1fl/fl;Gfap-cre with 5xFAD;Bace1fl/fl mice to generate 5xFAD;Bace1fl/fl;Gfap-cre and 5xFAD;Bace1fl/fl mice for comparison. We used P75 female mice from each genotype, as female 5xFAD mice show less variations in amyloid pathology [34]. Thioflavin-S staining of fixed brain sections revealed an overall less amyloid depositions in 5xFAD;Bace1fl/fl;Gfap-cre mouse brains compared to 5xFAD;Bace1fl/fl mouse controls (Supplemental Figure 4). In an enlarged view, it was clear that less Aβ plaques were formed in the 5xFAD;Bace1fl/fl;Gfap-cre cortex compared to controls (Fig. 9A), and was confirmed by quantification (Fig. 9B). Although, levels of Aβ plaques appeared to be reduced in the subiculum and hippocampus of 5xFAD;Bace1fl/fl;Gfap-cre mice, further quantification showed that this decrease in hippocampus was, overall, not significant (Fig. 9B).

Fig. 9figure 9

Targeted astrocytic deletion of Bace1 reduces amyloid plaques by increasing Aβ clearance. A Thioflavin-S staining of amyloid plaques from saggital brain sections of 5xFAD;Bace1fl/fl;Gfap-cre and 5xFAD;Bace1fl/fl. Scale bar indicates 100 µm (B) Quantification of Thioflavin-S positive amyloid plaques calculated by counting serial sagittal sections, which were selected at 10-section intervals (N = 7 for 5xFAD;Bace1fl/fl;Gfap-cre and 5xFAD;Bace1fl/fl). C Western blot of cortical lysates from 5xFAD;Bace1fl/fl;Gfap-cre and 5xFAD;Bace1fl/fl mice. Images indicate APP full length, APP C-terminal C99 (detected by 6E10) and C99/C83 bands (detected by APP A8717 antibody). Actin was included as loading control

We also examined the effect of astrocytic BACE1 on total amyloid processing and generation by Western blot (Fig. 9C). We found that total levels of full length APP, C99, and C83 cleavage substrates appeared to be unchanged across cortical samples. This most likely indicates that brain Aβ levels are mainly resulted from neuronal sources while the contribution of astrocytic Aβ levels are minimal. It is noted that neurons have higher levels of BACE1 and APP than glial cells. Most likely, astrocytic BACE1 deletion induced IR signaling and increased CLU levels, which contribute to the enhanced glial Aβ clearance.

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