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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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Effects of CD2AP haploinsufficiency on Aβ plaque deposition 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. (A) Representative images for Aβ staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (B) Quantification of Aβ plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex. (C) Representative images of Thioflavin S staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (D) Quantification of fibrillar plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex.
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Fig6: Effects of CD2AP haploinsufficiency on Aβ plaque deposition 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. (A) Representative images for Aβ staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (B) Quantification of Aβ plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex. (C) Representative images of Thioflavin S staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (D) Quantification of fibrillar plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex.

Mentions: To determine whether CD2AP haploinsufficiency affects the morphology or other properties of the Aβ plaques, we stained the tissue with biotinylated anti-Aβ1–13 monoclonal antibody HJ3.4B (Figure 6A) or Thioflavin S (Figure 6C) which stains fibrillar forms of Aβ plaques. We did not observe any significant changes in plaque distribution, individual plaque size or plaque morphology associated with CD2AP gene status. We further quantified the% area covered with plaques in the cortex. The results demonstrated that neither Aβ immunostained plaques (Figure 6B) nor fibrillar plaques (Figure 6D) were different in mice with CD2AP haploinsufficiency. Taken together, although CD2AP haploinsufficiency lowered the Aβ42/Aβ40 ratio in PBS fraction of female PS1APP mice, the majority of our data demonstrate that CD2AP haploinsufficiency did not cause changes in Aβ accumulation.Figure 6


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 Aβ plaque deposition 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. (A) Representative images for Aβ staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (B) Quantification of Aβ plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex. (C) Representative images of Thioflavin S staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (D) Quantification of fibrillar plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex.
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Related In: Results  -  Collection

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Fig6: Effects of CD2AP haploinsufficiency on Aβ plaque deposition 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. (A) Representative images for Aβ staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (B) Quantification of Aβ plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex. (C) Representative images of Thioflavin S staining in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex (Scale bar =400 μm). (D) Quantification of fibrillar plaque load in PS1APP/CD2AP+/+ and PS1APP/CD2AP+/− cortex.
Mentions: To determine whether CD2AP haploinsufficiency affects the morphology or other properties of the Aβ plaques, we stained the tissue with biotinylated anti-Aβ1–13 monoclonal antibody HJ3.4B (Figure 6A) or Thioflavin S (Figure 6C) which stains fibrillar forms of Aβ plaques. We did not observe any significant changes in plaque distribution, individual plaque size or plaque morphology associated with CD2AP gene status. We further quantified the% area covered with plaques in the cortex. The results demonstrated that neither Aβ immunostained plaques (Figure 6B) nor fibrillar plaques (Figure 6D) were different in mice with CD2AP haploinsufficiency. Taken together, although CD2AP haploinsufficiency lowered the Aβ42/Aβ40 ratio in PBS fraction of female PS1APP mice, the majority of our data demonstrate that CD2AP haploinsufficiency did not cause changes in Aβ accumulation.Figure 6

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