Targeting Lysosomal Dysfunction to Alleviate Plaque Deposition in an Alzheimer Disease Model

Neu1 deficiency leads to increased release of Aβ42.

“We also confirmed that Neu1 deficiency led to increased release of Ab42 (Figure 2H), a process facilitated by excessive lysosomal exocytosis in these mice 38 .”

• cites [38] sourceSupported

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Overexpression of PPCA under the CSF1R promoter has restricted expression in the microglial/monocytic lineage.

“We have reported earlier that transgenic mice overexpressing PPCA under the control of the CSFR1 promoter have restricted expression of the protein in the microglial/monocytic lineage 44 .”

• cites [44] sourceSupported

Lysosomal exocytosis secretes lysosomal contents, including Aβ peptides and active enzymes, into the extracellular space.

“Additionally, we find that Neu1 dysregulation leads to a marked increase in lysosomal exocytosis, a mechanism through which lysosomal contents, including Aβ peptides and active enzymes, are secreted into the extracellular space 35,38,39 .”

• cites [35] sourcePartly supported

  The Introduction describes lysosomal exocytosis as expelling lysosomal contents, and the Results section on Neu1-/- mice reports extracellular release of active lysosomal hydrolases. Our copy does not identify Aβ peptides among the released contents, so that specific example is not supported by this source. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [38] sourceSupported
• cites [39] sourceSupported

5xFAD/CSFR mice show a significantly higher extent of lysosomal exocytosis compared to 5xFAD mice, regardless of gender.

“Interestingly, we still measured a significantly higher extent of lysosomal exocytosis in 5xFAD/CSFR mice compared to 5xFAD mice, regardless of gender.”

β-HEX activity is elevated in serum of AD patients.

“along with elevated b-HEX activity in serum (Figure 1D).”

Neu1 deficiency reduces levels of both pro and mature TACE regardless of gender or background.

“Neu1 deficiency reduced the levels of both pro and mature TACE, regardless of gender or background, which would effectively limit the non-amyloidogenic cleavage pathway in 5xFAD/Neu1 -/-mice (Figure S2H and S2I).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Female 5xFAD/CSFR mice exhibit higher sialylated b-CTF levels than male counterparts.

“In general, female 5xFAD/CSFR mice consistently exhibited higher levels of both total and sialylated APP and b-CTF compared to their male counterparts (Figure 4F, 4G, S4H, and S4I).”

In female 5xFAD/Neu1-/- mice, levels of sialylated NCT increase.

“but it had a unique effect in female 5xFAD/Neu1 -/-mice, where the levels of sialylated NCT increased (Figure 2L).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Under normal Neu1, β-Hex activity is higher in female 5xFAD mice than WT controls.

“Notably, under normal Neu1, b-Hex activity was higher in female 5xFAD mice than WT controls (Figure 2E), suggesting an influence of the 5xFAD background on lysosomal biology in relation to Neu1.”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Female mice within the 5xFAD background consistently exhibit higher levels of β-Hex activity compared to males.

“Additionally, female mice within the 5xFAD background consistently exhibited higher levels of b-Hex activity compared to males, implying a gender-specific difference in lysosomal function (Figure 2E).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Female 5xFAD/CSFR mice exhibit higher total b-CTF levels than male counterparts.

“In general, female 5xFAD/CSFR mice consistently exhibited higher levels of both total and sialylated APP and b-CTF compared to their male counterparts (Figure 4F, 4G, S4H, and S4I).”

In females, the most notable difference in PPCA turnover occurs in 5xFAD/Neu1-/- mice, with a clear delay evident by 8 hours after CHX treatment.

“In females, the most notable difference in turnover rate occurred in 5xFAD/Neu1 -/-mice, where a clear delay in PPCA turnover was evident by 8 hours after CHX treatment (Figure 3A).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Neu1 deficiency slows BACE1 turnover regardless of gender or background.

“Regarding BACE1, Neu1 deficiency slowed its turnover, regardless of gender or background (Figure 3F).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Sialylated APP levels are increased in 5xFAD/CSFR mice compared to 5xFAD controls, irrespective of gender.

“However, sialylated levels of APP and b-CTF were increased in 5xFAD/CSFR mice compared to 5xFAD controls, irrespective of gender (Figure 4F and 4G).”

Neu1 deficiency tends to decrease total NCT levels regardless of gender or background.

“With regard to NCT, Neu1 deficiency tended to decrease total NCT levels, regardless of gender or background (Figure S2K), but it had a unique effect in female 5xFAD/Neu1 -/-mice, where the levels of sialylated NCT increased (Figure 2L).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Neu1 deficiency in human AD brains and 5xFAD/Neu1-/- mice leads to sialic acid retention on APP and its secretases.

“Neu1 deficiency in human AD brains and 5xFAD/Neu1 -/-mice leads to sialic acid retention on APP and its secretases, enhancing amyloidogenic cleavage and Aβ42 production.”

APP and β-CTF are elevated in Neu1-deficient mice, primarily in females within the 5xFAD background regardless of Neu1 status.

“When characterizing APP, we found that both APP and b-CTF were elevated in Neu1-deficient mice.This effect was observed primarily in females within the 5xFAD background, regardless of Neu1 status (Figure S2F and S2G).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Neu1 activity is significantly reduced in 5xFAD/Neu1-/- mice.

“We began by quantifying and characterizing the plaque burden in the brains of 5xFAD/Neu1 -/-mice where Neu1 activity was significantly reduced (Figure S2A).”

Sialylated pro-TACE levels are higher in 5xFAD/CSFR mice compared to 5xFAD controls, irrespective of gender.

“Additionally, levels of sialylated pro-TACE were higher in 5xFAD/CSFR mice compared to 5xFAD controls, irrespective of gender, with male mice consistently exhibiting higher levels than their female counterparts (Figure 4I).”

AD patients, but not controls, show a significant increase in serum β-HEX activity with age.

“In addition, AD patients, but not controls, showed a significant increase in serum b-HEX activity with age (Figure 1E)”

Total levels of mouse PPCA and human PPCA are increased in 5xFAD/CSFR mice compared to controls, specifically 5xFAD and CSRF mice.

“although the total levels of mouse PPCA (moPPCA) and huPPCA were increased in 5xFAD/CSFR mice compared to controls, specifically 5xFAD and CSRF mice (Figure S4E and S4F).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

PPCA enzyme activity is generally higher in all Neu1-deficient animals irrespective of background.

“In addition, PPCA enzyme activity was generally higher in all Neu1-deficient animal, irrespective of their background, suggesting a link between increased sialylation of PPCA and enhanced enzyme activity (Figure S2E).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

AAV-mediated co-expression of NEU1 and PPCA in 5XFAD brains reduces amyloid pathology.

“Additionally, AAV-mediated co-expression of NEU1 and PPCA in 5XFAD brains demonstrates therapeutic potential by reducing amyloid pathology.”

Total pro-TACE levels remain unchanged in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender.

“We found that total pro-TACE levels remained unchanged, while total mature TACE levels were higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender (Figure S4J and S4K).”

LAMP1 staining is increased in hippocampal sections of AD patients.

“In AD patients, we observed increased LAMP1 staining in hippocampal sections (Figure 1C)”

Levels of sialylated moPPCA and huPPCA are reduced compared to controls.

“In contrast, the levels of sialylated moPPCA and huPPCA were reduced compared to controls (Figure 4 and S4G).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

In 5xFAD/CSFR mice, no significant differences in plaque burden, plaque number, or average plaque size are detected compared to 5xFAD counterparts, regardless of gender.

“When 5xFAD mice were crossed with these PPCA expressing transgenic mice (5xFAD/CSFR), no significant differences in plaque burden, plaque number, or average plaque size were detected compared to 5xFAD counterparts, regardless of gender (Figure 4A-4C, and S4A).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Unlike AD patients, 5xFAD/Neu1-/- mice show elevated total PPCA levels compared to 5xFAD controls, regardless of gender.

“However, unlike AD patients, these mice also showed elevated total PPCA levels, compared to 5xFAD controls, with these differences observed regardless of gender (Figure 2D and S2D).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Female mice consistently display higher levels of extracellular b-Hex activity, irrespective of the background.

“Notably, female mice consistently displayed higher levels of extracellular b-Hex activity, irrespective of the background (Figure 4E).”

Female 5xFAD/CSFR mice exhibit higher total APP levels than male counterparts.

“In general, female 5xFAD/CSFR mice consistently exhibited higher levels of both total and sialylated APP and b-CTF compared to their male counterparts (Figure 4F, 4G, S4H, and S4I).”

Neu1 deficiency consistently increases levels of sialylated APP and β-CTF independent of total protein levels, genetic background, or sex.

“However, Neu1 deficiency consistently increased the levels of sialylated APP and b-CTF, independent of total protein levels, genetic background, or sex.”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Total levels of APP do not differ between 5xFAD and 5xFAD/CSFR mice, regardless of gender.

“Total levels of APP did not differ between 5xFAD and 5xFAD/CSFR mice, while b-CTF levels were reduced, regardless of gender (Figure S4H and S4I).”

Sialylated b-CTF levels are increased in 5xFAD/CSFR mice compared to 5xFAD controls, irrespective of gender.

“However, sialylated levels of APP and b-CTF were increased in 5xFAD/CSFR mice compared to 5xFAD controls, irrespective of gender (Figure 4F and 4G).”

5xFAD/Neu1-/- mice exhibit higher levels of sialylated PPCA.

“Similar to AD patients, 5xFAD/Neu1 -/-mice exhibited higher levels of sialylated PPCA.”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

In male mice, Neu1 deficiency accelerates pro-TACE turnover regardless of background.

“In contrast, Neu1deficient male mice showed accelerated pro-TACE turnover regardless of background (Figure 3D).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Total NEU1 protein levels are slightly reduced in AD brain samples compared to controls.

“The total NEU1 protein levels were slightly reduced in the AD brain samples compared to the controls (Figure 1A and S1A).”

The 5xFAD background itself consistently elevates sialylated APP and β-CTF levels.

“In addition, the 5xFAD background itself also consistently elevated these levels (Figure 2F and 2G).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Total mature TACE levels are higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender.

“We found that total pro-TACE levels remained unchanged, while total mature TACE levels were higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender (Figure S4J and S4K).”

Neu1 deficiency increases the average number of plaques per brain area in the 5xFAD brain regardless of gender.

“Next, we showed that Neu1 deficiency increased the average number of plaques per brain area in the 5xFAD brain, regardless of gender (Figure 2B).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

β-CTF processing varies depending on background, gender, and Neu1 levels.

“The processing of b-CTF varied depending on background, gender, and Neu1 levels (Figure 3C and S3C).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Overexpression of PPCA has no significant impact on total brain Neu1 or PPCA enzyme activity.

“Interestingly, overexpression of PPCA had no significant impact on total brain Neu1 or PPCA enzyme activity (Figure S4C and S4D), although the total levels of mouse PPCA (moPPCA) and huPPCA were increased in 5xFAD/CSFR mice compared to controls, specifically 5xFAD and CSRF mice (Figure S4E and S4F).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

5xFAD/CSFR mice release significantly less Ab42 in the CSF compared to 5xFAD controls, regardless of gender.

“Sialylation of APP has been shown to enhance its processing 23 , but when we measured the levels of Ab42 in the CSF, we found that 5xFAD/CSFR mice released significantly less amyloid compared to 5xFAD controls, regardless of gender (Figure 4H).”

Neu1 deficiency increases levels of sialylated pro and mature TACE regardless of gender or background.

“Conversely, however, Neu1 deficiency increased the levels of sialylated pro and mature TACE, regardless of gender or background (Figure 2I and 2J).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

b-CTF levels are reduced in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender.

“Total levels of APP did not differ between 5xFAD and 5xFAD/CSFR mice, while b-CTF levels were reduced, regardless of gender (Figure S4H and S4I).”

Total β-CTF is higher in AD patients compared to controls.

“We confirmed that this was also the case in AD patients, who exhibited higher levels of total b-CTF compared to controls (Figure S1D).”

Female AD patients are more affected in β-HEX activity.

“Further analysis of b-HEX activity in AD patients' samples revealed that female patients were more affected (Figure 1D).”

PPCA turnover is slower in Neu1-/-, 5xFAD, and 5xFAD/Neu1-/- mice regardless of gender.

“For PPCA, we observed slower turnover in Neu1 -/-, 5xFAD, and 5xFAD/Neu1 -/-mice, regardless of gender (Figure 3A).”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

No differences are observed in sialylated NCT levels between AD patients and controls.

“No differences were observed in the levels of sialylated NCT (Figure 1J) and pro-TACE was undetectable in our assay.”

Sialylated NCT levels are highest in female 5xFAD/GFAP mice across all comparisons.

“However, sialylated NCT levels were highest in female 5xFAD/GFAP mice across all comparisons (Figure 6L).”

Sialylated moPPCA levels are significantly reduced in 5xFAD/GNIP mice relative to controls.

“in line with observations in 5xFAD/CSFR and 5xFAD/GFAP mice, both sialylated moPPCA and huPPCA levels were significantly reduced in 5xFAD/GNIP mice relative to controls (Figure 7D and S7G).”

Total BACE1 protein levels are decreased in female 5xFAD/GFAP mice compared to 5xFAD controls.

“As for BACE1, total protein levels were decreased in female 5xFAD/GFAP mice compared to 5xFAD controls, while no significant differences were observed in males (Figure S6L).”

5xFAD/CSFR and 5xFAD/GFAP mice display comparable biochemical profiles of APP family proteins.

“Remarkably, 5xFAD/CSFR and 5xFAD/GFAP mice displayed similar plaque morphology and comparable biochemical profiles of APP family proteins, setting them apart from 5sFAD/NSE mice.”

PPCA overexpression in microglia or astrocytes altered enzyme sialylation and Aβ secretion.

“In contrast, overexpression of PPCA in microglia or astrocytes altered enzyme sialylation and Aβ secretion but did not significantly affect plaque accumulation.”

Sialylated huPPCA levels are significantly reduced in 5xFAD/GNIP mice relative to controls.

“in line with observations in 5xFAD/CSFR and 5xFAD/GFAP mice, both sialylated moPPCA and huPPCA levels were significantly reduced in 5xFAD/GNIP mice relative to controls (Figure 7D and S7G).”

Extracellular b-Hex activity in the CSF is increased in male 5xFAD/GFAP mice compared to their respective 5xFAD controls.

“Extracellular b-Hex activity in the CSF was decreased in female 5xFAD/GFAP mice but increased in male 5xFAD/GFAP mice, compared to their respective 5xFAD controls (Figure 6E).”

Sialylated b-CTF levels are higher in female 5xFAD/NSE mice compared to controls.

“However, sialylated b-CTF levels were higher in female 5XFAD/NSE mice and lower in male mice, compared to controls (Figure 5F and 5G).”

Total BACE1 levels are lower in male 5xFAD/CSFR mice compared to 5xFAD controls.

“Total levels of BACE1 were unchanged between female 5xFAD/CSFR and 5xFAD mice but were lower in male 5xFAD/CSFR mice compared to 5xFAD controls (Figure S4L).”

Male 5xFAD mice exhibit significantly higher sialylated mature TACE levels than females.

“Notably, this gender difference resulted in comparable levels of sialylated mature TACE between female and male 5xFAD/GFAP mice, while male 5xFAD mice exhibited significantly higher levels than females (Figure 6J).”

Sialylated BACE1 levels are unchanged in male 5xFAD/NSE mice compared to controls.

“Total levels of BACE1 were lower in both male and female 5xFAD/NSE mice, although sialylated BACE1 levels were increased in female 5xFAD/NSE mice and unchanged in males, compared to controls (Figure 5 and S5L).”

There are no significant differences between 5xFAD and 5xFAD/GFAP animals in average plaque size, irrespective of sex.

“When these transgenic mice were crossed into the 5xFAD line to generate 5xFAD/GFAP mice, we found no significant differences between 5xFAD and 5xFAD/GFAP animals in total plaque burden, average plaque number per brain area, average plaque size, or total brain area, irrespective of sex (Figures 6A-6C, S6A and S6B).”

Total b-CTF levels do not differ between 5xFAD/GNIP and 5xFAD mice in either sex.

“Downstream of APP, total b-CTF levels did not differ between 5xFAD/GNIP and 5xFAD mice in either sex.”

Sialylated mature TACE levels are lower in male 5xFAD/CSFR mice compared to 5xFAD controls.

“This pattern slightly differed for mature TACE, where levels of sialylated mature TACE were still higher in female 5xFAD/CSFR mice but were lower in male 5xFAD/CSFR mice compared to 5xFAD controls.”

Total pro-TACE levels do not significantly differ between the sexes in 5xFAD/NSE mice.

“Total levels of pro-TACE did not significantly differ between the sexes in 5xFAD/NSE mice, but mature TACE levels were higher, regardless of gender, compared to controls (Figure S5J and S5K).”

Total b-CTF levels are higher in female 5xFAD/NSE mice compared to 5xFAD controls.

“A similar trend was seen for b-CTF, with higher total levels in female 5xFAD/NSE mice, but lower levels in males, compared to 5xFAD controls (Figure S5I).”

Sialylated BACE1 levels are increased in female 5xFAD/GNIP mice compared to 5xFAD controls.

“Consistent with all other models characterized, levels of sialylated BACE1 were increased in female 5xFAD/GNIP mice but remained unchanged in males compared to 5xFAD controls (Figure 7K).”

Total pro-TACE levels are similar between 5xFAD/GFAP and 5xFAD mice.

“Regarding TACE, total levels of the pro-form were similar between 5xFAD/GFAP and 5xFAD mice, while levels of the mature form were significantly higher in 5xFAD/GFAP mice, irrespective of sex.”

Female mice exhibited higher baseline levels of sialylated APP and secretases compared to male mice.

“Female mice exhibited higher baseline levels of sialylated APP and secretases, more pronounced lysosomal exocytosis activity, and greater plaque burden compared to male mice.”

There is a clear and consistent gender disparity in NEU1/PPCA-related processes.

“Our data also underscore a clear and consistent gender disparity in NEU1/PPCArelated processes.”

There is no significant difference in sialylated APP levels between female 5xFAD/GFAP and 5xFAD mice.

“While females consistently had higher sialylated APP levels overall, there was no significant difference between female 5xFAD/GFAP and 5xFAD mice.”

Total NCT levels show no change between female 5xFAD/CSFR and 5xFAD mice.

“Total levels of NCT mirrored BACE1, showing no change between female 5xFAD/CSFR and 5xFAD mice but lower levels in male 5xFAD/CSFR mice (Figure S4M).”

Ab42 concentrations in the CSF are significantly lower in 5xFAD/GFAP mice compared to 5xFAD controls, regardless of sex.

“Similar to what was observed in 5xFAD/CSFR mice, Ab42 concentrations in the CSF were significantly lower in 5xFAD/GFAP mice compared to 5xFAD controls, regardless of sex (Figure 6H).”

Total BACE1 levels are decreased in female 5xFAD/GNIP mice compared to 5xFAD controls.

“In terms of BACE1, total levels were decreased in female 5xFAD/GNIP mice, but increased in males, relative to 5XFAD controls (Figure S7L).”

Female 5xFAD/NSE mice tend to have fewer plaques per brain area unit compared to 5xFAD controls.

“For the average number of plaques per brain area unit, we found that female 5xFAD/NSE mice tended to have fewer plaques compared to 5xFAD controls, while no difference was observed in males (Figure 5B).”

PPCA overexpression appears to increase Neu1 activity.

“Interestingly, while PPCA overexpression did not obviously alter PPCA enzyme activity levels, it appeared to increase Neu1 activity (Figure S5C and S5D).”

Total pro-TACE levels are largely unchanged between 5xFAD/GNIP and 5xFAD mice.

“Total levels of pro-TACE and mature TACE were largely unchanged between 5xFAD/GNIP and 5xFAD mice, with the exception of a significant reduction in mature TACE levels in female 5xFAD/GNIP mice (Figure S7J and S7K).”

Sialylated APP levels tend to be higher in female 5xFAD/GNIP mice compared to controls.

“Sialylated APP levels followed a similar trend -tending to be higher in females 5xFAD/GNIP mice but significantly reduced in males relative to controls (Figure 7F).”

b-Hex enzyme activity in the CSF is only slightly increased in female 5xFAD/NSE mice compared to 5xFAD controls.

“Interestingly, b-Hex enzyme activity in the CSF was only slightly increased in female 5xFAD/NSE mice, while it was significantly higher in male 5xFAD/NSE mice, compared to 5xFAD controls (Figure 5E).”

Ab42 concentrations are significantly higher in the CSF of 5xFAD/NSE mice compared to 5xFAD.

“While Ab42 release was significantly reduced in 5xFAD/CSFR mice, Ab42 concentrations were significantly higher in the CSF of 5xFAD/NSE mice compared to 5xFAD (Figure 5H).”

In the 5xFAD/GFAP group, females have lower total BACE1 levels compared to males.

“However, in the 5xFAD/GFAP group, females had lower total BACE1 levels compared to males (Figure S6L).”

No differences in total APP protein expression are observed in male 5xFAD/NSE mice compared to 5xFAD controls.

“Total APP protein expression was enhanced in female 5xFAD/NSE mice, with no differences observed in males when compared to 5xFAD controls (Figure S5H).”

PPCA is subject to Neu1-mediated de-sialylation.

“PPCA itself is subject to Neu1-mediated de-sialylation, and its activity and turnover are similarly altered in Neu1-deficient contexts.”

5xFAD/GNIP mice have lower levels of huPPCA compared to 5xFAD controls, regardless of gender.

“The absence of increased PPCA activity may be explained by the opposing trends in PPCA protein levels: 5xFAD/GNIP mice had higher levels of moPPCA but lower levels of huPPCA, compared to 5xFAD controls, regardless of gender (Figure S7E and S7F).”

Sialylated pro-TACE levels are elevated in 5xFAD/GFAP mice relative to 5xFAD controls.

“In contrast, levels of sialylated pro-TACE were elevated in 5xFAD/GFAP mice relative to 5xFAD controls, with males showing higher levels than females (Figure 6I).”

Sialylated pro-TACE levels are significantly reduced in 5xFAD/GNIP mice compared to 5xFAD controls, regardless of gender.

“In contrast to 5xFAD/Neu1 -/-, 5xFAD/CSFR, and 5xFAD/GFAP mice, but closely resembling the 5xFAD/NSE model, levels of sialylated pro-TACE and mature TACE were significantly reduced in 5xFAD/GNIP mice compared to 5xFAD controls, regardless of gender (Figure 7I and 7J).”

No significant differences in plaque density (average number of plaques per unit brain area) are found between 5xFAD/GNIP and 5xFAD mice overall.

“Regarding plaque density, measured as the average number of plaques per unit brain area, no significant differences were found between 5xFAD/GNIP and 5xFAD mice overall.”

Overexpression of PPCA under the GFAP promoter results in elevated b-Hex activity in male mice, regardless of the presence or absence of the 5xFAD transgene.

“However, overexpression of PPCA under the GFAP promoter resulted in elevated b-Hex activity in male mice, regardless of the presence or absence of the 5xFAD transgene (Figure 6E).”

Total PPCA protein levels are significantly higher in 5xFAD/NSE mice.

“Furthermore, the total PPCA protein levels were significantly higher; moPPCA expression was higher in 5xFAD/NSE mice compared to 5xFAD controls and huPPCA expression was similarly higher in 5xFAD/NSE mice compared to NSE controls, regardless of gender (Figures S5E and S5F).”

Total b-CTF levels are lower in male 5xFAD/NSE mice compared to 5xFAD controls.

“A similar trend was seen for b-CTF, with higher total levels in female 5xFAD/NSE mice, but lower levels in males, compared to 5xFAD controls (Figure S5I).”

Sialylated mature TACE levels are significantly reduced in male 5xFAD/GFAP mice compared to their respective 5xFAD controls.

“For the mature form, sialylated TACE levels tended to be higher in female 5xFAD/GFAP mice but were significantly reduced in male mice, compared to their respective 5xFAD controls (Figure 6J).”

Sialylated mature TACE levels are comparable between female and male 5xFAD/GFAP mice.

“Notably, this gender difference resulted in comparable levels of sialylated mature TACE between female and male 5xFAD/GFAP mice, while male 5xFAD mice exhibited significantly higher levels than females (Figure 6J).”

Average plaque size is slightly reduced in 5xFAD/NSE mice compared to 5xFAD controls, regardless of gender.

“Regarding average plaque size, we measured a slight reduction in 5xFAD/NSE mice compared to 5xFAD controls, regardless of gender (Figure 5C).”

Mature TACE levels are higher in 5xFAD/NSE mice compared to controls, regardless of gender.

“Total levels of pro-TACE did not significantly differ between the sexes in 5xFAD/NSE mice, but mature TACE levels were higher, regardless of gender, compared to controls (Figure S5J and S5K).”

Total APP levels are elevated in female 5xFAD/GNIP mice compared to 5xFAD controls.

“Total APP levels were elevated in female 5xFAD/GNIP mice but remained unchanged in males compared to 5xFAD controls (Figure S7H).”

Extracellular b-Hex activity in the CSF is decreased in female 5xFAD/GFAP mice compared to their respective 5xFAD controls.

“Extracellular b-Hex activity in the CSF was decreased in female 5xFAD/GFAP mice but increased in male 5xFAD/GFAP mice, compared to their respective 5xFAD controls (Figure 6E).”

Sialylated mature TACE levels tend to be higher in female 5xFAD/GFAP mice compared to their respective 5xFAD controls.

“For the mature form, sialylated TACE levels tended to be higher in female 5xFAD/GFAP mice but were significantly reduced in male mice, compared to their respective 5xFAD controls (Figure 6J).”

Neu1 enzymatic activity levels are elevated in 5xFAD/GFAP mice relative to 5xFAD controls.

“Interestingly, the enzymatic activity levels of both Neu1 and PPCA were elevated in 5xFAD/GFAP mice relative to 5xFAD controls (Figures S6C and S6D).”

5xFAD/GNIP mice are the only transgenic model in this study that does not show any differences in CSF Ab42 concentrations relative to 5xFAD mice, regardless of gender.

“Remarkably, 5xFAD/GNIP mice were the only transgenic model in this study that did not show any differences in CSF Ab42 concentrations relative to 5xFAD mice, regardless of gender (Figure 7H).”

Sialylation acts as a regulatory signal for APP cleavage and secretase activity.

“Here, we show that sialylation acts as a regulatory signal for APP cleavage and secretase activity.”

In AD patients and Neu1-deficient mice, extracellular β-Hex activity is elevated, indicating heightened lysosomal exocytosis.

“In both AD patients and Neu1-deficient mice, we observed elevated levels of extracellular β-Hex activity, a marker of heightened lysosomal exocytosis activity.”

Male 5xFAD/NSE mice show decreased levels of sialylated pro-TACE compared to 5xFAD mice.

“In comparison to 5xFAD mice, female 5xFAD/NSE mice showed increased levels of sialylated pro-TACE, while males showed a decrease.”

Total BACE1 levels are increased in male 5xFAD/GNIP mice compared to 5xFAD controls.

“In terms of BACE1, total levels were decreased in female 5xFAD/GNIP mice, but increased in males, relative to 5XFAD controls (Figure S7L).”

Female 5xFAD/NSE mice show a marked reduction in total plaque area (DAB staining) compared to 5xFAD controls.

“In terms of total plaque area, female 5xFAD/NSE mice showed a marked reduction in DAB staining, while male mice showed only a slight decrease compared to 5xFAD controls (Figure 5A).”

Total NCT levels are lower in male 5xFAD/CSFR mice compared to 5xFAD controls.

“Total levels of NCT mirrored BACE1, showing no change between female 5xFAD/CSFR and 5xFAD mice but lower levels in male 5xFAD/CSFR mice (Figure S4M).”

huPPCA expression is higher in 5xFAD/NSE mice compared to NSE controls, regardless of gender.

“Furthermore, the total PPCA protein levels were significantly higher; moPPCA expression was higher in 5xFAD/NSE mice compared to 5xFAD controls and huPPCA expression was similarly higher in 5xFAD/NSE mice compared to NSE controls, regardless of gender (Figures S5E and S5F).”

Sialylated NCT levels are consistently higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender.

“Sialylated NCT levels were consistently higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender (Figure 4L).”

Sialylated APP levels are significantly higher in male 5xFAD/GFAP mice compared to male 5xFAD controls.

“In contrast, sialylated APP levels were significantly higher in male 5xFAD/GFAP mice compared to male 5XFAD controls (Figure 6F).”

moPPCA expression is higher in 5xFAD/NSE mice compared to 5xFAD controls, regardless of gender.

“Furthermore, the total PPCA protein levels were significantly higher; moPPCA expression was higher in 5xFAD/NSE mice compared to 5xFAD controls and huPPCA expression was similarly higher in 5xFAD/NSE mice compared to NSE controls, regardless of gender (Figures S5E and S5F).”

There are no significant differences between 5xFAD and 5xFAD/GFAP animals in total plaque burden, irrespective of sex.

“When these transgenic mice were crossed into the 5xFAD line to generate 5xFAD/GFAP mice, we found no significant differences between 5xFAD and 5xFAD/GFAP animals in total plaque burden, average plaque number per brain area, average plaque size, or total brain area, irrespective of sex (Figures 6A-6C, S6A and S6B).”

Neu1 dysregulation leads to a marked increase in lysosomal exocytosis.

“Additionally, we find that Neu1 dysregulation leads to a marked increase in lysosomal exocytosis, a mechanism through which lysosomal contents, including Aβ peptides and active enzymes, are secreted into the extracellular space 35,38,39 .”

Total BACE1 levels are unchanged between female 5xFAD/CSFR and 5xFAD mice.

“Total levels of BACE1 were unchanged between female 5xFAD/CSFR and 5xFAD mice but were lower in male 5xFAD/CSFR mice compared to 5xFAD controls (Figure S4L).”

No difference in average plaque size is observed in male 5xFAD/GNIP mice compared to 5xFAD controls.

“Consistent with the reduction in total plaque burden, average plaque size was significantly reduced in female 5xFAD/GNIP mice compared to 5xFAD controls, while no difference was observed in males (Figure 7C).”

PPCA enzymatic activity levels are elevated in 5xFAD/GFAP mice relative to 5xFAD controls.

“Interestingly, the enzymatic activity levels of both Neu1 and PPCA were elevated in 5xFAD/GFAP mice relative to 5xFAD controls (Figures S6C and S6D).”

Sialylated b-CTF levels are modestly increased in female 5xFAD/GNIP mice compared to controls.

“However, sialylated b-CTF levels were modestly increased in female 5xFAD/GNIP mice and significantly decreased in males compared to their respective controls (Figure 7G and S7I).”

Sialylated APP levels are significantly reduced in male 5xFAD/GNIP mice compared to controls.

“Sialylated APP levels followed a similar trend -tending to be higher in females 5xFAD/GNIP mice but significantly reduced in males relative to controls (Figure 7F).”

moPPCA levels are higher in 5xFAD/GFAP mice compared to GFAP transgenic controls.

“This increase was accompanied by higher levels of moPPCA in 5xFAD/GFAP mice, while levels of huPPCA were reduced when compared to GFAP transgenic controls (Figures S6E and S6F).”

Total BACE1 levels are lower in both male and female 5xFAD/NSE mice compared to controls.

“Total levels of BACE1 were lower in both male and female 5xFAD/NSE mice, although sialylated BACE1 levels were increased in female 5xFAD/NSE mice and unchanged in males, compared to controls (Figure 5 and S5L).”

5xFAD/GNIP mice lack significant changes in extracellular b-Hex activity compared to 5xFAD controls.

“However, a unique facet of 5xFAD/GNIP mice was the lack of significant changes in extracellular b-Hex activity compared to 5xFAD controls (Figure 7E).”

There is no difference in sialylated BACE1 levels between female and male 5xFAD mice.

“Additionally, while there was no difference in sialylated BACE1 levels between female and male 5xFAD mice, female 5xFAD/GFAP displayed significantly higher levels than their male counterparts (Figure 6K).”

Sialylated NCT levels are lower in male 5xFAD/NSE mice compared to 5xFAD controls.

“Sialylated NCT levels were higher in female 5xFAD/NSE mice and lower in males, compared to 5xFAD controls (Figure 5L and S5M).”

Sialylated BACE1 levels are higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender.

“Contrary to this, levels of sialylated BACE1 were higher in 5xFAD/CSFR mice compared to 5xFAD controls, regardless of gender, with levels in females being higher compared to males (Figure 4K).”

De-sialylation through Neu1 dampens secretase enzymatic function.

“Increased sialylation enhances secretase enzymatic function, while de-sialylation through Neu1 dampens it.”

Total APP protein expression is enhanced in female 5xFAD/NSE mice compared to 5xFAD controls.

“Total APP protein expression was enhanced in female 5xFAD/NSE mice, with no differences observed in males when compared to 5xFAD controls (Figure S5H).”

APP and its processing enzymes are not the only substrates of Neu1.

“Importantly, we reveal that APP and its processing enzymes are not the only substrates of Neu1.”

Sialylated BACE1 levels are increased in female 5xFAD/NSE mice compared to controls.

“Total levels of BACE1 were lower in both male and female 5xFAD/NSE mice, although sialylated BACE1 levels were increased in female 5xFAD/NSE mice and unchanged in males, compared to controls (Figure 5 and S5L).”

Female mice tend to display higher plaque burdens than male counterparts within the same genotype.

“However, female mice tended to display higher plaque burdens, a greater number of plaques per area, and larger plaque sizes compared to their male counterparts within the same genotype (Figures 6A-6C).”

Average plaque size is significantly reduced in female 5xFAD/GNIP mice compared to 5xFAD controls.

“Consistent with the reduction in total plaque burden, average plaque size was significantly reduced in female 5xFAD/GNIP mice compared to 5xFAD controls, while no difference was observed in males (Figure 7C).”

5xFAD/CSFR and 5xFAD/GFAP mice display similar plaque morphology.

“Remarkably, 5xFAD/CSFR and 5xFAD/GFAP mice displayed similar plaque morphology and comparable biochemical profiles of APP family proteins, setting them apart from 5sFAD/NSE mice.”

Total APP levels remain unchanged in male 5xFAD/GNIP mice compared to 5xFAD controls.

“Total APP levels were elevated in female 5xFAD/GNIP mice but remained unchanged in males compared to 5xFAD controls (Figure S7H).”

The AAV-based approach proved effective in mice.

“Lastly, although our AAV-based approach proved effective in mice, optimization of vector delivery, distribution, and duration of expression will be essential for clinical translation.”

Within each genotype, females consistently exhibit a higher plaque count than their male counterparts.

“However, within each genotype, females consistently exhibited a higher plaque count than their male counterparts (Figure 7B).”

No significant differences in total BACE1 protein levels are observed in male 5xFAD/GFAP mice compared to 5xFAD controls.

“As for BACE1, total protein levels were decreased in female 5xFAD/GFAP mice compared to 5xFAD controls, while no significant differences were observed in males (Figure S6L).”

No difference in average number of plaques per brain area unit is observed in male 5xFAD/NSE mice compared to 5xFAD controls.

“For the average number of plaques per brain area unit, we found that female 5xFAD/NSE mice tended to have fewer plaques compared to 5xFAD controls, while no difference was observed in males (Figure 5B).”

Sialylated b-CTF levels are lower in male 5xFAD/NSE mice compared to controls.

“However, sialylated b-CTF levels were higher in female 5XFAD/NSE mice and lower in male mice, compared to controls (Figure 5F and 5G).”

Female 5xFAD/NSE mice show increased levels of sialylated pro-TACE compared to 5xFAD mice.

“In comparison to 5xFAD mice, female 5xFAD/NSE mice showed increased levels of sialylated pro-TACE, while males showed a decrease.”

Sialylated NCT levels are higher in female 5xFAD/NSE mice compared to 5xFAD controls.

“Sialylated NCT levels were higher in female 5xFAD/NSE mice and lower in males, compared to 5xFAD controls (Figure 5L and S5M).”

Sialylated moPPCA levels are lower in 5xFAD/NSE mice compared to controls, regardless of gender.

“Similar to the observations in 5xFAD/CSFR mice, sialylated moPPCA and huPPCA levels were lower in 5xFAD/NSE mice compared to controls, regardless of gender; however, these differences were less pronounced than those observed in 5xFAD/CSFR mice (Figure 5D and S5G).”

Total b-CTF levels are reduced in 5xFAD/GFAP mice compared to 5xFAD mice, across both sexes.

“Regarding b-CTF, total levels were reduced in 5xFAD/GFAP mice compared to 5xFAD mice, across both sexes.”

Sialylated mature TACE levels are significantly reduced in 5xFAD/GNIP mice compared to 5xFAD controls, regardless of gender.

“In contrast to 5xFAD/Neu1 -/-, 5xFAD/CSFR, and 5xFAD/GFAP mice, but closely resembling the 5xFAD/NSE model, levels of sialylated pro-TACE and mature TACE were significantly reduced in 5xFAD/GNIP mice compared to 5xFAD controls, regardless of gender (Figure 7I and 7J).”

Female mice exhibited greater plaque burden compared to male mice.

“Female mice exhibited higher baseline levels of sialylated APP and secretases, more pronounced lysosomal exocytosis activity, and greater plaque burden compared to male mice.”

Mature TACE levels are significantly reduced in female 5xFAD/GNIP mice compared to 5xFAD controls.

“Total levels of pro-TACE and mature TACE were largely unchanged between 5xFAD/GNIP and 5xFAD mice, with the exception of a significant reduction in mature TACE levels in female 5xFAD/GNIP mice (Figure S7J and S7K).”

Females consistently have higher sialylated APP levels overall.

“While females consistently had higher sialylated APP levels overall, there was no significant difference between female 5xFAD/GFAP and 5xFAD mice.”

In 5xFAD/NSE mice, the effect on plaque characterization measurements is greater in females than in males compared to 5xFAD controls.

“When we crossed 5xFAD mice with mice overexpressing PPCA under the NSE promoter (5xFAD/NSE), we observed a greater effect in females than in males for all plaque characterization measurements, compared to 5xFAD controls (Figure 5A-5C and S5A).”

There are no significant differences between 5xFAD and 5xFAD/GFAP animals in total brain area, irrespective of sex.

“When these transgenic mice were crossed into the 5xFAD line to generate 5xFAD/GFAP mice, we found no significant differences between 5xFAD and 5xFAD/GFAP animals in total plaque burden, average plaque number per brain area, average plaque size, or total brain area, irrespective of sex (Figures 6A-6C, S6A and S6B).”

Female 5xFAD/GFAP mice display significantly higher sialylated BACE1 levels than their male counterparts.

“Additionally, while there was no difference in sialylated BACE1 levels between female and male 5xFAD mice, female 5xFAD/GFAP displayed significantly higher levels than their male counterparts (Figure 6K).”

Female mice exhibited more pronounced lysosomal exocytosis activity compared to male mice.

“Female mice exhibited higher baseline levels of sialylated APP and secretases, more pronounced lysosomal exocytosis activity, and greater plaque burden compared to male mice.”

5xFAD/GNIP mice have increased levels of Neu1 enzyme activity.

“Similar to what was seen in 5xFAD/NSE mice, 5xFAD/GNIP mice had increased levels of Neu1 enzyme activity but showed no change in PPCA activity (Figure S7C and S7D).”

Female mice tend to display a greater number of plaques per area than male counterparts within the same genotype.

“However, female mice tended to display higher plaque burdens, a greater number of plaques per area, and larger plaque sizes compared to their male counterparts within the same genotype (Figures 6A-6C).”

Sialylated BACE1 levels remain unchanged in male 5xFAD/GNIP mice compared to 5xFAD controls.

“Consistent with all other models characterized, levels of sialylated BACE1 were increased in female 5xFAD/GNIP mice but remained unchanged in males compared to 5xFAD controls (Figure 7K).”

Sialylated mature TACE levels are decreased in male 5xFAD/NSE mice compared to controls.

“Additionally, the levels of sialylated mature TACE remained unchanged in female 5xFAD/NSE mice but were decreased in males (Figure 5I and 5J).”

In female 5xFAD/GNIP mice, total plaque burden is significantly reduced compared to 5xFAD controls.

“In female 5xFAD/GNIP mice, total plaque burden was significantly reduced compared to 5xFAD controls, while no differences were observed between males of the two genotypes (Figure 7A and S7A), a contrast to findings in 5xFAD/Neu1 -/-mice.”

Sialylated huPPCA levels are lower in 5xFAD/NSE mice compared to controls, regardless of gender.

“Similar to the observations in 5xFAD/CSFR mice, sialylated moPPCA and huPPCA levels were lower in 5xFAD/NSE mice compared to controls, regardless of gender; however, these differences were less pronounced than those observed in 5xFAD/CSFR mice (Figure 5D and S5G).”

Among 5xFAD mice, no gender differences are observed in total BACE1 levels.

“Among 5XFAD mice, no gender differences were observed in total BACE1 levels.”

Total NCT levels are comparable between 5xFAD/GFAP and 5xFAD mice.

“Total levels of NCT were comparable between 5xFAD/GFAP and 5xFAD mice, with no significant differences between sexes within the same genotype (Figure S6M).”

b-Hex enzyme activity in the CSF is significantly higher in male 5xFAD/NSE mice compared to 5xFAD controls.

“Interestingly, b-Hex enzyme activity in the CSF was only slightly increased in female 5xFAD/NSE mice, while it was significantly higher in male 5xFAD/NSE mice, compared to 5xFAD controls (Figure 5E).”

Total NCT levels do not differ between female 5xFAD and 5xFAD/NSE mice.

“For NCT, total levels did not differ between female 5xFAD and 5xFAD/NSE mice but were lower in male 5xFAD/NSE mice.”

Total mature TACE levels are significantly higher in 5xFAD/GFAP mice compared to 5xFAD mice, irrespective of sex.

“Regarding TACE, total levels of the pro-form were similar between 5xFAD/GFAP and 5xFAD mice, while levels of the mature form were significantly higher in 5xFAD/GFAP mice, irrespective of sex.”

Within the same genotype group, female 5xFAD mice have higher b-Hex activity than males.

“Within the same genotype group, female 5xFAD mice hade higher b-Hex activity than males.”

PPCA overexpression in microglia or astrocytes did not significantly affect plaque accumulation.

“In contrast, overexpression of PPCA in microglia or astrocytes altered enzyme sialylation and Aβ secretion but did not significantly affect plaque accumulation.”

Sialylated mature TACE levels are higher in female 5xFAD/CSFR mice compared to 5xFAD controls.

“This pattern slightly differed for mature TACE, where levels of sialylated mature TACE were still higher in female 5xFAD/CSFR mice but were lower in male 5xFAD/CSFR mice compared to 5xFAD controls.”

Increased sialylation enhances secretase enzymatic function.

“Increased sialylation enhances secretase enzymatic function, while de-sialylation through Neu1 dampens it.”

huPPCA levels are reduced in 5xFAD/GFAP mice compared to GFAP transgenic controls.

“This increase was accompanied by higher levels of moPPCA in 5xFAD/GFAP mice, while levels of huPPCA were reduced when compared to GFAP transgenic controls (Figures S6E and S6F).”

5xFAD/GNIP mice show no change in PPCA activity.

“Similar to what was seen in 5xFAD/NSE mice, 5xFAD/GNIP mice had increased levels of Neu1 enzyme activity but showed no change in PPCA activity (Figure S7C and S7D).”

PPCA overexpression does not obviously alter PPCA enzyme activity levels.

“Interestingly, while PPCA overexpression did not obviously alter PPCA enzyme activity levels, it appeared to increase Neu1 activity (Figure S5C and S5D).”

The differences in sialylated moPPCA and huPPCA levels in 5xFAD/NSE mice are less pronounced than those observed in 5xFAD/CSFR mice.

“Similar to the observations in 5xFAD/CSFR mice, sialylated moPPCA and huPPCA levels were lower in 5xFAD/NSE mice compared to controls, regardless of gender; however, these differences were less pronounced than those observed in 5xFAD/CSFR mice (Figure 5D and S5G).”

Sialylated mature TACE levels remain unchanged in female 5xFAD/NSE mice compared to controls.

“Additionally, the levels of sialylated mature TACE remained unchanged in female 5xFAD/NSE mice but were decreased in males (Figure 5I and 5J).”

Total NCT levels are lower in male 5xFAD/NSE mice compared to 5xFAD controls.

“For NCT, total levels did not differ between female 5xFAD and 5xFAD/NSE mice but were lower in male 5xFAD/NSE mice.”

Sialylated APP levels do not differ between 5xFAD/NSE and 5xFAD mice, regardless of gender.

“Interestingly, sialylated APP levels did not differ between 5xFAD/NSE and 5xFAD mice, regardless of gender.”

Male 5xFAD/NSE mice show only a slight decrease in total plaque area compared to 5xFAD controls.

“In terms of total plaque area, female 5xFAD/NSE mice showed a marked reduction in DAB staining, while male mice showed only a slight decrease compared to 5xFAD controls (Figure 5A).”

These sex differences persisted across genetic backgrounds and experimental manipulations.

“These differences persisted across genetic backgrounds and experimental manipulations, suggesting a biologically intrinsic basis for the observed sex bias in AD prevalence and severity.”

There are no significant differences between 5xFAD and 5xFAD/GFAP animals in average plaque number per brain area, irrespective of sex.

“When these transgenic mice were crossed into the 5xFAD line to generate 5xFAD/GFAP mice, we found no significant differences between 5xFAD and 5xFAD/GFAP animals in total plaque burden, average plaque number per brain area, average plaque size, or total brain area, irrespective of sex (Figures 6A-6C, S6A and S6B).”

Female mice tend to display larger plaque sizes than male counterparts within the same genotype.

“However, female mice tended to display higher plaque burdens, a greater number of plaques per area, and larger plaque sizes compared to their male counterparts within the same genotype (Figures 6A-6C).”

Sialylated b-CTF levels are significantly decreased in male 5xFAD/GNIP mice compared to controls.

“However, sialylated b-CTF levels were modestly increased in female 5xFAD/GNIP mice and significantly decreased in males compared to their respective controls (Figure 7G and S7I).”

No differences in total plaque burden are observed between male 5xFAD/GNIP and 5xFAD mice.

“In female 5xFAD/GNIP mice, total plaque burden was significantly reduced compared to 5xFAD controls, while no differences were observed between males of the two genotypes (Figure 7A and S7A), a contrast to findings in 5xFAD/Neu1 -/-mice.”

5xFAD/GNIP mice have higher levels of moPPCA compared to 5xFAD controls, regardless of gender.

“The absence of increased PPCA activity may be explained by the opposing trends in PPCA protein levels: 5xFAD/GNIP mice had higher levels of moPPCA but lower levels of huPPCA, compared to 5xFAD controls, regardless of gender (Figure S7E and S7F).”

Sialylated PPCA may be more enzymatically active than desialylated PPCA.

“These findings suggest that sialylated PPCA may be more enzymatically active than desialylated PPCA.”

Lysosomal exocytosis may still be contributing to the pathological development in these mice, possibly by releasing toxic Ab42 peptides.

“This indicates that lysosomal exocytosis may still be contributing to the pathological development in these mice, possibly by releasing toxic Ab42 peptides.”

Amyloidogenic processing of APP is reduced in 5xFAD/CSFR mice.

“This further suggests that amyloidogenic processing of APP is reduced in 5xFAD/CSFR mice.”

Neu1 and PPCA are critical regulators of APP metabolism.

“This study identifies two members of the lysosomal multi-enzyme complex (LMC), neuraminidase 1 (Neu1) and protective protein/cathepsin A (PPCA), as a critical regulators of APP metabolism.”

PPCA overexpression in the myeloid population may reduce amyloidogenic APP processing.

“This suggests that PPCA overexpression in the myeloid population may reduce amyloidogenic APP processing.”

Lysosomal exocytosis is enhanced in AD patients as a result of NEU1 downregulation.

“These findings indicate that lysosomal exocytosis is enhanced in AD patients as a result of NEU1 downregulation.”

Neu1 deficiency increases lysosomal exocytosis, contributing to extracellular Aβ release and neuroinflammation.

“Additionally, Neu1 deficiency increases lysosomal exocytosis, contributing to extracellular Aβ release and neuroinflammation.”

There is a novel bidirectional dependency between Neu1 and PPCA.

“These findings reveal a novel bidirectional dependency between Neu1 and PPCA, underscoring their cooperative role in maintaining lysosomal homeostasis.”

PPCA is a substrate of NEU1.

“These findings also indicate that PPCA is a substrate of NEU1, a correlation that has not yet been defined.”

Neu1 deficiency enhances amyloidogenic cleavage and Aβ42 production.

“Neu1 deficiency in human AD brains and 5xFAD/Neu1 -/-mice leads to sialic acid retention on APP and its secretases, enhancing amyloidogenic cleavage and Aβ42 production.”

The proteolytic processing of PPCA, APP and the secretase proteins is influenced independently and differentially by the expression of the 5xFAD transgenes, the levels of Neu1 present, and the gender of the mouse.

“Overall, the CHX turnover analyses revealed that the proteolytic processing of PPCA, APP and the secretase proteins is influenced independently and differentially by the expression of the 5xFAD transgenes, the levels of Neu1 present, and the gender of the mouse.”

• AssumptionRests on an assumption: The 5xFAD mouse model recapitulates key aspects of AD pathology relevant to lysosomal dysfunction and amyloidosis.

Overexpression of huPPCA protein in microglia has little impact on the plaque pathology that develops in the brain parenchyma.

“Altogether, these data demonstrate that overexpression of huPPCA protein in microglia influences the biochemical properties of secretase enzymes and subunits but has little impact on the plaque pathology that develops in the brain parenchyma.”

Increased Neu1 activity likely contributes to the significantly reduced levels of sialylated moPPCA and huPPCA observed in 5xFAD/GFAP mice relative to controls.

“The increased Neu1 activity likely contributed to the significantly reduced levels of sialylated moPPCA and huPPCA observed in 5xFAD/GFAP mice relative to the appropriate controls (Figure 6D and S6G).”

Co-overexpression of NEU1 and PPCA yields more favorable outcomes than overexpression of PPCA alone.

“Taken together, these results suggest that co-overexpression of NEU1 and PPCA yields more favorable outcomes than overexpression of PPCA alone, highlighting the importance of maintaining a functional balance between these two interdependent enzymes.”

There is a coordinated interplay between neuronal and glial cells in the regulation of LMC function.

“These results point to a coordinated interplay between neuronal and glial cells in the regulation of LMC function and suggest that glial-targeted interventions may be more effective for modulating extracellular Aβ clearance or immune responses, rather than directly influencing primary APP processing.”

There is a relationship between the level of sialylation on b-CTF and the amount of Ab42 released into the CSF.

“These findings suggest a relationship between the level of sialylation on b-CTF and the amount of Ab42 released into the CSF.”

Neu1 and PPCA may serve as a critical node that connects proteostasis with immune signaling in AD.

“Our findings support this hypothesis by linking elevated lysosomal exocytosis to increased extracellular Aβ, suggesting that Neu1 and PPCA may serve as a critical node that connects proteostasis with immune signaling in AD.”

Overexpression of huPPCA protein in microglia influences the biochemical properties of secretase enzymes and subunits.

“Altogether, these data demonstrate that overexpression of huPPCA protein in microglia influences the biochemical properties of secretase enzymes and subunits but has little impact on the plaque pathology that develops in the brain parenchyma.”

Neu1 and PPCA form a tightly coordinated relationship in which each protein stabilizes and regulates the other within the LMC.

“Our data instead suggest a tightly coordinated relationship, in which each protein stabilizes and regulates the other, forming a dynamic enzymatic partnership within the LMC.”

Overexpressing PPCA under the NSE promoter effectively reduces plaque pathology in 5xFAD mice.

“Overall, overexpressing PPCA under the NSE promoter effectively reduced plaque pathology in 5xFAD mice.”

The unique and distinct changes in the expression and sialylation patterns of APP family proteins may underlie the observed significant reduction in plaque pathology in female 5xFAD/GNIP mice.

“These unique and distinct changes in the expression and sialylation patterns of APP family of proteins may underlie the observed significant reduction in plaque pathology in female 5xFAD/GNIP mice.”

The observed sex bias in AD prevalence and severity has a biologically intrinsic basis.

“These differences persisted across genetic backgrounds and experimental manipulations, suggesting a biologically intrinsic basis for the observed sex bias in AD prevalence and severity.”

Differences in Neu1 activity alone cannot fully explain the pronounced gender-specific variations observed in b-Hex enzyme activity.

“However, the differences in Neu1 activity alone cannot fully explain the pronounced gender-specific variations observed in b-Hex enzyme activity, used to monitor the extent of lysosomal exocytosis.”

Increased Neu1 activity alone is sufficient to drive a reduction in the sialylation of TACE proteins.

“Both 5xFAD/NSE and 5xFAD/GNIP mice shared the distinctive feature of elevated Neu1 activity without a corresponding increase in PPCA activity, suggesting that increased Neu1 activity alone is sufficient to drive a reduction in the sialylation of TACE proteins.”

Neuronal APP metabolism plays a central role in plaque pathology.

“Neuron-specific expression (under the NSE promoter) was the most effective at reducing plaque burden, especially in female mice, underscoring the central role of neuronal APP metabolism in plaque pathology.”

The absence of increased PPCA activity in 5xFAD/GNIP mice may be explained by the opposing trends in PPCA protein levels.

“The absence of increased PPCA activity may be explained by the opposing trends in PPCA protein levels: 5xFAD/GNIP mice had higher levels of moPPCA but lower levels of huPPCA, compared to 5xFAD controls, regardless of gender (Figure S7E and S7F).”