Severe respiratory impairment is a prominent feature of Rett syndrome, an X-linked disorder caused by mutations in methyl CpG-binding protein 2 (MECP2). Despite MECP2's ubiquitous expression, respiratory anomalies are attributed to neuronal dysfunction. Here, we show that neutral lipids accumulate in mouse Mecp2-mutant lungs, whereas surfactant phospholipids decrease. Conditional deletion of Mecp2 from lipid-producing alveolar epithelial 2 (AE2) cells causes aberrant lung lipids and respiratory symptoms, whereas deletion of Mecp2 from hindbrain neurons results in distinct respiratory abnormalities. Single-cell RNA sequencing of AE2 cells suggests lipid production and storage increase at the expense of phospholipid synthesis. Lipid production enzymes are confirmed as direct targets of MECP2-directed nuclear receptor co-repressor 1/2 transcriptional repression. Remarkably, lipid-lowering fluvastatin improves respiratory anomalies in Mecp2-mutant mice. These data implicate autonomous pulmonary loss of MECP2 in respiratory symptoms for the first time and have immediate impacts on patient care.

Respiratory Syncytial Virus (RSV) is the leading cause of acute lower respiratory infections in young children and infection has been linked to the development of persistent lung disease in the form of wheezing and asthma. Despite substantial research efforts, there are no RSV vaccines currently available and an effective monoclonal antibody targeting the RSV fusion protein (palivizumab) is of limited general use given the associated expense. Therefore, the development of novel approaches to prevent RSV infection is highly desirable to improve pediatric health globally. We have developed a method to generate alveolar-like macrophages (ALMs) from pluripotent stem cells. These ALMs have shown potential to promote airway innate immunity and tissue repair and so we hypothesized that ALMs could be used as a strategy to prevent RSV infection. Here, we demonstrate that ALMs are not productively infected by RSV and prevent the infection of epithelial cells. Prevention of epithelial infection was mediated by two different mechanisms: phagocytosis of RSV particles and release of an antiviral soluble factor different from type I interferon. Furthermore, intratracheal administration of ALMs protected mice from subsequent virus-induced weight loss and decreased lung viral titres and inflammation, indicating that ALMs can impair the pathogenesis of RSV infection. Our results support a prophylactic role for ALMs in the setting of RSV infection and warrant further studies on stem cell-derived ALMs as a novel cell-based therapy for pulmonary viral infections.

The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

Nucleolin is an essential cellular receptor to human respiratory syncytial virus (RSV). Pharmacological targeting of the nucleolin RNA binding domain RBD1,2 can inhibit RSV infections in vitro and in vivo; however, the site(s) on RBD1,2 which interact with RSV are not known. We undertook a series of experiments designed to: document RSV-nucleolin co-localization on the surface of polarized MDCK cells using immunogold electron microscopy, to identify domains on nucleolin that physically interact with RSV using biochemical methods and determine their biological effects on RSV infection in vitro, and to carry out structural analysis toward informing future RSV drug development. Results of immunogold transmission and scanning electron microscopy showed RSV-nucleolin co-localization on the cell surface, as would be expected for a viral receptor. RSV, through its fusion protein (RSV-F), physically interacts with RBD1,2 and these interactions can be competitively inhibited by treatment with Palivizumab or recombinant RBD1,2. Treatment with synthetic peptides derived from two 12-mer domains of RBD1,2 inhibited RSV infection in vitro, with structural analysis suggesting these domains are potentially feasible for targeting in drug development. In conclusion, the identification and characterization of domains of nucleolin that interact with RSV provide the essential groundwork toward informing design of novel nucleolin-targeting compounds in RSV drug development.

Bronchopulmonary dysplasia (BPD) remains a major respiratory illness in extremely premature infants. The biological mechanisms leading to BPD are not fully understood, although an arrest in lung development has been implicated. The current study aimed to investigate the occurrence of autophagy in the developing mouse lung and its regulatory role in airway branching and terminal sacculi formation. We found 2 windows of epithelial autophagy activation in the developing mouse lung, both resulting from AMPK activation. Inhibition of AMPK-mediated autophagy led to reduced lung branching in vitro. Conditional deletion of beclin 1 (Becn1) in mouse lung epithelial cells (Becn1Epi-KO), either at early (E10.5) or late (E16.5) gestation, resulted in lethal respiratory distress at birth or shortly after. E10.5 Becn1Epi-KO lungs displayed reduced airway branching and sacculi formation accompanied by impaired vascularization, excessive epithelial cell death, reduced mesenchymal thinning of the interstitial walls, and delayed epithelial maturation. E16.5 Becn1Epi-KO lungs had reduced terminal air sac formation and vascularization and delayed distal epithelial differentiation, a pathology similar to that seen in infants with BPD. Taken together, our findings demonstrate that intrinsic autophagy is an important regulator of lung development and morphogenesis and may contribute to the BPD phenotype when impaired.

Rationale: Premature infants subjected to mechanical ventilation (MV) are prone to lung injury that may result in bronchopulmonary dysplasia. MV causes epithelial cell death and halts alveolar development. The exact mechanism of MV-induced epithelial cell death is unknown. Objectives: To determine the contribution of autophagy to MV-induced epithelial cell death in newborn rat lungs. Methods: Newborn rat lungs and fetal rat lung epithelial (FRLE) cells were exposed to MV and cyclic stretch, respectively, and were then analyzed by immunoblotting and mass spectrometry for autophagy, apoptosis, and bioactive sphingolipids. Measurements and Main Results: Both MV and stretch first induce autophagy (ATG 5-12 [autophagy related 5-12] and LC3B-II [microtubule-associated proteins 1A/1B light chain 3B-II] formation) followed by extrinsic apoptosis (cleaved CASP8/3 [caspase-8/3] and PARP [poly(ADP-ribose) polymerase] formation). Stretch-induced apoptosis was attenuated by inhibiting autophagy. Coimmunoprecipitation revealed that stretch promoted an interaction between LC3B and the FAS (first apoptosis signal) cell death receptor in FRLE cells. Ceramide levels, in particular C16 ceramide, were rapidly elevated in response to ventilation and stretch, and C16 ceramide treatment of FRLE cells induced autophagy and apoptosis in a temporal pattern similar to that seen with MV and stretch. SMPD1 (sphingomyelin phosphodiesterase 1) was activated by ventilation and stretch, and its inhibition prevented ceramide production, LC3B-II formation, LC3B/first apoptosis signal interaction, caspase-3 activation, and, ultimately, FLRE cell death. SMPD1 inhibition also attenuated ventilation-induced autophagy and apoptosis in newborn rats. Conclusions: Ventilation-induced ceramides promote autophagy-mediated cell death, and identifies SMPD1 as a potential therapeutic target for the treatment of ventilation-induced lung injury in newborns.

Glycogen synthesis is vital, malstructure resulting in precipitation and accumulation into neurotoxic polyglucosan bodies (PBs). One well-understood mechanism of PB generation is glycogen branching enzyme deficiency (GBED). Less understood is Lafora disease (LD), resulting from absence of the glycogen phosphatase laforin or the E3 ubiquitin ligase malin, and accumulation of hyperphosphorylated PBs. LD afforded first insight that glycogen sphericity depends on more than adequate branching activity. Unexpectedly, deficiencies of the Linear Ubiquitin Chain Assembly Complex (LUBAC) components RBCK1 and HOIP result in PBs in muscle tissues. Here we analyzed nervous system phenotypes of mice lacking RBCK1 and find profuse PB accumulations in brain and spinal cord with extensive neurodegeneration and neurobehavioral deficits. Brain glycogen in these mice is characterized by long chains and hyperphosphorylation, similar to LD. Like in LD, glycogen synthase and branching enzyme are unaltered. Regional PB distribution mirrors LD and not GBED. Perisynaptic PB localization is unlike LD or GBED. The results indicate that RBCK1 is part of a system supplementing laforin-malin in regulating glycogen architecture including in unique neuronal locales.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor resulting in high rates of morbidity and mortality. A strategy to increase the efficacy of available drugs and enhance the delivery of chemotherapeutics through the blood brain barrier (BBB) is desperately needed. We investigated the potential of Cisplatin conjugated gold nanoparticle (GNP-UP-Cis) in combination with MR-guided Focused Ultrasound (MRgFUS) to intensify GBM treatment. Viability assays demonstrated that GNP-UP-Cis greatly inhibits the growth of GBM cells compared to free cisplatin and shows marked synergy with radiation therapy. Additionally, increased DNA damage through γH2AX phosphorylation was observed in GNP-UP-Cis treated cells, along with enhanced platinum concentrations. In vivo, GNP-UP-Cis greatly reduced the growth of GBM tumors and MRgFUS led to increased BBB permeability and GNP-drug delivery in brain tissue. Our studies suggest that GNP-Cis conjugates and MRgFUS can be used to focally enhance the delivery of targeted chemotherapeutics to brain tumors.

Lafora disease (LD) is an autosomal recessive late onset, progressive myoclonic epilepsy with a high prevalence in the miniature Wirehaired Dachshund. The disease is due to a mutation in the Epm2b gene which results in intracellular accumulation of abnormal glycogen (Lafora bodies). Recent breed-wide testing suggests that the carrier plus affected rate may be as high as 20%. A characteristic feature of the disease is spontaneous and reflex myoclonus; however clinical signs and disease progression are not well described. A survey was submitted to owners of MWHD which were homozygous for Epm2b mutation (breed club testing program) or had late onset reflex myoclonus and clinical diagnosis of LD. There were 27 dogs (11 male; 16 female) for analysis after young mutation-positive dogs that had yet to develop disease were excluded. Average age of onset of clinical signs was 6.94 years (3.5-12). The most common initial presenting sign was reflex and spontaneous myoclonus (77.8%). Other presenting signs included hypnic myoclonus (51.9%) and generalized seizures (40.7%). Less common presenting signs include focal seizures, "jaw smacking", "fly catching", "panic attacks", impaired vision, aggression and urinary incontinence. All these clinical signs may appear, and then increase in frequency and intensity over time. The myoclonus in particular becomes more severe and more refractory to treatment. Signs that developed later in the disease include dementia (51.9%), blindness (48.1%), aggression to people (25.9%) and dogs (33.3%), deafness (29.6%) and fecal (29.6%) and urinary (37.0%) incontinence as a result of loss of house training (disinhibited type behavior). Further prospective study is needed to further characterize the canine disease and to allow more specific therapeutic strategies and to tailor therapy as the disease progresses.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset, slowly progressive myopathy leading to lost ambulation after age 50 years.1 It is caused by mutations of the VMA21 gene, which encodes the assembly chaperone of the V-ATPase proton pump that acidifies organelles. Autophagosomal alkalinization impairs autophagy completion, which drives autophagosome proliferation and apparent damage to skeletal muscle.2 All known XMEA mutations reduce but do not eliminate VMA21 expression. Some cause greater VMA21 reduction than in classic XMEA and lead to a severe course with congenital/neonatal onset, extreme muscle wasting, and ventilation dependence. However, even with these mutations, no overt extramuscular disease is present.2–5 Given the vital role of V-ATPases in all cells, absence of manifest disease outside muscle is surprising. Possibly, extramuscular pathology is actually present but subclinical. In fact, patients with congenital/neonatal onset do have increased liver function tests, suggesting latent hepatopathy.

Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy due to mutations in the EPM2A (laforin) and EPM2B (malin) genes, with no substantial genotype-phenotype differences between the two. Founder effects and recurrent mutations are common, and mostly isolated to specific ethnic groups and/or geographical locations. Pathologically, LD is characterized by distinctive polyglucosans, which are formations of abnormal glycogen. Polyglucosans, or Lafora bodies (LB) are typically found in the brain, periportal hepatocytes of the liver, skeletal and cardiac myocytes, and in the eccrine duct and apocrine myoepithelial cells of sweat glands. Mouse models of the disease and other naturally occurring animal models have similar pathology and phenotype. Hypotheses of LB formation remain controversial, with compelling evidence and caveats for each hypothesis. However, it is clear that the laforin and malin functions regulating glycogen structure are key.With the exception of a few missense mutations LD is clinically homogeneous, with onset in adolescence. Symptoms begin with seizures, and neurological decline follows soon after. The disease course is progressive and fatal, with death occurring within 10 years of onset. Antiepileptic drugs are mostly non-effective, with none having a major influence on the progression of cognitive and behavioral symptoms. Diagnosis and genetic counseling are important aspects of LD, and social support is essential in disease management.Future therapeutics for LD will revolve around the pathogenesics of the disease. Currently, efforts at identifying compounds or approaches to reduce brain glycogen synthesis appear to be highly promising.

RATIONALE:Abnormal alveolar macrophages (AM) are found in chronic obstructive pulmonary disease, asthma, cystic fibrosis, and adenosine deaminase deficiency (ADA(-/-)). There is no specific treatment strategy to compensate for these innate immune abnormalities. Recent findings suggest AMs are of early embryonic or fetal origin. Pluripotent stem cells (PSCs) as a source of embryonic-derived AMs for therapeutic use in acute and chronic airway diseases has yet to be investigated.OBJECTIVES:To determine if embryonic Myb(-/-) alveolar-like macrophages have therapeutic value on pulmonary transplantation in acute and chronic airway diseases.METHODS:Directed differentiation of murine PSCs was used in factor-defined media to produce expandable embryonic macrophages conditioned to an alveolar-like phenotype with granulocyte-macrophage colony-stimulating factor. AMs were partially depleted in mice to create an acute lung injury. To model a chronic lung disease, ADA(-/-) mice were used. Alveolar-like macrophages were intratracheally transplanted to the injured animals and therapeutic potential was determined.MEASUREMENTS AND MAIN RESULTS:The differentiation protocol is highly efficient and adaptable to human PSCs. The PSC macrophages are phenotypically like AMs both functionally and by ligand marker characterization. They engulf bacteria and apoptotic cells and are better phagocytes than bone marrow-derived macrophages. In vivo, these macrophages remain in healthy airways for at least 4 weeks, can engulf neutrophils during acute lung injury, enhance pulmonary tissue repair, and promote survival in ADA(-/-) mice. Animals receiving the macrophages do not develop abnormal pathology or teratomas.CONCLUSIONS:PSCs are a reliable source to produce therapeutically active alveolar-like macrophages to treat airway disease.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

Efficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM) scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

Reciprocal signaling between the lung mesenchyme and epithelium is crucial for differentiation and branching morphogenesis. We hypothesized that the combination of signaling pathways comprising early epithelial-mesenchymal interactions and a 3D spatial environment are necessary for an efficient induction of embryonic and induced pluripotent stem cells (ESCs and iPSCs) into a lung cell phenotype with hallmarks of the distal niche. Aggregating early, but not late, embryonic lung mesenchyme with endoderm-induced mouse ESCs and iPSCs for 6 days resulted in organization into tubular structures and differentiation of the tubular lining cells to an NKX2-1(+)/SOX2(-)/SOX9(+)/proSFTPC(+) lineage. Over 80% of the endoderm-induced cells committed to an NKX2-1(+) lineage. Electron microscopy analysis demonstrated numerous multivesicular bodies and glycogen deposits in the tubular lining cells, characteristic features of type II epithelial cell progenitors. Using soluble FGFR2 receptor antagonists, we demonstrate that reciprocal fibroblast growth factor (FGF) 2, 7, and 10 signaling is essential for differentiation of endoderm-induced cells to an NKX2-1(+)/proSFTPC(+) phenotype within 3D aggregates. Only FGF2 was able to commit endoderm-induced cells in monolayer cultures to an NKX2-1(+) lineage, however with a significant lower efficiency (∼16%) than seen with mesenchyme. Thus, while FGF2 signaling alone can induce a primed population of ESCs and iPSCs, the cells do not differentiate to distal lung epithelial progenitors with the same efficiency and level of maturity that is achieved when the complex tissue and 3D environment of the developing lung is more accurately recapitulated.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

Cystic fibrosis (CF) is caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene resulting in a loss of Cl− channel function, disrupting ion and fluid homeostasis, leading to severe lung disease with airway obstruction due to mucus plugging and inflammation. The most common CFTR mutation, F508del, occurs in 90% of patients causing the mutant CFTR protein to misfold and trigger an endoplasmic reticulum based recycling response. Despite extensive research into the pathobiology of CF lung disease, little attention has been paid to the cellular changes accounting for the pathogenesis of CF lung disease. Here we report a novel finding of intracellular retention and accumulation of a cleaved fragment of F508del CFTR in concert with autophagic like phagolysosomes in the airway epithelium of patients with F508del CFTR. Aggregates consisting of poly-ubiquitinylated fragments of only the N-terminal domain of F508del CFTR but not the full-length molecule accumulate to appreciable levels. Importantly, these undegraded intracytoplasmic aggregates representing the NT-NBD1 domain of F508del CFTR were found in ciliated, in basal, and in pulmonary neuroendocrine cells. Aggregates were found in both native lung tissues and ex-vivo primary cultures of bronchial epithelial cells from CF donors, but not in normal control lungs. Our findings present a new, heretofore, unrecognized innate CF gene related cell defect and a potential contributing factor to the pathogenesis of CF lung disease. Mutant CFTR intracytoplasmic aggregates could be analogous to the accumulation of misfolded proteins in other degenerative disorders and in pulmonary “conformational protein-associated” diseases. Consequently, potential alterations to the functional integrity of airway epithelium and regenerative capacity may represent a critical new element in the pathogenesis of CF lung disease.

Ubiquitin ligases regulate quantities and activities of target proteins, often pleiotropically. The malin ubiquitin E3 ligase is reported to regulate autophagy, the misfolded protein response, microRNA silencing, Wnt signaling, neuronatin‐mediated endoplasmic reticulum stress, and the laforin glycogen phosphatase. Malin deficiency causes Lafora disease, pathologically characterized by neurodegeneration and accumulations of malformed glycogen (Lafora bodies). We show that reducing glycogen production in malin‐deficient mice by genetically removing PTG, a glycogen synthesis activator protein, nearly completely eliminates Lafora bodies and rescues the neurodegeneration, myoclonus, seizure susceptibility, and behavioral abnormality. Glycogen synthesis downregulation is a potential therapy for the fatal adolescence onset epilepsy Lafora disease. Ann Neurol 2014;75:442–446

Lung development-associated diseases are major causes of morbidity and lethality in preterm infants and children. Access to the lung progenitor/stem cell populations controlling pulmonary development during embryogenesis and early postnatal years is essential to understand the molecular basis of such diseases. Using a Nkx2-1(mCherry) reporter mouse, we have identified and captured Nkx2-1-expressing lung progenitor cells from the proximal lung epithelium during fetal development. These cells formed clonal spheres in semisolid culture that could be maintained in vitro and demonstrated self-renewal and expansion capabilities over multiple passages. In-vitro-derived Nkx2-1-expressing clonal spheres differentiated into a polarized epithelium comprised of multiple cell lineages, including basal and secretory cells, that could repopulate decellularized lung scaffolds. Nkx2-1 expression thus defines a fetal lung epithelial progenitor cell population that can be used as a model system to study pulmonary development and associated pediatric diseases.