Pathogenesis of Lethal Aspiration Pneumonia in Mecp2-null Mouse Model for Rett Syndrome

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder mainly caused by mutations in the gene encoding the transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2), located on the X chromosome. Many RTT patients have breathing abnormalities, such as apnea and breathing irregularity, and respiratory infection is the most common cause of death in these individuals. Previous studies showed that MeCP2 is highly expressed in the lung, but its role in pulmonary function remains unknown. In this study, we found that MeCP2 deficiency affects pulmonary gene expression and structures. We also found that Mecp2-null mice, which also have breathing problems, often exhibit inflammatory lung injury. These injuries occurred in specific sites in the lung lobes. In addition, polarizable foreign materials were identified in the injured lungs of Mecp2-null mice. These results indicated that aspiration might be a cause of inflammatory lung injury in Mecp2-null mice. On the other hand, MeCP2 deficiency affected the expression of several neuromodulator genes in the lower brainstem. Among them, neuropeptide substance P (SP) immunostaining was reduced in Mecp2-null brainstem. These findings suggest that alteration of SP expression in brainstem may be involved in autonomic dysregulation, and may be one of the causes of aspiration in Mecp2-null mice.

Figure 1

Lung abnormalities in Mecp2-null mice. (a) Incidence of abnormalities in the indicated organs and tissues in wild-type (Mecp2 ^+/y, black columns; n = 22), Mecp2-null (Mecp2 ^null/y, red columns; n = 30), and dead Mecp2-null (Mecp2 ^null/y Death, gray columns; n = 34) mice. (b) Age (days) at which Mecp2-null mice died or were sacrificed. Data are expressed as means ± SE. (c) Body weight (g), measured at sacrifice. Data are expressed as means ± SE. **p < 0.01 versus wild-type mice. (d) Gross morphology of lung from wild-type (left panels) and Mecp2-null (right panels) mice. Dashed yellow lines indicate injured area. RA, right anterior; RM, right middle; RP, right posterior; RAc, right accessory; and LA, left anterior lobe. (e) Schematic diagram indicating mouse lung lobes (ventral view). (f) Incidence of abnormalities in each pulmonary lobe of Mecp2-null lungs (n = 15).

Figure 2

Lung inflammation in Mecp2-null mice. (a) Morphology of BAL cells from wild-type (left panel) and Mecp2-null (right panel) mice. Cells in BALFs were centrifuged onto glass slides, dried in air, and stained with Wright-Giemsa. Scale bar indicates 200 μm. (b) Cell populations in BALFs. Plots showed the indicated cell counts in BALFs obtained from wild-type (n = 8) and Mecp2-null mice (n = 9). SEG, Segmented Neutrophils; MONO, Monocytes; LYMPHO, Lymphocytes; and EOSINO, Eosinophils. (c) Representative H&E stained sections of the right anterior lobe in wild-type (left panel) and Mecp2-null (right panel) lungs. Scale bars, 200 μm. (d) Representative immunofluorescence images showing the distribution of IbaI signals in wild-type (left panels) and Mecp2-null (right panels) lung tissues. Cryosections of right anterior lung lobes obtained from 8-week-old wild-type and Mecp2-null mice were immunostained for IbaI (red) and Pdpn (green), and counterstained with Hoechst 33342 (blue). Scale bar, 100 μm. (e) H&E-stained sections of Mecp2-null lungs observed under bright-field (left panel) and polarization (right panel). Scale bar, 50 μm.

Figure 3

Histological analysis of lungs from Mecp2-null mice. (a) Representative immunofluorescence images showing the distribution of MeCP2, ABCA3, and Pdpn signals in wild-type and Mecp2-null lungs. Cryosections of right anterior lobes obtained from 8-week-old wild-type (left panel) and Mecp2-null (right panel) mice were immunostained for MeCP2 (green, upper panel or red, lower panel), ABCA3 (red), and Pdpn (green), and then counterstained with Hoechst 33342 (blue). MeCP2 signals were detected in wild-type lung tissues. Scale bar, 50 μm. (b) Representative H&E stained lung sections of wild-type (left panel) and Mecp2-null (right panel) mice. Scale bar, 100 μm. The graph at right shows the mean alveolar chord lengths in wild-type (n = 6) and Mecp2-null (n = 6) lungs. (c) Representative immunofluorescence images showing the distribution of ABCA3 and Pdpn signals in wild-type (left panels) and Mecp2-null (right panels) lungs. Scale bar, 50 μm. The graph shows the percentage of ABCA3-positive cells in wild-type (n = 6) and Mecp2-null (n = 6) mice lungs. Data are expressed as means ± SE. *p < 0.05 and **p < 0.01 versus wild-type mice.

Figure 4

Endogenous gene expression in wild-type and Mecp2-null mice lungs. Whole lungs of wild-type and Mecp2-null mice were harvested, and qRT-PCR was performed using primers against the indicated genes (a, lung-specific marker genes; b, inflammation-related genes). The graphs show the relative levels of the indicated mRNAs in wild-type (black columns, n = 6), normal Mecp2-null (red columns, n = 8), and abnormal Mecp2-null (orange columns, n = 6) lungs harvested from 7–10 week-old mice. The level of each mRNA was normalized to the level of Rps18. All data were normalized to the corresponding values from wild-type mice, defined as 1.0. Data are expressed as means ± SE. *p < 0.05 and **p < 0.01 versus wild-type samples; ^#p < 0.05 versus Mecp2-null samples.

Figure 5

Endogenous gene expression in wild-type and Mecp2-null mice brainstem. Whole brain and brainstem of wild-type and Mecp2-null mice were harvested, and qRT-PCR was performed using primers for the indicated genes (a, whole brain; b; brainstem). The graphs show the relative tissue levels of the indicated mRNAs in wild-type (black columns, n = 8), normal Mecp2-null (red columns, n = 9), and abnormal Mecp2-null (orange columns, n = 8) organs harvested from 7–10 week-old mice. The level of each mRNA was normalized to the corresponding level of Rps18. All data were normalized to the value from wild-type mice, defined as 1.0. Data are expressed as means ± SE. *p < 0.05 and **p < 0.01 versus wild-type mice samples.

Figure 6

SP immunoreactivity in wild-type and Mecp2-null mouse brainstem. Representative images of distribution of SP-ir structures in the brainstem: PB in the pons (a), and rostral NTS (b), caudal NTS (c), and DMX (d) in the medulla oblongata. Cryosections of brainstem obtained from 7–10-week-old wild-type (left panels), normal Mecp2-null (middle panels), and abnormal Mecp2-null (right panels) mice were immunostained for SP (red) and TH (green), and counterstained with Hoechst 33342 (blue, merged image panels) (Supplementary Figure S10). Dorsal is up. Vertical dotted lines indicate the median. White dotted outlines identify areas of the PB (a), NTS (b and c), and DMX (d). V4: fourth ventricle; PB: parabrachial nucleus; LC: locus coeruleus; NTS: nucleus of the solitary tract; AP: area postrema; DMX: dorsal motor nucleus of vagus nerve; cc: central canal. Asterisks indicate the nucleus ambiguous (AMB). Scale bars, 300 μm. (e) Comparison of the areas of SP-ir in the indicated regions of the brainstem in wild-type (black columns, n = 4), normal Mecp2-null (red columns, n = 3), and abnormal Mecp2-null (orange columns, n = 4) mice (Supplementary Figures S11 and S12). (f) Comparison of the percentage of the SP-ir area in DMX regions in wild-type (black columns, n = 4), normal Mecp2-null (red columns, n = 3), and abnormal Mecp2-null (orange columns, n = 4) mice. Data are expressed as means ± SE. *p < 0.05 and **p < 0.01 versus wild-type samples; ^#p < 0.05 versus Mecp2-null samples.