Lipocalin 2: novel component of proinflammatory signaling in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is associated with an altered immune response, resulting in chronic increased inflammatory cytokine production with a prominent role of TNF-α. TNF-α signals are mediated by two receptors: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Signaling through TNFR2 is associated with neuroprotection, whereas signaling through TNFR1 is generally proinflammatory and proapoptotic. Here, we have identified a TNF-α-induced proinflammatory agent, lipocalin 2 (Lcn2) via gene array in murine primary cortical neurons. Further investigation showed that Lcn2 protein production and secretion were activated solely upon TNFR1 stimulation when primary murine neurons, astrocytes, and microglia were treated with TNFR1 and TNFR2 agonistic antibodies. Lcn2 was found to be significantly decreased in CSF of human patients with mild cognitive impairment and AD and increased in brain regions associated with AD pathology in human postmortem brain tissue. Mechanistic studies in cultures of primary cortical neurons showed that Lcn2 sensitizes nerve cells to β-amyloid toxicity. Moreover, Lcn2 silences a TNFR2-mediated protective neuronal signaling cascade in neurons, pivotal for TNF-α-mediated neuroprotection. The present study introduces Lcn2 as a molecular actor in neuroinflammation in early clinical stages of AD.

Figure 1.

Regulatory network for TNF-α-dependent transcripts in mouse cortical neurons. Transcripts with an abundance of 2-fold or greater in TNF-α-treated (24 h) compared with control treated primary cortical neurons by 2-color microarray analysis (10 transcripts; see Table 2) were used as the input for analysis of potential networks using the signal transduction knowledge environment of IPA. Network involves 9 TNF-α-regulated transcripts and 4 additional components with IPA-confirmed direct or indirect links to them. TNF-α-regulated transcripts are depicted in gray, linked components in white. Circular lines above symbols indicate autoregulation, connecting lines without arrows indicate direct protein interaction, and dashed and solid arrows indicate indirect (e.g., regulation of mRNA levels) and direct (e.g., enzymatic) activation.

Figure 2.

TNF-α-mediated Lcn2 production in primary neuronal cells. A–C) Expression of intracellular and secreted Lcn2 protein levels in primary cortical neurons (A), astrocytes (B), and microglia (C) at different time intervals of TNF-α (100 ng/ml) treatment was determined by Western blot. D–F) Effect of TNFR1- and TNFR2-mediated signaling on Lcn2 expression in primary cortical neurons (D), astrocytes (E), and microglia (F) was assessed by Western blot by treating the cells with either TNF-α (100 ng/ml), TNFR1 agonistic antibody (2 μg/ml), or TNFR2 agonistic antibody (20 μg/ml) for 36 h. Lcn2 expression was normalized to actin before the percentage relative to control was calculated. Bars indicate the mean ± se protein expression as a percentage relative to that for untreated controls. *P < 0.05; **P < 0.005; ***P < 0.0001.

Figure 3.

Expression of Lcn2 in human samples. A) Serum Lcn2 concentrations among control (n=26), MCI (n=28), and AD (n=26) groups. B) Differences in CSF Lcn2 concentrations among control (n=26), MCI (n=28), and AD (n=26) groups. Bars indicate mean ± se protein concentrations in the different study groups. *P < 0.05; **P < 0.005. C) Lcn2 expression in homogenated cerebellum, VMPC, occipital lobe, entorhinal cortex, and hippocampus of control (n=10) and AD (n=10) brain tissues, determined by Western blot analysis. Lcn2 expression was normalized to actin before the percentage relative to control was calculated. Bars indicate mean ± se ratio of protein expression in nondementia control and AD brain tissue. *P < 0.05; **P < 0.005. D) Immunohistochemical staining of Lcn2 of pyramidal neurons in the CA1 hippocampus region of a patient with AD compared with that of a control subject without dementia.

Figure 4.

Influence of Lcn 2 on the TNFR2-mediated signaling pathway in primary cortical neurons. Primary cortical neurons were incubated with TNF-α (100 ng/ml), TNFR1 agonistic antibody (2 μM), or TNFR2 agonistic antibody (20 μM) and also coincubated with recombinant murine Lcn2 (200 ng/ml) for 24 h. Western blotting was used to quantify protein expression. A) Neurons were analyzed for PKB/Akt phosphorylation at the Ser473 site. B) NF-κB activation was determined via analysis of phosphorylation of nuclear p65 at Ser536. C) Caspase 3 activation was determined by the ratio of cleaved caspase 3 levels to caspase 3 levels. Lcn2 mediated PTEN up-regulation. D) Intracellular PTEN expression in primary cortical neurons treated with murine Lcn2 (200 ng/ml) at different time intervals was determined via Western blot. E) Effect of Lcn2 on TNF-α-mediated PTEN regulation. F) Role of PTEN in Lcn2-mediated TNFR2 cosignaling-induced caspase 3 activation was assessed by pretreating the indicated neurons with PTEN inhibitor VO-OHpic for 1 h and subsequent incubation with either TNF-α (100 ng/ml) or TNFR2 agonistic antibody (20 μM) with or without Lcn2 (200 ng/ml) for 24 h. Measured protein expressions were normalized to actin before the percentage relative to control was calculated. Bars indicate mean ± se protein expression as a percentage relative to that of untreated control subjects. *P < 0.05; **P < 0.005; ***P < 0.0001.

Figure 5.

Effect of Lcn2 on TNF-α-mediated neuronal protection against Aβ and glutamate-induced neuron toxicity. A) Primary cortical neurons were preincubated for 24 h with TNF-α (100 ng/ml), TNFR2 agonistic antibody (20 μM), and Lcn2 (200 ng/ml) or TNF-α (100 ng/ml) and TNFR2 agonistic antibody (20 μM) together with Lcn2 (200 ng/ml). After the 24-h incubation period, Aβ_1–42 (25 μM) was added, and cell viability was determined 24 h later with the MTT assay. B) Neurons were also pretreated as indicated with TNF-α (100 ng/ml), Lcn2 (200 ng/ml), and TNFR2 agonistic antibody (20 μM) and/or TNF-α (100 ng/ml) and TNFR2 agonistic antibody (20 μM) together with Lcn2 (200 ng/ml) for 24 h and then challenged with 50 μM glutamate (concentration that induced ≈50% cell death) for 1 h. Neuronal viability was determined 24 h after the glutamate challenge using the MTT assay. C) Primary cortical neurons were preincubated with Lcn2 at different concentrations for 24 h, and Aβ_1–42 (25 μM) was then added, after which neuronal viability was determined using the MTT assay. 24p3R expression was normalized to actin before the percentage relative to control was calculated. Bars indicate mean ± se cell viability as a percentage relative to that for untreated control subjects. *P < 0.05; **P < 0.005; ***P < 0.0001. D) Expression of the Lcn2 receptor 24p3R in primary cortical neurons after a 24-h incubation with TNF-α. Bars indicate mean ± se protein expression as a percentage relative to that for the untreated control subjects. **P < 0.005.

Figure 6.

Lcn2-mediated sensitivity of neurons toward glutamate and Aβ_1–42 toxicity. Lcn2 expression is induced via TNFR1-specific signaling in primary cortical neurons, astrocytes, and microglia. Lcn2 can silence TNFR2-mediated phosphorylation of Akt and phosphorylation of p65 via the up-regulation of PTEN and therefore inhibit TNFR2-mediated neuroprotection against the AD-associated excitotoxic factors.