Identification and Characterization of Elevated Expression of Transferrin and Its Receptor TfR1 in Mouse Models of Depression

Abstract

Depression has become one of the severe mental disorders threatening global human health. In this study, we first used the proteomics approach to obtain the differentially expressed proteins in the liver between naive control and chronic social defeat stress (CSDS) induced depressed mice. We have identified the upregulation of iron binding protein transferrin (TF) in the liver, the peripheral blood, and the brain in CSDS-exposed mice. Furthermore, bioinformatics analysis of the Gene Expression Omnibus (GEO) database from various mouse models of depression revealed the significantly upregulated transcripts of TF and its receptor TfR1 in multiple brain regions in depressed mice. We also used the recombinant TF administration via the tail vein to detect its permeability through the blood-brain barrier (BBB). We demonstrated the permeability of peripheral TF into the brain through the BBB. Together, these results identified the elevated expression of TF and its receptor TfR1 in both peripheral liver and the central brain in CSDS-induced depressed mice, and peripheral administration of TF can be transported into the brain through the BBB. Therefore, our data provide a compelling information for understanding the potential role and mechanisms of the cross-talk between the liver and the brain in stress-induced depression.

Keywords: bioinformatics; blood-brain barrier (BBB); chronic social defeat stress (CSDS); depression; mental disorders; proteomics; transferrin (TF); transferrin receptor 1 (TfR1).

Figure 1

Chronic social defeat stress (CSDS) paradigm and depression-like phenotype in susceptible mice. (A) Experimental design of chronic social defeat stress (CSDS) using the resident intruder paradigm. (B) The social interaction ratio of susceptible mice compared to non-stressed controls (n = 19), p < 0.0001. (C) Representative trajectory heat maps of social interaction test between non-stressed controls (top) and susceptible mice (bottom). (D) The concentration of serum cortisol in non-stressed controls (n = 10) and susceptible mice (n = 12), p = 0.016. (E) The sucrose water preference test (SPT) in non-stressed controls (n = 12) and susceptible mice (n = 12), p = 0.0004. (F) The immobility time of the forced swimming test (FST) in non-stressed controls (n = 12) and susceptible mice (n = 12), p = 0.0059. (G) The immobility time of the tail-suspension test (TST) in non-stressed controls (n = 12) and susceptible mice (n = 12), p = 0.005. Unpaired t test. All data represent mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Identification of differential expression of protein in the livers in CSDS induced susceptible mice. (A) Schematic diagram of two-dimensional electrophoresis and mass spectrometry. (B) Representative 2-DE gel pattern of differentially expressed protein between non-stressed control and susceptible mice indicated by identical names, respectively. (C) qRT-PCR analysis of differentially expressed proteins at transcriptional level in liver RNA extractions from non-stressed controls (n = 3) and susceptible mice (n = 3). Hspa9: heat shock protein 9, Hspa5: heat shock protein 5, TF: transferrin, Cps1: carbamoyl phosphate synthase 1, Pnp: purine nucleoside phosphorylase, Hmgb1: High mobility group protein 1, Apcs: serum amyloid P component, PGM1: phosphoglucosidase-1, PMM2: phosphomannomutase 2, INMT: indoleethylamine N-methyltransferase. Unpaired t test. All data represent mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

The elevated expression of TF and TfR1 in peripheral tissue induced by CSDS. (A–C) Representative immunoblot and quantitative analysis showing the elevated expression of TF and its receptor TfR1 in susceptible liver lysate induced by CSDS, normalized to β-actin. CSDS induced the significantly upregulated expression of TF (p = 0.0040) and TfR1 (p = 0.0286) in liver in susceptible mice (n = 4) compared to non-stressed controls (n = 4). (D) Representative standard curve of TF ELISA detection kit. (E) The concentration of TF in liver lysates detected by ELISA kit from non-stressed controls (n = 10), resilient mice (n = 10), and susceptible mice (n = 10), (F = 6.934, p = 0.0037), multiple comparison (C-S, p = 0.0031). (F) The concentration of TF in the serum detected by ELISA kit from non-stressed controls (n = 10), resilient mice (n = 10), and susceptible mice (n = 10), (F = 14.9, p < 0.0001), multiple comparison (Control vs. Resilient, p = 0.0296; Control vs. Susceptible, p < 0.0001; Resilient vs. Susceptible, p = 0.0280). All data represent mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Delivery of exogenous TF peripherally across the blood brain barrier into the brain. (A) Schematic diagram of red transferrin conjugate delivery and imaging. (B) Red fluorescence signals were detected in multiple tissues including liver, heart, spleen, lung and kidney of injected mice (n = 3). (C) Red fluorescence signals were detected in the central brain including mPFC, vHip, dHip, Hb and BLA of injected mice (n = 3). mPFC, medial prefrontal cortex; vHip, ventral hippocampus; dHip, dorsal hippocampus; Hb: habenular; BLA, basolateral amygdala.

Figure 5

Elevated expression of TF in the mPFC and hippocampus induced by CSDS. (A–C) Representative immunoblot and quantitative analysis showing the elevated expression of TF and its receptor TfR1 in mPFC induced by CSDS, normalized to β-actin. CSDS induced the significantly upregulated expression of TF (p = 0.0179) and TfR1 (p = 0.0308) in mPFC in susceptible mice (n = 4) compared to non-stressed controls (n = 4). (D–F) Representative immunoblot and quantitative analysis showing the elevated expression of TF, but not its receptor TfR1 in the hippocampus induced by CSDS, normalized to β-actin. CSDS induced the significantly upregulated expression of TF (p = 0.0415) in the hippocampus in susceptible mice (n = 4) compared to non-stressed controls (n = 4). The expression of TfR1 did not change significantly (p = 0.6172) in the hippocampus in susceptible mice (n = 4) compared to non-stressed controls (n = 4). Unpaired t test. All data represent mean ± SEM. * p < 0.05, n.s., not significant.