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

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.


Inventory of supplemental Items -Supplementary Tables
Related to Figure 1 and 2 - Table S1 Related to Methods - Table S2 -   Table S1. Results of blood biochemistry analysis of Mecp2-null mice.

Supplementary Figure Legends
Air bubbles are indicated by white arrowheads. lu; lung, li; liver, st; stomach.

Macroscopic observation of the larynx in wild-type and Mecp2-null mice.
Representative images of the larynx in wild-type (left panels) and abnormal Mecp2-null (right panels) mice at the age of 9-10 weeks.

Immunofluorescence of Iba I in wild-type and Mecp2-null lungs.
Representative immunofluorescence images showing the distribution of Iba I signals in wild-type and Mecp2-null lung tissues. Cryosections of right anterior lung lobe obtained from 8-week-old wild-type (left panels) and Mecp2-null (right panels) mice were immunostained for Iba I (red) and Pdpn (green), and counterstained with Hoechst 33342 (blue). Scale bars indicate 100 μm.

Mecp2-null lungs.
Representative immunofluorescence images showing the distribution of MeCP2 and ABCA3 signals in wild-type and Mecp2-null lungs. Cryosections of right anterior lobes obtained from 8-week-old wild-type (left panels) and Mecp2-null (right panels) mice were immunostained for MeCP2 (green) and ABCA3 (red), and counterstained with Hoechst 33342 (blue). MeCP2 signals were detected in wild-type lung tissues.
Double-label immunofluorescence also confirmed that nuclear MeCP2 signals were detected in ABCA3-positive ATII cells. Scale bars indicate 50 μm.

Double-immunofluorescence staining for MeCP2 and Pdpn in wild-type and
Mecp2-null lungs.

Representative immunofluorescence images showing the distribution of MeCP2 and
Pdpn signals in wild-type and Mecp2-null lungs. Cryosections of right anterior lobes obtained from 8-week-old wild-type (left panels) and Mecp2-null (right panels) mice were immunostained for MeCP2 (red) and Pdpn (green), and counterstained with Hoechst 33342 (blue). Double-label immunofluorescence indicted that nuclear MeCP2 signals were detected in Pdpn-positive ATI cells. Scale bars indicate 50 μm.

Double-immunofluorescence staining for ABCA3 and Pdpn in wild-type and
Mecp2-null lungs.

Representative immunofluorescence images showing the distribution of 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 ABCA3 (red), Pdpn (green), and counterstained with Hoechst 33342 (blue). Scale bars indicate 50 μm.

LacZ gene expression in lungs after intranasal administration.
Macroscopic views of lungs of wild-type and Mecp2-null mice after intranasal administration of adenoviral vectors (Ad.LacZ or Ad.dE1.3), with or without anesthesia.