Correction to Supporting Information for Talantova et al., Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss

NEUROSCIENCE Correction to Supporting Information for “Aβ induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss,” by Maria Talantova, Sara Sanz-Blasco, Xiaofei Zhang, Peng Xia, Mohd Waseem Akhtar, Shu-ichi Okamoto, Gustavo Dziewczapolski, Tomohiro Nakamura, Gang Cao, Alexander E. Pratt, Yeon-Joo Kang, Shichun Tu, Elena Molokanova, Scott R. McKercher, Samuel Andrew Hires, Hagit Sason, David G. Stouffer, Matthew W. Buczynski, James P. Solomon, Sarah Michael, Evan T. Powers, Jeffery W. Kelly, Amanda Roberts, Gary Tong, Traci Fang-Newmeyer, James Parker, Emily A. Holland, Dongxian Zhang, Nobuki Nakanishi, H.-S. Vincent Chen, Herman Wolosker, Yuqiang Wang, Loren H. Parsons, Rajesh Ambasudhan, Eliezer Masliah, Stephen F. Heinemann, Juan C. Piña-Crespo, and Stuart A. Lipton, which appeared in issue 27, July 2, 2013, of Proc Natl Acad Sci USA (110: E2518–E2527; first published June 17, 2013; 10.1073/pnas.1306832110).

The authors note that Fig. S1 and its corresponding legend appeared incorrectly. This error does not affect the conclusions of the article. The online version has been corrected.

Fig. S1.

Aβ preparations and effect on astrocyte glutamate release. (A) Regularization analysis representing the size distribution of particles in synthetic Aβ_1–42 preparations, as measured by dynamic light scattering. Peaks represent different-sized particles after Aβ_1–42 was oligomerized (Materials and Methods). The peaks at 1.35 ± 0.15 nm and 4.49 ± 0.66 nm likely represent residual monomers, and these peaks together comprise 1.1% of the total light scattering intensity. The peak at 18.2 ± 3.7 nm, likely representing small oligomers of Aβ_1–42, comprises ∼5% of the total light scattering intensity and ∼15% of the soluble fraction (which was the fraction used in the physiological experiments in the present study). The peak located at just under 100 nm likely represents soluble amyloid fibrils. The soluble fraction, obtained after centrifugation no longer contained peaks at >100 nm, which likely represent large amyloid fibrils. (B) Immunoblot characterization of monomeric and oligomeric synthetic Aβ_1–42 peptides under denaturing (SDS) conditions. Monomeric (Left; Mono) or oligomeric (Right; Olig) Aβ_1–42 peptides (∼88 ng each) were electrophoresed and immunoblotted using clone 6E10 monoclonal antibody against Aβ. The monomeric Aβ preparation revealed only one predominant immunoreactive band corresponding to the size of Aβ monomer. The oligomeric Aβ preparation displayed multiple immunoreactive bands corresponding to the size of Aβ monomers (M), dimers (D), trimers (T), and dodecamers (56 kDa). Relative molecular masses (kDa) are shown at left. (C) Naturally-occurring oligomeric Aβ detected in brain extracts by immunoblot analysis of postmortem human brains with late-onset Alzheimer’s disease. (Left) Control IgG antibody pull down. (Right) Aβ 82E1 monoclonal antibody pull down. By this analysis, primarily dimers and trimers of Aβ_1–42 were observed. (D) Glutamate release induced by low concentrations of synthetic Aβ_1–42 oligomers. Normalized ratio of FRET intensity induced by 325 pM of oligomeric Aβ_1–42 represents glutamate release from purified rat astrocytes. Glutamate standard (Glu) represents the nearly saturated FRET response to ≥30 µM glutamate. Data are shown as mean ± SEM; n = 32 cells analyzed in 5 experiments. (E) HPLC measurements of glutamate released from mouse astrocyte cultures incubated with Aβ oligomers. Astrocytes were incubated with 250 nM oligomeric Aβ_1–42 for 30 min. Medium was then collected and filtered, and HPLC measurements performed. n = 9 samples analyzed; *P < 0.05). (F) Aβ-induced glutamate release from astrocytes is not dependent on microglia/macrophages. Synthetic oligomeric Aβ_25–35 (250 nM) in the presence of l-leucine methyl ester (7.5 mM for 24 h) to deplete monocytoid cells still induced release of glutamate in purified rat astrocyte cultures, as monitored by measurement of the normalized FRET ratio. Glutamate standard (Glu) represents the nearly-saturated FRET response to ≥30 µM glutamate. Values are mean ± SEM; n = 24 cells analyzed in three experiments. (G) Aβ_25–35 (10 µM) peptide induces glutamate release from purified rat astrocytes by measurement of the normalized FRET ratio. Glutamate standard (Glu) represents the nearly-saturated FRET response to ≥30 μM glutamate. Data are shown as mean ± SEM; n = 61 cells analyzed in six experiments. (H) Oligomeric Aβ_1–42 (500 nM) induced glutamate release from purified astrocytes in the presence of the metabotropic glutamate receptor antagonist (S)-α-methyl-4-carboxyphenylglycine (S-MCPG; 500 µM). Data are shown as mean ± SEM; n = 15 cells analyzed in three experiments. (I) In vivo microdialysis showed that basal levels of extracellular glutamate were higher in the hippocampus of ∼12-mo-old transgenic mice overexpressing human APP (hAPP tg) than in age-matched WT mice (mean + SEM; n = 4; *P < 0.05). (J) Memantine (10 µM) manifests a minor effect on glutamate release from astrocytes induced by 250 nM oligomeric Aβ_1–42, as monitored by the FRET probe. The figure shows the area under the curve for FRET ratios for mouse astrocytes exposed to oligomerized Aβ_1–42 alone (n = 19 cells) or to Aβ_1–42 in the presence of 10 µM memantine (n = 12 cells). Values represent mean + SEM from three different experiments for each condition. FRET ratios were normalized to 1 and plotted as a function of time for the duration of the response. Although there was a trend for memantine to decrease glutamate release in the face of Aβ_1–42 exposure, it did not reach statistical significance. n.s., not significant.