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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

CD2AP shRNAs decreased extracellular Aβ levels in cell culture. N2a-695 cells were transfected with CD2AP shRNAs or control shRNA. (A-C) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell culture medium. (D-F) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell lysates (n = 5/group; *, p < 0.05, **, p < 0.01, ***, p < 0.001, one-way ANOVA followed by Tukey test).
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Fig1: CD2AP shRNAs decreased extracellular Aβ levels in cell culture. N2a-695 cells were transfected with CD2AP shRNAs or control shRNA. (A-C) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell culture medium. (D-F) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell lysates (n = 5/group; *, p < 0.05, **, p < 0.01, ***, p < 0.001, one-way ANOVA followed by Tukey test).

Mentions: APP processing is regulated by endocytosis. Given that CD2AP plays an important role in regulating endocytosis, we first tested whether CD2AP has an effect on Aβ synthesis or Aβ release in cultured cells. We used CD2AP shRNA to knockdown CD2AP levels (Figures 1, 2A) in neuroblastoma N2a-APP 695 cells and measured Aβ in the cell lysates and Aβ released into the cell culture medium. The results showed that CD2AP sh RNA1 (Sh1) significantly decreased both Aβ40 and Aβ42 secreted into the culture medium by about 20 ~ 30% (Figure 1A,B) while the Aβ40 and Aβ42 in cell lysates were not affected (Figure 1D,E). CD2AP sh2 had greater effects on the Aβ42 in the medium as compared to CD2AP sh1 (Figure 1A,B). The Aβ40 and Aβ42 in cell lysates were increased about 30% by CD2AP sh2 (Figure 1D,E). Interestingly, both CD2AP sh1 and CD2AP sh2 decreased the Aβ42/ Aβ40 ratio in cell culture medium (Figure 1C). However, the Aβ42/ Aβ40 ratios in cell lysates were unaltered (Figure 1F). We further examined the total APP and APP on the cell surface in these N2a cells. The results showed that CD2AP sh1 and sh2 did not change total APP levels in N2a cells (Figure 2B,D). However, membrane APP levels were decreased by CD2AP sh1 and sh2 (Figure 2B,C). In cells, nascent APP is post-translationally modified and transported from the endoplasmic reticulum to the plasma membrane [15]. To be proteolytically cleaved into Aβ, APP must be internalized from the cell surface into the cell and transported to endosomes where β-secretase and γ-secretase complexes cleave APP to produce Aβ [7,16]. In the current study, knocking down CD2AP in N2a-APP695 cells may decrease APP on cell surface which would result in less APP getting into endosomes and less Aβ being released into cell culture medium.Figure 1


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)

CD2AP shRNAs decreased extracellular Aβ levels in cell culture. N2a-695 cells were transfected with CD2AP shRNAs or control shRNA. (A-C) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell culture medium. (D-F) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell lysates (n = 5/group; *, p < 0.05, **, p < 0.01, ***, p < 0.001, one-way ANOVA followed by Tukey test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: CD2AP shRNAs decreased extracellular Aβ levels in cell culture. N2a-695 cells were transfected with CD2AP shRNAs or control shRNA. (A-C) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell culture medium. (D-F) Aβ40, Aβ42 and Aβ42/Aβ40 ratio in the cell lysates (n = 5/group; *, p < 0.05, **, p < 0.01, ***, p < 0.001, one-way ANOVA followed by Tukey test).
Mentions: APP processing is regulated by endocytosis. Given that CD2AP plays an important role in regulating endocytosis, we first tested whether CD2AP has an effect on Aβ synthesis or Aβ release in cultured cells. We used CD2AP shRNA to knockdown CD2AP levels (Figures 1, 2A) in neuroblastoma N2a-APP 695 cells and measured Aβ in the cell lysates and Aβ released into the cell culture medium. The results showed that CD2AP sh RNA1 (Sh1) significantly decreased both Aβ40 and Aβ42 secreted into the culture medium by about 20 ~ 30% (Figure 1A,B) while the Aβ40 and Aβ42 in cell lysates were not affected (Figure 1D,E). CD2AP sh2 had greater effects on the Aβ42 in the medium as compared to CD2AP sh1 (Figure 1A,B). The Aβ40 and Aβ42 in cell lysates were increased about 30% by CD2AP sh2 (Figure 1D,E). Interestingly, both CD2AP sh1 and CD2AP sh2 decreased the Aβ42/ Aβ40 ratio in cell culture medium (Figure 1C). However, the Aβ42/ Aβ40 ratios in cell lysates were unaltered (Figure 1F). We further examined the total APP and APP on the cell surface in these N2a cells. The results showed that CD2AP sh1 and sh2 did not change total APP levels in N2a cells (Figure 2B,D). However, membrane APP levels were decreased by CD2AP sh1 and sh2 (Figure 2B,C). In cells, nascent APP is post-translationally modified and transported from the endoplasmic reticulum to the plasma membrane [15]. To be proteolytically cleaved into Aβ, APP must be internalized from the cell surface into the cell and transported to endosomes where β-secretase and γ-secretase complexes cleave APP to produce Aβ [7,16]. In the current study, knocking down CD2AP in N2a-APP695 cells may decrease APP on cell surface which would result in less APP getting into endosomes and less Aβ being released into cell culture medium.Figure 1

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