Differential regulation of Abeta42-induced neuronal C1q synthesis and microglial activation - PubMed (original) (raw)

Differential regulation of Abeta42-induced neuronal C1q synthesis and microglial activation

Rong Fan et al. J Neuroinflammation. 2005.

Abstract

Expression of C1q, an early component of the classical complement pathway, has been shown to be induced in neurons in hippocampal slices, following accumulation of exogenous Abeta42. Microglial activation was also detected by surface marker expression and cytokine production. To determine whether C1q induction was correlated with intraneuronal Abeta and/or microglial activation, D-(-)-2-amino-5-phosphonovaleric acid (APV, an NMDA receptor antagonist) and glycine-arginine-glycine-aspartic acid-serine-proline peptide (RGD, an integrin receptor antagonist), which blocks and enhances Abeta42 uptake, respectively, were assessed for their effect on neuronal C1q synthesis and microglial activation. APV inhibited, and RGD enhanced, microglial activation and neuronal C1q expression. However, addition of Abeta10-20 to slice cultures significantly reduced Abeta42 uptake and microglial activation, but did not alter the Abeta42-induced neuronal C1q expression. Furthermore, Abeta10-20 alone triggered C1q production in neurons, demonstrating that neither neuronal Abeta42 accumulation, nor microglial activation is required for neuronal C1q upregulation. These data are compatible with the hypothesis that multiple receptors are involved in Abeta injury and signaling in neurons. Some lead to neuronal C1q induction, whereas other(s) lead to intraneuronal accumulation of Abeta and/or stimulation of microglia.

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Figures

Figure 1

Figure 1

APV inhibited Aβ uptake, neuronal C1q production, and microglial activation. Slices were treated with no peptide (a, b, c), 30 μM Aβ 42 (d, e, f), or 30 μM Aβ 42 + 50 μM APV (g, h, i) for 3 days with fresh reagents added daily. Immunohistochemistry for Aβ (4G8, a, d, g), C1q (anti-rat C1q, b, e, h), and microglia (CD45, c, f, i) was performed on fixed and sectioned slices. Scale bar = 50 μm. Results are representative of three separately performed experiments. j. Immunoreactivity of Aβ (open bar), C1q (black bar), or CD45 (striped bar) was quantified as described in Materials and Methods. Values are the mean ± SD (error bars) from images taken from 8 slices (2 sections per slice) in 3 independent experiments (* p < 0.0001 compared to Aβ, Anova single factor test).

Figure 2

Figure 2

Inhibition of Aβ-induced C1q synthesis by APV. a. C1q and β-actin mRNAs were assessed by RT-PCR in slices after 3 days of no peptide, 30 μM Aβ, or 30 μM Aβ + 50 μM APV treatment. Results are from one experiment representative of two independent experiments. b. Slices were treated with no peptide (open bar), 30 μM Aβ (black bar), or 30 μM Aβ + 50 μM APV (striped bar) daily for 3 days. 3 or 4 slices that had received same treatment were pooled, extracted and proteins analyzed by ELISA. Data are presented as percentage of control in ng C1q/mg total protein (mean ± SD of three independent experiments, **p = 0.01 compared to Aβ, one-tailed paired t-test).

Figure 3

Figure 3

RGD enhanced Aβ uptake, neuronal C1q expression, and microglial activation. Hippocampal slices were treated with no peptide (a, b, c), 10 μM Aβ 42 (d, e, f), or 10 μM Aβ 42 + 2 mM RGD (g, h, i) for 3 days with fresh peptides added daily. Immunohistochemistry for Aβ (4G8, a, d, g), C1q (anti-rat C1q, b, e, h), and microglia (CD45, c, f, i) was performed on fixed slice sections. Scale bar = 50 μm. Results are representative of three separately performed experiments. j. Immunoreactivities of Aβ (open bar), C1q (black bar), or CD45 (striped bar) were quantified as described in Materials and Methods. Values are the mean ± SD (error bars) from images taken from 8 slices (2 sections per slice) in 3 independent experiments (* p < 0.0001, compared to Aβ, Anova single factor test).

Figure 4

Figure 4

Enhancement of Aβ-induced C1q synthesis by RGD. a. C1q and β-actin mRNAs were assessed by RT-PCR in slices after 3 days of no peptide, 10 μM Aβ, or 10 μM Aβ + 2 mM RGD treatment. Results are from one experiment representative of two independent experiments. b. Slices were treated with no peptide (open bar), 10 μM Aβ (black bar), or 10 μM Aβ + 2 mM RGD (striped bar) daily for 3 days. 3 or 4 slices that had received same treatment were pooled, extracted and proteins analyzed by ELISA. Data are presented as percentage of control in ng C1q/mg total protein (mean ± SD of three independent experiments, **p = 0.06 compared to Aβ, one-tailed paired t-test).

Figure 5

Figure 5

Aβ10–20 blocked Aβ42 uptake, microglial activation, but not neuronal C1q induction. Slices were treated with no peptide (a, b, c), 10 μM Aβ 42 (d, e, f), 10 μM Aβ 42 + 30 μM Aβ 10–20 (g, h, i) or 30 μM Aβ 10–20 (j, k, l) for 3 days with fresh peptides added daily. Immunohistochemistry for Aβ (4G8, a, d, g, j), C1q (anti-rat C1q, b, e, h, k), and microglia (CD45, c, f, i, l) was performed on fixed and sectioned slices. Results are representative of three independent experiments. Scale bar = 50 μm. m. Immunoreactivities of Aβ (open bar), C1q (black bar), or CD45 (striped bar) were quantified as described in Materials and Methods. Values are the mean ± SD (error bars) from images taken from 8 slices (2 sections per slice) in 3 independent experiments. Microglial activation by Aβ42 was significantly inhibited by Aβ10–20 (* p < 0.0001, compared to either Aβ42 + Aβ10–20 or Aβ10–20, Anova single factor test).

Figure 6

Figure 6

a. Aβ10–20 inhibited Aβ42-induced C1q and CD40 mRNA elevation, but not that of MCSF. C1q, MCSF, CD40, and β-actin mRNAs were assessed by RT-PCR in slices treated for 3 days with no peptide, 10 μM Aβ 42, 30 μM Aβ 10–20, or 10 μM Aβ 42 + 30 μM Aβ 10–20. Results are from one experiment representative of two independent experiments. b. APV blocked MCSF, CD40, and IL-8 mRNA induction triggered by Aβ42. RT-PCR for MCSF, CD40, IL-8, and β-actin were performed on RNA extracted from slices treated with no peptide (control), 30 μM Aβ 42, or 30 μM Aβ42 + 50 μM APV for 3 days. Results are from one experiment representative of two separate experiments.

Figure 7

Figure 7

Model of Aβ interaction with neurons and microglia in slice cultures. Exogenous Aβ peptide interacts with neuronal receptors leads to at least two separate consequences, in one of which C1q expression is upregulated in neurons. A second receptor mediates the secretion of certain modulatory molecules, which lead to microglial activation involving the expression of CD45, CR3, CD40, and IL-8. This does not exclude the direct interactions of Aβ with receptor(s) on microglia that may also contribute to microglial activation.

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