Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. We combine genetic manipulation, imaging and quantitative dendritic architecture analysis in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dα7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter we demonstrate that AP-1 transcriptional activity is downstream of Dα7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. Our results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown cause that is characterized by progressive fibrotic lung remodeling. An abnormal emergence of airway epithelial‐like cells within the alveolar compartments of the lung, herein termed bronchiolization, is often observed in IPF. However, the origin of this dysfunctional distal lung epithelium remains unknown due to a lack of suitable human model systems. In this study, we established a human induced pluripotent stem cell (iPSC)‐derived air‐liquid interface (ALI) model of alveolar epithelial type II (ATII)‐like cell differentiation that allows us to investigate alveolar epithelial progenitor cell differentiation in vitro. We treated this system with an IPF‐relevant cocktail (IPF‐RC) to mimic the pro‐fibrotic cytokine milieu present in IPF lungs. Stimulation with IPF‐RC during differentiation increases secretion of IPF biomarkers and RNA sequencing (RNA‐seq) of these cultures reveals significant overlap with human IPF patient data. IPF‐RC treatment further impairs ATII differentiation by driving a shift toward an airway epithelial‐like expression signature, providing evidence that a pro‐fibrotic cytokine environment can influence the proximo‐distal differentiation pattern of human lung epithelial cells. In conclusion, we show for the first time, the establishment of a human model system that recapitulates aspects of IPF‐associated bronchiolization of the lung epithelium in vitro .

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Idiopathic pulmonary fibrosis (IPF) is a lethal disease with substantial unmet medical needs. While aberrant epithelial remodeling is a key factor in IPF progression, the molecular mechanisms behind this process remain elusive. Harnessing a 3D patient‐derived organoid model and multi‐omics approach, the first inventory of the connection between metabolic alteration, chromatin accessibility, and transcriptional regulation in IPF aberrant epithelial remodeling is provided. This remodeling is characterized by an increase in chromatin accessibility, particularly at JUNB motif‐enriched promoter regions proximal to transcription start sites of metabolic and pro‐fibrotic genes. Mechanistically, JUNB undergoes O‐linked β‐N‐acetylglucosamine modification (O‐GlcNAcylation), a critical step in modulating pro‐fibrotic responses to chronic injury. This modification is pivotal in fostering the emergence of aberrant epithelial basal cells in the alveolar niche, a proposed driver of IPF pathology. Specific deletion of O‐GlcNAcylation sites on JUNB attenuates the metaplastic differentiation of basal cells, thereby aiding in the restoration of the alveolar lineage. Together, the findings reveal a novel link between metabolic dysregulation and cell fate regulation at the chromatin level in fibrosis, mediated by the O‐GlcNAc‐JUNB axis, suggesting avenues for the development of new therapeutic strategies in IPF.