LRRC25 expression during physiological aging and in mouse models of Alzheimer's disease and iPSC-derived neurons

Abstract

The leucine-rich repeat-containing protein 25 (LRRC25) is relatively a novel protein with no information on its role in neuronal or brain function. A recent study suggested LRRC25 is a potential risk factor for Alzheimer's disease (AD). As a first step to understanding LRRC25's role in the brain and AD, we found LRRC25 is expressed in both cell membranes and cytoplasm in a punctuate appearance in astrocytes, microglia, and neurons in cell lines as well as mouse brain. We also found that LRRC25 expression is both age- and brain region-dependent and that 1-day-old (1D) pups expressed the least amount of LRRC25 protein compared to adult ages. In the APΔE9 mice, immunoblot quantified LRRC25 protein levels were increased by 166% (**p < 0.01) in the cortex (CX) and by 215% (***p < 0.001) in the hippocampus (HP) relative to wild-type (WT) controls. Both the brainstem (BS) and cerebellum (CB) showed no significant alterations. In the 3xTg mice, only CX showed an increase of LRRC25 protein by 91% (*p < 0.05) when compared to WT controls although the increased trend was noted in the other brain regions. In the AD patient brains also LRRC25 protein levels were increased by 153% (***p < 0.001) when compared to normal control (NC) subjects. Finally, LRRC25 expression in the iPSC-derived neurons quantified by immunofluorescence was increased by 181% (**p < 0.01) in AD-derived neurons when compared to NC-derived neurons. Thus increased LRRC25 protein in multiple models of AD suggests that LRRC25 may play a pathogenic role in either Aβ or tau pathology in AD. The mechanism for the increased levels of LRRC25 in AD is unknown at present, but a previous study showed that LRRC25 levels also increase during neonatal hypoxic-ischemia neuronal damage. Based on the evidence that autophagy is highly dysregulated in AD, the increased LRRC25 levels may be due to decreased autophagic degradation of LRRC25. Increased LRRC25 in turn may regulate the stability or activity of key enzymes involved in either Aβ or hyperphosphorylated tau generation and thus may contribute to increased plaques and neurofibrillary tangles.

Keywords: 3xTg mice; APΔE9 mice; Alzheimer’s disease; LRRC25; aging; brain regions; iPSC neurons; immunohistochemistry.

FIGURE 1

LRRC25 is expressed in astrocyte-like cells, microglia, and neuronal cell lines. The CCF-STTG1, HMC3, and undifferentiated NT2 cells were immunocytochemically stained with LRRC25 antibody and visualized the subcellular LRRC25 expression. There is a clear indication of the membranous localization of LRRC25 and punctuate appearance in the cytoplasm (red) in all three cell types, and DAPI-stained nuclei (blue) appear to show no LRRC25 signals. The scale bar is 10 μm.

FIGURE 2

Demonstration of LRRC25 expression by astrocytes, microglia, and neurons in the adult mouse brains using cell-type specific markers. Polyclonal LRRC25 antibody was co-stained with monoclonal antibodies against GFAP (astrocyte), IBA1 (microglia), and Neun (neuron), followed by DAPI staining for nuclei (blue). There is a clear expression of LRRC25 (red) in the plasma membranes and a punctuate appearance in the cytoplasm of all three cell types examined, with a clear absence in the nuclei. The astrocyte and microglial cell images were acquired in the cortex region, whereas neuron images were acquired in the CA2 region of the hippocampus. The scale bar is 5 μm.

FIGURE 3

Differential expression levels of LRRC25 protein at different ages and in different brain regions. The brain regions such as cortex [CX, (A,B)], hippocampus [HP, (C,D)], cerebellum [BS, (E,F)], and brainstem [CB, (G,H)] were isolated on 1 day (1D), 1 month (1M), 1 year (1Y), 1.5 years (1.5Y), and 2 years (2Y) and brain lysates were subjected to immunoblotting. 1D expressed the lowest levels of LRRC25 in all brain regions, and relative expression levels at other ages were compared to 1D. The two lanes for each time point represent that samples were run in duplicates. Data were statistically analyzed by Analysis of Variance (ANOVA) followed by Tukey’s Multiple Comparisons Test. *p < 0.05, **p < 0.01, and ***p < 0.001, Data are mean + SEM, n = 3 per group.

FIGURE 4

Different regions of APΔE9 mice show a robust increase in the levels of actin-normalized LRRC25 protein levels relative to wild-type (WT) controls. Relative to WT, the increased LRRC25 protein was 166.171% in the cortex [CX (A,B)] and 215.407% in the hippocampus [HP (C,D)], while the cerebellum [CB (E,F)] and brainstem [BS (G,H)] showed no significant (ns) changes in the APΔE9 mice relative to non-transgenic WT controls. Data were statistically analyzed by paired Student’s t-test. **p < 0.01 and ***p < 0.001, Data are mean + SEM, n = 3 per group.

FIGURE 5

The cortical brain region of 3xTg mice shows significantly increased LRRC25 protein levels relative to wild-type (WT) controls. Image quantified and actin-normalized LRRC25 protein levels were compared among 3xTg and WT control mice. Relative to WT, the 3xTg mice cortex [CX (A,B)] showed 91.256% increased LRRC25 protein levels, but no changes in the hippocampus [HP (C,D)], cerebellum [CB (E,F)] and brainstem [BS (G,H)] were noted. Data were statistically analyzed by paired t-test. *p < 0.05. Data are mean + SEM, n = 3 per group.

FIGURE 6

LRRC25 protein levels are robustly increased in Alzheimer’s disease (AD) brains. (A) Protein lysates were prepared from normal control (NC) subjects and AD patient brains, immunoblotted. (B) Quantification by ImageJ showed an increase of 153% in AD compared to NC. ***p < 0.001. Data are mean + SEM, n = 4 per group.

FIGURE 7

A robust increase in the LRRC25 fluorescence intensity in the fibroblast-derived iPSC neurons from AD patients when compared to iPSC neurons derived from normal control (NC) subjects. iPSC cells were grown and differentiated into mature neurons until 16 days in vitro (16DIV) and immunocytochemically stained for LRRC25, NeuN (red) as a marker of neurons, and MAP2 (red) as a marker of dendrites. (A) Shows Neun-positive mature neurons. (B) Shows MAP2-positive dendrites. (C) Positive staining of LRRC25 (green) in Neun-expressing cells (red) suggests LRRC25 is expressed in iPSC neurons. (D) Quantification of LRRC25 immunofluorescence showed an increase of 181% in AD-derived iPSC neurons when compared to NC-derived neurons. Data were statistically analyzed by paired t-test. **p < 0.01, Data are mean + SEM, n = 4 per group. The scale bar is 10 μm in (A,B) and 5 μm in (C,D).