Overexpression of Swedish mutant APP in aged astrocytes attenuates excitatory synaptic transmission

Abstract

Amyloid precursor protein (APP), a type I transmembrane protein, has different aspects, namely, performs essential physiological functions and produces β-amyloid peptide (Aβ). Overexpression of neuronal APP is responsible for synaptic dysfunction. In the central nervous system, astrocytes - a major glial cell type - have an important role in the regulation of synaptic transmission. Although APP is expressed in astrocytes, it remains unclear whether astrocytic overexpression of mutant APP affects synaptic transmission. In this study, the effect of astrocytic overexpression of a mutant APP on the excitatory synaptic transmission was investigated using coculture system of the transgenic (Tg) cortical astrocytes that express the human APP695 polypeptide with the double mutation K670N + M671L found in a large Swedish family with early onset Alzheimer's disease, and wild-type hippocampal neuron. Significant secretion of Aβ 1-40 and 1-42 was observed in cultured cortical astrocytes from the Tg2576 transgenic mouse that genetically overexpresses Swedish mutant APP. Under the condition, Tg astrocytes did not affect excitatory synaptic transmission of cocultured wild-type neurons. However, aged Tg astrocytes cultured for 9 weeks elicited a significant decrease in excitatory synaptic transmission in cocultured neurons. Moreover, a reduction in the number of readily releasable synaptic vesicles accompanied a decrease in the number of excitatory synapses in neurons cocultured with aged Tg astrocytes. These observations indicate that astrocytic expression of the mutant APP is involved in the downregulation of synaptic transmission with age.

Keywords: Aging; alzheimer's disease; amyloid; astrocyte; synaptic release.

Figure 1

Amyloid precursor protein (APP) is expressed in cultured cortical astrocytes from wild‐type (WT) and transgenic (Tg) 2576 mice. (A) Relative APP mRNA levels in the astrocytes from WT (9 cultures) and Tg2576 mice (7 cultures). Note that APP mRNA was overexpressed in Tg2576 astrocyte culture. (B) Western blot analysis in WT (4 cultures) or Tg2576 (4 cultures) astrocytes. Note that APP was overexpressed in Tg2576 astrocyte culture. (C) Quantification of Aβ 1–40 (WT: 7 cultures, Tg: 7 cultures) and Aβ 1–42 (WT: 8 cultures, Tg: 8 cultures) in cultured WT or Tg astrocyte‐conditioned medium. The data are shown as mean ± SEM.

Figure 2

Excitatory synaptic transmission is not affected by the transgenic (Tg) astrocyte coculture. (A) Representative traces of evoked excitatory postsynaptic currents (EPSCs) recorded from autaptic hippocampal neurons cocultured with wild‐type (WT) or Tg astrocytes. Depolarization artifacts caused by the generated action currents have been removed for clarity of presentation. (B) Average amplitudes of the evoked EPSCs in neurons cocultured with either WT (n = 176/10 cultures) or Tg astrocytes (n = 177/10 cultures). (C) Representative mEPSC traces in neurons cocultured with WT or Tg astrocytes. (D) An averaged mEPSC amplitude in neurons cocultured with either WT (n = 176/10 cultures) or Tg astrocytes (n = 176/10 cultures). (E) An averaged mEPSC frequency in neurons cocultured with either WT (n = 176/10 cultures) or Tg astrocytes (n = 176/10 cultures). The data are shown as mean  ± SEM.

Figure 3

Astrocytes cultured for long periods show detectable signs of aging. (A) Representative bright‐field images of WT and Tg2576 astrocytes mass‐cultured for 5 or 9 weeks in vitro. Scale bars represent 200 μm. (B) Normalized IL‐6 mRNA expression in large‐scale WT and Tg2576 astrocytes cultured for 5 or 9 weeks in vitro (8 cultures at each age). *P < 0.05, **P < 0.01 versus 5‐week WT astrocytes. The data are shown as mean ± SEM. (C) Quantification of Aβ 1–40 (WT: 5 cultures; Tg: 5 cultures) and Aβ 1–42 (WT: 5 cultures; Tg: 5 cultures) in the culture medium of aged WT or aged Tg astrocyte‐conditioned culture medium. The data are shown as mean ± SEM.

Figure 4

Excitatory synaptic transmission is attenuated in coculture with aged transgenic (Tg) astrocytes. (A) Representative traces of evoked excitatory postsynaptic currents (EPSCs) recorded from a single autaptic hippocampal neuron cocultured with either wild‐type (WT) or Tg astrocytes that had been cultured for 9 weeks. Depolarization artifacts caused by the generated action currents have been removed for clarity of presentation. (B) Average amplitudes of the evoked EPSCs in neurons cocultured with either aged WT (n = 139/9 cultures) or aged Tg astrocytes (n = 144/9 cultures). ***P < 0.001. (C) Representative mEPSC traces in neurons cocultured with either aged WT or aged Tg astrocytes. (D) An averaged mEPSC amplitude in neurons cocultured with either aged WT (n = 104/9 cultures) or aged Tg astrocytes (n = 120/9 cultures). (E) An averaged mEPSC frequency in neurons cocultured with either aged WT (n = 104/9 cultures) or aged Tg astrocytes (n = 120/9 cultures). *P <0.05. The data are shown as mean ±SEM.

Figure 5

The number of excitatory synapses is decreased in coculture with aged transgenic (Tg) astrocytes. (A) Representative traces of response to 0.5 mol/L hypertonic sucrose solution (5 sec, black bar) in an autapse neuron cocultured with either the aged wild‐type (WT) or aged Tg astrocytes. (B) Number of synaptic vesicles in readily releasable pool of autaptic neurons cocultured with either aged WT (n = 103/9 cultures) or aged Tg astrocytes (n = 117/9 cultures). (C) P_vr in single autaptic neurons cocultured with either aged WT (n = 135/9 cultures) or aged Tg astrocytes (n = 138/9 cultures). (D) Paired pulse ratio (EPSC ₂/EPSC ₁) in single autaptic neurons cocultured with either aged WT (n = 83/5 cultures) or aged Tg astrocytes (n = 86/5 cultures). (E) Representative images of autaptic neurons immunostained for the excitatory nerve terminal marker VGLUT 1 (red) and the dendritic marker MAP 2 (green). Parts of images in the top row (scale bar 50 μm) are enlarged in the bottom row (scale bar 5 μm). (F) The number of VGLUT 1–positive synaptic puncta in autaptic neurons cocultured with either aged WT (n = 44/4 cultures) or aged Tg astrocytes (n = 50/4 cultures). (G) An averaged size of synaptic puncta labeled with VGLUT 1 in individual autaptic neurons. The data were obtained from the samples used in (F). The data are shown as mean ± SEM.