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Effects of CD2-associated protein deficiency on amyloid-β in neuroblastoma cells and in an APP transgenic mouse model.

Liao F, Jiang H, Srivatsan S, Xiao Q, Lefton KB, Yamada K, Mahan TE, Lee JM, Shaw AS, Holtzman DM - Mol Neurodegener (2015)

Bottom Line: Our data show that suppressing CD2AP expression using shRNA in N2a-APP695 cells results in decreased cell membrane amyloid precursor protein, decreased Aβ release and a lower Aβ42/Aβ40 ratio.In 1-month old PS1APP mice, complete loss of CD2AP in brain resulted in a decreased Aβ42/Aβ40 ratio in brain tissue lysates while there was no effect on Aβ deposition or accumulation in PS1APP mice expressing one copy of CD2AP.The effect of CD2-Associated Protein on Aβ metabolism is subtle in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Hope Center for Neurological Disorders, Charles F. and Joanne Knight Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA. liaof@neuro.wustl.edu.

ABSTRACT

Background: CD2-associated protein (CD2AP) is an SH3-containing scaffold adaptor protein which regulates the actin cytoskeleton. Recently, CD2AP was identified as a genetic risk factor for Alzheimer's disease (AD) by several genome-wide association studies. One of the hallmarks of AD is the accumulation of aggregated forms of Amyloid-β (Aβ) in the brain. In humans, CD2AP AD susceptibility locus (rs9349407) is associated with an increased plaque burden. Aβ production is highly regulated by endocytosis and is influenced by lysosomal function. Lysosomal trafficking is influenced by CD2AP. In this study, we decreased CD2AP levels in N2a neuroblastoma cultures and PS1APP mice and analyzed Aβ levels and plaque burden.

Results: Our data show that suppressing CD2AP expression using shRNA in N2a-APP695 cells results in decreased cell membrane amyloid precursor protein, decreased Aβ release and a lower Aβ42/Aβ40 ratio. CD2AP protein is expressed in the brain as detected by western blot, and the expression level is dependent on gene dosage. In 1-month old PS1APP mice, complete loss of CD2AP in brain resulted in a decreased Aβ42/Aβ40 ratio in brain tissue lysates while there was no effect on Aβ deposition or accumulation in PS1APP mice expressing one copy of CD2AP.

Conclusion: CD2-Associated Protein affects Aβ levels and Aβ42/Aβ40 ratio in vitro. The effect of CD2-Associated Protein on Aβ metabolism is subtle in vivo.

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Related in: MedlinePlus

Effects of CD2AP haploinsufficiency on tissue Aβ levels in 7-month old PS1APP mice. PS1APP/CD2AP+/+ (n = 19 total, 10 females and 9 males) and PS1APP/CD2AP+/− (n = 18 total, 9 females and 9 males) mice were sacrificed. Aβ40, Aβ42 and Aβ42/Aβ40 ratio in PBS fraction, 1% triton fraction and Guanidine fraction (*, p < 0.05, Student’s t-test).
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Fig5: Effects of CD2AP haploinsufficiency on tissue Aβ levels in 7-month old PS1APP mice. PS1APP/CD2AP+/+ (n = 19 total, 10 females and 9 males) and PS1APP/CD2AP+/− (n = 18 total, 9 females and 9 males) mice were sacrificed. Aβ40, Aβ42 and Aβ42/Aβ40 ratio in PBS fraction, 1% triton fraction and Guanidine fraction (*, p < 0.05, Student’s t-test).

Mentions: Shifting the ratio of Aβ42/Aβ40 results in an altered time course of plaque deposition in both humans [19,20] and mice [25]. Since we observed changes in the Aβ42/Aβ40 ratio in cultured cells and 1-month old PS1APP mice, we next asked whether CD2AP deficiency affects amyloid plaque load in older PS1APP mice. CD2AP−/− mice have a ~ 6-week life-span due to renal failure [14] while the average plaque onset age in PS1APP mice is at ~4-month. Therefore, we were not able to assess plaque deposition in PS1APP/CD2AP−/− mice. Mice with CD2AP haploinsufficiency (CD2AP+/−) live a normal life span but express ~50% less CD2AP in the brain compared to CD2AP+/+ mice (Figure 3A,B). We therefore asked whether a ~50% reduction of CD2AP levels affects Aβ pathology in PS1APP mice. We generated PS1APP/CD2AP+/− and PS1APP/CD2AP+/+ mice and characterized their Aβ pathology at the age of 7-months. We first measured Aβ levels in the cortical tissue lysates. The results showed no difference in the absolute level of Aβ40 and Aβ42 in the PBS (soluble forms of Aβ), Triton or Guanidine (insoluble forms of Aβ) brain fractions between PS1APP/CD2AP+/− and PS1APP/CD2AP+/+ groups (Figure 5). In the PBS soluble fraction, the Aβ42/Aβ40 ratio in the female PS1APP/CD2AP+/− group was significantly lower than that in female PS1APP/CD2AP+/+ group (p < 0.05, Student’s t-test). However, the effects on males tended to trend in the opposite direction (Figure 5). In the Triton and Guanidine fractions, there was no change in the Aβ42/Aβ40 ratio associated with CD2AP gene status (Figure 5).Figure 5


Effects of CD2-associated protein deficiency on amyloid-β in neuroblastoma cells and in an APP transgenic mouse model.

Liao F, Jiang H, Srivatsan S, Xiao Q, Lefton KB, Yamada K, Mahan TE, Lee JM, Shaw AS, Holtzman DM - Mol Neurodegener (2015)

Effects of CD2AP haploinsufficiency on tissue Aβ levels in 7-month old PS1APP mice. PS1APP/CD2AP+/+ (n = 19 total, 10 females and 9 males) and PS1APP/CD2AP+/− (n = 18 total, 9 females and 9 males) mice were sacrificed. Aβ40, Aβ42 and Aβ42/Aβ40 ratio in PBS fraction, 1% triton fraction and Guanidine fraction (*, p < 0.05, Student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374406&req=5

Fig5: Effects of CD2AP haploinsufficiency on tissue Aβ levels in 7-month old PS1APP mice. PS1APP/CD2AP+/+ (n = 19 total, 10 females and 9 males) and PS1APP/CD2AP+/− (n = 18 total, 9 females and 9 males) mice were sacrificed. Aβ40, Aβ42 and Aβ42/Aβ40 ratio in PBS fraction, 1% triton fraction and Guanidine fraction (*, p < 0.05, Student’s t-test).
Mentions: Shifting the ratio of Aβ42/Aβ40 results in an altered time course of plaque deposition in both humans [19,20] and mice [25]. Since we observed changes in the Aβ42/Aβ40 ratio in cultured cells and 1-month old PS1APP mice, we next asked whether CD2AP deficiency affects amyloid plaque load in older PS1APP mice. CD2AP−/− mice have a ~ 6-week life-span due to renal failure [14] while the average plaque onset age in PS1APP mice is at ~4-month. Therefore, we were not able to assess plaque deposition in PS1APP/CD2AP−/− mice. Mice with CD2AP haploinsufficiency (CD2AP+/−) live a normal life span but express ~50% less CD2AP in the brain compared to CD2AP+/+ mice (Figure 3A,B). We therefore asked whether a ~50% reduction of CD2AP levels affects Aβ pathology in PS1APP mice. We generated PS1APP/CD2AP+/− and PS1APP/CD2AP+/+ mice and characterized their Aβ pathology at the age of 7-months. We first measured Aβ levels in the cortical tissue lysates. The results showed no difference in the absolute level of Aβ40 and Aβ42 in the PBS (soluble forms of Aβ), Triton or Guanidine (insoluble forms of Aβ) brain fractions between PS1APP/CD2AP+/− and PS1APP/CD2AP+/+ groups (Figure 5). In the PBS soluble fraction, the Aβ42/Aβ40 ratio in the female PS1APP/CD2AP+/− group was significantly lower than that in female PS1APP/CD2AP+/+ group (p < 0.05, Student’s t-test). However, the effects on males tended to trend in the opposite direction (Figure 5). In the Triton and Guanidine fractions, there was no change in the Aβ42/Aβ40 ratio associated with CD2AP gene status (Figure 5).Figure 5

Bottom Line: Our data show that suppressing CD2AP expression using shRNA in N2a-APP695 cells results in decreased cell membrane amyloid precursor protein, decreased Aβ release and a lower Aβ42/Aβ40 ratio.In 1-month old PS1APP mice, complete loss of CD2AP in brain resulted in a decreased Aβ42/Aβ40 ratio in brain tissue lysates while there was no effect on Aβ deposition or accumulation in PS1APP mice expressing one copy of CD2AP.The effect of CD2-Associated Protein on Aβ metabolism is subtle in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Hope Center for Neurological Disorders, Charles F. and Joanne Knight Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA. liaof@neuro.wustl.edu.

ABSTRACT

Background: CD2-associated protein (CD2AP) is an SH3-containing scaffold adaptor protein which regulates the actin cytoskeleton. Recently, CD2AP was identified as a genetic risk factor for Alzheimer's disease (AD) by several genome-wide association studies. One of the hallmarks of AD is the accumulation of aggregated forms of Amyloid-β (Aβ) in the brain. In humans, CD2AP AD susceptibility locus (rs9349407) is associated with an increased plaque burden. Aβ production is highly regulated by endocytosis and is influenced by lysosomal function. Lysosomal trafficking is influenced by CD2AP. In this study, we decreased CD2AP levels in N2a neuroblastoma cultures and PS1APP mice and analyzed Aβ levels and plaque burden.

Results: Our data show that suppressing CD2AP expression using shRNA in N2a-APP695 cells results in decreased cell membrane amyloid precursor protein, decreased Aβ release and a lower Aβ42/Aβ40 ratio. CD2AP protein is expressed in the brain as detected by western blot, and the expression level is dependent on gene dosage. In 1-month old PS1APP mice, complete loss of CD2AP in brain resulted in a decreased Aβ42/Aβ40 ratio in brain tissue lysates while there was no effect on Aβ deposition or accumulation in PS1APP mice expressing one copy of CD2AP.

Conclusion: CD2-Associated Protein affects Aβ levels and Aβ42/Aβ40 ratio in vitro. The effect of CD2-Associated Protein on Aβ metabolism is subtle in vivo.

Show MeSH
Related in: MedlinePlus