Haploinsufficiency of lysosomal enzyme genes in Alzheimer's disease

Abstract

There is growing evidence suggesting that the lysosome or lysosome dysfunction is associated with Alzheimer's disease (AD). Pathway analysis of post mortem brain-derived proteomic data from AD patients shows that the lysosomal system is perturbed relative to similarly aged unaffected controls. However, it is unclear if these changes contributed to the pathogenesis or are a response to the disease. Consistent with the hypothesis that lysosome dysfunction contributes to AD pathogenesis, whole genome sequencing data indicate that heterozygous pathogenic mutations and predicted protein-damaging variants in multiple lysosomal enzyme genes are enriched in AD patients compared to matched controls. Heterozygous loss-of-function mutations in the palmitoyl protein thioesterase-1 (PPT1), α-L-iduronidase (IDUA), β-glucuronidase (GUSB), N-acetylglucosaminidase (NAGLU), and galactocerebrosidase (GALC) genes have a gene-dosage effect on Aβ₄₀ levels in brain interstitial fluid in C57BL/6 mice and significantly increase Aβ plaque formation in the 5xFAD mouse model of AD, thus providing in vivo validation of the human genetic data. A more detailed analysis of PPT1 heterozygosity in 18-month-old mice revealed changes in α-, β-, and γ-secretases that favor an amyloidogenic pathway. Proteomic changes in brain tissue from aged PPT1 heterozygous sheep are consistent with both the mouse data and the potential activation of AD pathways. Finally, CNS-directed, AAV-mediated gene therapy significantly decreased Aβ plaques, increased life span, and improved behavioral performance in 5xFAD/PPT1+/- mice. Collectively, these data strongly suggest that heterozygosity of multiple lysosomal enzyme genes represent risk factors for AD and may identify precise therapeutic targets for a subset of genetically-defined AD patients.

Fig. 1.

Heterozygosity of PPT1 exacerbates the AD-like phenotype of the 5xFAD mouse. (A) Heterozygosity of PPT1 decreases brain PPT1 activity by approximately half and homozygous deficiency completely eliminates enzyme activity. (B) The median life span of 5xFAD (blue line) and PPT1 heterozygous mice (black line) on a C57Bl/6 background is 24 months. In contrast, the median life span of 5xFAD/PPT1+/− mice (red line) is approximately 10 months. (C) There is an obvious and statistically significant (D) increase in Aβ plaques in seven-month-old 5xFAD/PPT1+/− mice compared to age-matched 5xFAD mice. The levels of detergent-insoluble Aβ₄₀ (E) and Aβ₄₂ (F) in 5xFAF/PPT1+/− mice are significantly greater than those in age-matched 5xFAD mice. **p<0.01, ****p<0.0001.

Fig. 2.

Heterozygosity of PPT1, alone, affects Aβ processing and the processing enzymes. There is a clear gene-dosage effect of PPT1 on ISF Aβ levels. (A) The level of ISF Aβ₄₀ is significantly decreased in 3.5-month-old PPT1 heterozygous animals compared to age-matched wild type animals. The level of ISF Aβ₄₀ is further decreased in PPT1 homozygous deficient mice compared to heterozygous animals. (B) Quantitative Western blots of APP, ADAM 10, Presenilin-1, and BACE are shown [Note: a single Western blot was probed with APP, ADAM10, Presenilin-1, and GAPDH antibodies; a second Western blot (below the dashed line) was probed with BACE and GAPDH antibodies]. (C) The levels of amyloid precursor protein [APP (CT695)] in 18-month-old PPT1 heterozygous animals are not significantly different from age matched wild type mice. (D) The levels of α-secretase (ADAM10) are significantly decreased in PPT1 heterozygous mice. In contrast, the levels of both β-secretase (E) and γ-secretase (F) are significantly increased in PPT1 heterozygous animals compared to wild type mice. *p<0.05, **p<0.01, ***p<0.001.

Fig. 3.

CNS-directed, AAV-mediated gene therapy improves the AD phenotype in 5xFAD/PPT1+/− mice. (A) Intracranial injection of an AAV9-hPPT1 vector in newborn (PND 1-2) 5xFAD/PPT1+/− mice significantly decreases the Aβ plaque load at 7 months-of-age compared to uninjected 5xFAD/PPT1+/− mice. (B) The level of PPT1 activity in AAV-hPPT1-injected mice is approximately 2-fold greater than uninjected mice. (C) 5xFAD/PPT1+/− mice receiving intraparenchymal injections of AAV-hPPT1 (PPT, IP) at 3.5-months of age performed significantly better in the Y-maze behavioral test compared to 5xFAD/PPT1+/− mice receiving an ICV injection of AAV9-GFP (GFP, ICV) at the same age. Although, on average, mice receiving an ICV injection of AAV9-hPPT1 (PPT, ICV) performed better than AAV9-GFP-injected animals, it did not reach statistical significance. (D) The level of PPT1 activity in 17-month-old 5xFAD/PPT1+/− mice receiving an ICV or an intraparenchymal injection was significantly greater than 5xFAD/PPT1+/− mice injected with AAV9-GFP. (E) 5xFAD/PPT1+/− mice receiving intraparenchymal injections of AAV-hPPT1 (green line) at 3.5-months of age had a significantly extended life span compared to untreated 5xFAD/PPT1+/− animals (red line). The life span of 5xFAD/PPT1+/− mice receiving an ICV injection of AAV9-hPPT1 (blue line) was significantly greater than untreated mice, and mice receiving an intraparenchymal injection of virus. The levels of detergent-insoluble Aβ₄₀ (F) and Aβ₄₂ (G) were significantly decreased in 17 month-old 5xFAD/PPT1+/− mice injected with AAV9-hPPT1 by either route compared with 9 month-old 5xFAD/PPT1+/− mice injected ICV with AAV9-GFP. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 4.

Two additional lysosomal enzyme genes identified in the human genetic analysis affect Aβ metabolism and exacerbate the AD phenotype of 5xFAD mice. Three and one half-month-old mice that are heterozygous for the α-L-iduronidase (IDUA) gene (IDUA+/−) have approximately 50% enzyme activity (A) and significantly decreased ISF Aβ levels (B) compared to age-matched wild type (WT) mice. Heterozygosity of IDUA also significantly increased the Aβ plaque load (C) and the level of detergent-insoluble Aβ₄₂ in 5xFAD mice (E). Although the mean level of detergent-insoluble Aβ₄₀ was greater than that observed in 5xFAD mice, it did not reach statistical significance (D). Three and one half-month-old mice that are heterozygous for the galactocerebrosidase (GALC) gene (GALC+/−) have approximately 50% enzyme activity (F) and significantly decreased ISF Aβ levels (G) compared to age-matched wild type mice. Heterozygosity of GALC also significantly increased the Aβ plaque load (H) and the level of detergent-insoluble Aβ₄₀ (I) and Aβ₄₂ (J) in 5xFAD mice (I, J). Note: The ELISA assays and Aβ immunostaining for the 5xFAD, 5xFAD/IDUA+/−, 5xFAD/GALC+/−, and 5xFAD/GUSB+/− animals were performed at the same time with the same reagents to ensure consistency for direct comparisons. Therefore, the values for the 5xFAD/IDUA+/−, 5xFAD/GALC+/−, and 5xFAD/GUSB+/− animals were compared to the same set of data from the 5xFAD mice (see Figures 4C, D, E, H, I, J, 5C, D, and E). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig. 5.

Two lysosomal enzyme genes that were not identified in the human genetic analysis affect Aβ metabolism and exacerbate the AD phenotype of 5xFAD mice. Three and one half-month-old mice that are heterozygous for the N-acetylglucosaminidase (NAGLU) gene (NAGLU+/−) have approximately half of the enzyme activity (A) and significantly decreased ISF Aβ levels (B) compared to age-matched wild type mice (WT). Heterozygosity of NAGLU also significantly increased the Aβ plaque load (C) and the level of detergent-insoluble Aβ₄₀ (D) and Aβ₄₂ (E) in 5xFAD mice. Three and one half-month-old mice that are heterozygous for the β-glucuronidase (GUSB) gene (GUSB+/−) have approximately 50% enzyme activity (F) and significantly decreased ISF Aβ levels (G) compared to age-matched wild type mice. Heterozygosity of GUSB also significantly increased the Aβ plaque load (H) and the level of detergent-insoluble Aβ₄₀ (I) and Aβ₄₂ (J) in 5xFAD mice. *p<0.05, **p<0.01, ****p<0.0001.