<i>Cx3cr1</i> promotes Aβ(1–42)-induced cytotoxicity, preferentially in hippocampal neurons.

<p>(A) Aβ(1–42)-induced cell loss, measured by β-tubulin staining patterns of mature microglia-depleted cultured hippocampal neurons from C57Bl/6 (WT) or <i>Cx3cr1</i>^<i>-/-</i> (KO) mice. Scale bar, 10 μm. (B) Aβ(1–42)-induced cytotoxicity, quantified biochemically as % of maximal LDH release in microglial-depleted hippocampal and mixed cortical neurons. Maximum LDH release was defined using control neurons, or microglia respectively, treated with 2% Triton X-100. Results are summarized as the mean ± SEM of cumulative data from 7 separate experiments repeated in triplicate for each genotype. **** signifies <i>P</i> = 0.0001, two-way ANOVA. (C) Aβ(1–42)-induced cell loss, measured by tubulin staining patterns of pure microglia. Scale bar, 10 μm. (D) Aβ(1–42)-induced cytotoxicity, quantified biochemically as % of maximal LDH release in pure microglia cultures. Maximum LDH release was defined using control neurons, or microglia respectively, treated with 2% Triton X-100. Results are summarized as mean ± SEM of cumulative data from 7 separate experiments repeated in triplicate for each genotype. ****<i>P</i> = 0.0001, Mann-Whitney.</p

Aβ-induced mEPSC depression in microglia-depleted mature <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons.

<p>¹ mEPSC frequency units are events/s (Hz).</p><p>² mEPSC amplitude units are pico-amperes (pA).</p><p>³ mEPSC tau decay time constant units are milliseconds (ms).</p><p>Aβ-induced mEPSC depression in microglia-depleted mature <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons.</p

Preparation and maintenance of mature hippocampal cultures results in glial depletion at DIV21.

<p>Hippocampal primary neurons prepared from C57Bl/6 mice on P0 using a densitometric gradient centrifugation protocol were treated with 2 μM cytosine arabinoside (AraC) on DIV3 to enrich for neurons and deplete microglia. Prior to treatment at DIV3, GFAP+ cell contamination (A) was 21% ± 5 and IBA1+ cell contamination (B) was < 0.003%. At DIV 21, GFAP+ cell contamination was 6% ± 0.5 and IBA1+ cell contamination was < 0.003%. Microglial depletion was confirmed by staining for Cd11b (data not shown). <i>n</i> = 3 experiments in each sample category, with 300 cells/experiment. Scale bars, 10 μm.</p

Aβ (1–42) aggregation kinetics are dictated by peptide source, concentration, and preparation protocol.

<p>(A) Experimental aggregation kinetics of synthetic and recombinant Aβ(1–42) as measured by ThT fluorescence intensity. 2 μM synthetic Aβ(1–42): non-HFIP (solid dark blue circles), HFIP Aβ (1–42) (solid dark green circles); 2 μM recombinant met-Aβ (1–42): non-HFIP (open dark blue circles), HFIP (open dark green circles). 200 nM synthetic Aβ(1–42): non-HFIP (solid light blue circles), HFIP (solid light green circles); 200 nM recombinant met-Aβ (1–42): non-HFIP (open light blue circles), HFIP (open light green circles). Graphed data represent average of 3 experiments conducted in triplicate. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t001" target="_blank">Table 1</a> for half-times and errors. (B) AFM images of non-HFIP and HFIP-monomerized synthetic Aβ(1–42). Panel scale bars, 4 μm. Inset scale bars, 400 nm. (C) AFM images of non-HFIP and HFIP-monomerized recombinant met-Aβ(1–42). Panel scale bars, 4 μm. Inset scale bars, 400 nm.</p

Aggregation state of synthetic Aβ(1–42) determines its ability to depress pre- or postsynaptic components of mEPSCs, and <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons are resistant to both effects.

<p>Pre- and post-synaptic components of mEPSCs are measured as mEPSC frequency and amplitude, respectively. (A inset) Recorded mEPSCs were abolished by 10 μM CNQX Arrow indicates time of CNQX administration. (A) Non-HFIP synthetic Aβ(1–42) in wild-type hippocampal neurons. (B) HFIP synthetic Aβ(1–42) in wild-type hippocampal neurons. (C) Non-HFIP synthetic Aβ(1–42) in <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons. (D) HFIP synthetic Aβ(1–42) in <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons. For (A) through (D): the top three tracings are representative tracings of multiple mEPSCs recorded in the conditions indicated above each tracing. The single mEPSC is a representative tracing of the average amplitude, rise- and decay-time of recordings from 6 independent experiments, each of which averaged recordings from 3 separate coverslips. Cumulative probabilities represent averages of all 6 experiments. For the bottom three graphs: control, solid black line; 200 nM Aβ(1–42), light blue line (A) and (D), and light green line (B) and (D); 2 μM Aβ(1–42), dark blue line (A) and (C) and dark green line (B) and (D). For (A)–(D), the scale bars for multiple mEPSC tracings: vertical, 20 pA and horizontal, 1 s; for multiple mEPSCs treated with CNQX: vertical, 50 pA and horizontal, 1 min; and for single mEPSC tracings: vertical, 20 pA and horizontal, 6 ms. See Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t003" target="_blank">3</a> for all parameters and <i>P</i> values. See Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.g006" target="_blank">6E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.g006" target="_blank">6F</a> for summary of comparison between parameters of wild-type and Cx3cr1-/- hippocampal neurons.</p

Aggregation kinetics of synthetic and recombinant Aβ (1–42) and Aβ (1–40).

<p>¹ T_1/2 beyond experimental window of 22 hours were not assessed</p><p>² Rec. met-Aβ refers to the recombinant peptide (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#sec002" target="_blank">methods</a> for full description).</p><p>³ IPP refers to peptide addition immediately post-purification without any pre-treatment preparation</p><p>⁴ All samples were aggregated in aCSF per materials and methods except for those denoted as prepared in sodium-phosphate (NaP) buffer</p><p>⁵ Sonication of Aβ (1–40) for 10 minutes in aCSF prior to addition to neuronal culture does not significantly change aggregation kinetics of the Aβ (1–40) monomer directly after purification</p><p>Aggregation kinetics of synthetic and recombinant Aβ (1–42) and Aβ (1–40).</p

Monomeric recombinant met-Aβ(1–40) does not suppress spontaneous AMPAR-dependent mEPSCs.

<p>(A) 2 μM non-HFIP-monomerized met-Aβ(1–40) (dashed black line), 2 μM HFIP-monomerized met-Aβ(1–40) (dashed light green line), and 2 μM direct met-Aβ(1–40) monomer (dashed light blue line) administration do not significantly alter amplitude, frequency, or decay times of AMPAR-dependent mEPSCs. Solid lines, wild type control per condition. The top five tracings are representative tracings of multiple mEPSCs recorded in the conditions indicated above each tracing. The single mEPSC is representative tracing of the average amplitude, rise- and decay-time of recordings from 6 independent experiments, each of which averaged recordings from 3 separate coverslips. Cumulative probabilities represent averages of all 6 experiments. (B) Comparative plots of mEPSCs after 2 μM non-HFIP-monomerized met-Aβ(1–40), 2 μM HFIP-monomerized met-Aβ(1–40), and 2 μM direct met-Aβ(1–40) monomer administration. Kolmogorov-Smirnov test for normality of data distribution. One-way ANOVA, post-hoc Bonferroni. (C) Recombinant met-Aβ(1–40) does not demonstrate aggregation by increased THT fluorescence until 15 hours of incubation under experimental conditions. T_1/2 for maximal THT fluorescence occurs at 16.1 ± 0.09 hours for 2 μM non-HFIP prepared met-Aβ (1–40) (light purple). 2 μM HFIP-prepared met-Aβ (1–40) (dark purple) does not demonstrate increased THT fluorescence in the experimental window of 22 hours. No 200 nM met-Aβ (1–40) demonstrates fibril formation. 200 nM met-Aβ (1–40), non-HFIP-monomerized (dark blue), HFIP-monomerized (light green). (D) Artificial CSF accelerates monomeric met-Aβ(1–40) aggregation as determined by increased ThT fluorescence but not during the experimental window for neuron treatment. T1/2 to maximal ThT fluorescence is 16.01 ± 0.01 hours for 2 μM met-Aβ(1–40) (red) purified monomer added to neurons immediately following purification. 200 nM met-Aβ(1–40) (orange) in artificial CSF does not demonstrate fibril formation. Purified recombinant met-Aβ(1–40) monomers in sodium-phosphate (NaP) buffer do not exhibit fibril formation, 200 nM (black), 2 μM (yellow).</p

Aggregation state of recombinant met-Aβ(1–42) determines its ability to depress pre- or postsynaptic components of mEPSCs, and <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons are resistant to both effects.

<p>(A) Non-HFIP recombinant met-Aβ(1–42) in wild-type hippocampal neurons. (B) HFIP recombinant met-Aβ(1–42) in wild-type hippocampal neurons. (C) Non-HFIP recombinant met-Aβ(1–42) in <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons. (D) HFIP recombinant met-Aβ(1–42) in <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons. For (A) through (D): the top three tracings are representative tracings of multiple mEPSCs recorded in the conditions indicated above each tracing. The single mEPSC is representative tracing of the average amplitude, rise- and decay-time of recordings from 6 independent experiments, each of which averaged recordings from 3 separate coverslips. Cumulative probabilities represent averages of all 6 experiments. For the bottom three graphs: control, solid black line; 200 nM Aβ(1–42), light blue line (A) and (D) and light green line (B) and (D); 2 μM Aβ(1–42), dark blue line (A) and (C) and dark green line (B) and (D). Recorded mEPSCs were abolished by 10 μM CNQX. For (A) through (D), the scale bars for multiple mEPSC tracings are: vertical, 20 pA and horizontal, 1 s; for multiple mEPSCs treated with CNQX: vertical, 50 pA and horizontal, 1 min; and for single mEPSC tracings: vertical, 20 pA and horizontal, 6 ms. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t002" target="_blank">Table 2</a> for all parameters and P values. (E) Summary of mEPSC amplitudes and frequencies for synthetic (solid black circles) and recombinant (solid gray circles) Aβ(1–42)-treated wild-type neurons. (F) Summary of mEPSC amplitudes and frequencies for synthetic (solid black circles) and recombinant (solid gray circles) Aβ(1–42)-treated <i>Cx3cr1</i>^<i>-/-</i> neurons. Data were inspected for normality of distribution of frequency and amplitude values by the Kolmogorov-Smirnov test, analyzed for statistical significance by two-way ANOVA by genotype and treatment condition, with posthoc Bonferroni correction for multiple testing. Aβ concentrations and preparation protocols are indicated on the x-axes. Control, incubated with aCSF only. Data represent mean ± SEM of 6 independent experiments in each condition. <i>P</i> values for difference in parameters after treatment with synthetic (s) or recombinant (r) Aβ are shown. See Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t003" target="_blank">3</a> for all parameters.</p

<i>Cx3cr1</i> knockout decreases baseline mEPSC amplitude and increases mEPSC frequency in hippocampal neurons.

<p>(A) Non-HFIP control aCSF in wild-type and <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons. (B) HFIP control artificial CSF in wild-type and <i>Cx3cr1</i>^<i>-/-</i> hippocampal neurons. Cumulative probabilities represent averages of 6 experiments. Wild-type, black line; <i>Cx3cr1</i>^<i>-/-</i>, red line. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127730#pone.0127730.t002" target="_blank">Table 2</a> for all parameters. Data were inspected for normality of distribution of frequency values by the Kolmogorov-Smirnov test, analyzed for statistical significance by two-way ANOVA by genotype and treatment condition, with post-hoc Bonferroni correction for multiple comparisons.</p

Aβ-induced mEPSC depression in microglia-depleted mature wild type hippocampal neurons.

<p>¹ mEPSC frequency units are events/s (Hz).</p><p>² mEPSC amplitude units are pico-amperes (pA).</p><p>³ mEPSC tau decay time constant units are milliseconds (ms).</p><p>Aβ-induced mEPSC depression in microglia-depleted mature wild type hippocampal neurons.</p