CB1 R and iNOS are distinct players promoting pulmonary fibrosis in Hermansky-Pudlak syndrome
Clinical and translational medicine
Cinar, R;Park, JK;Zawatsky, CN;Coffey, NJ;Bodine, SP;Abdalla, J;Yokoyama, T;Jourdan, T;Jay, L;Zuo, MXG;O'Brien, KJ;Huang, J;Mackie, K;Alimardanov, A;Iyer, MR;Gahl, WA;Kunos, G;Gochuico, BR;Malicdan, MCV;
PMID: 34323400 | DOI: 10.1002/ctm2.471
Hermansky-Pudlak syndrome (HPS) is a rare genetic disorder which, in its most common and severe form, HPS-1, leads to fatal adult-onset pulmonary fibrosis (PF) with no effective treatment. We evaluated the role of the endocannabinoid/CB1 R system and inducible nitric oxide synthase (iNOS) for dual-target therapeutic strategy using human bronchoalveolar lavage fluid (BALF), lung samples from patients with HPS and controls, HPS-PF patient-derived lung fibroblasts, and bleomycin-induced PF in pale ear mice (HPS1ep/ep ). We found overexpression of CB1 R and iNOS in fibrotic lungs of HPSPF patients and bleomycin-infused pale ear mice. The endocannabinoid anandamide was elevated in BALF and negatively correlated with pulmonary function parameters in HPSPF patients and pale ear mice with bleomycin-induced PF. Simultaneous targeting of CB1 R and iNOS by MRI-1867 yielded greater antifibrotic efficacy than inhibiting either target alone by attenuating critical pathologic pathways. Moreover, MRI-1867 treatment abrogated bleomycin-induced increases in lung levels of the profibrotic interleukin-11 via iNOS inhibition and reversed mitochondrial dysfunction via CB1 R inhibition. Dual inhibition of CB1 R and iNOS is an effective antifibrotic strategy for HPSPF.
Nabhan, AN;Webster, JD;Adams, JJ;Blazer, L;Everrett, C;Eidenschenk, C;Arlantico, A;Fleming, I;Brightbill, HD;Wolters, PJ;Modrusan, Z;Seshagiri, S;Angers, S;Sidhu, SS;Newton, K;Arron, JR;Dixit, VM;
PMID: 37321220 | DOI: 10.1016/j.cell.2023.05.022
Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.
Sottile SY, Hackett TA, Cai R, Ling L, Llano DA, Caspary DM.
PMID: 29061702 | DOI: 10.1523/JNEUROSCI.1795-17.2017
Acetylcholine is a potent neuromodulator capable of modifying patterns of acoustic information flow. In auditory cortex, cholinergic systems have been shown to increase salience/gain while suppressing extraneous information. However, the mechanism by which cholinergic circuits shape signal processing in the auditory thalamus (medial geniculate body, MGB) is poorly understood. The present study, in male Fischer Brown Norway rats, seeks to determine the location and function of presynaptic neuronal nicotinic acetylcholine receptors (nAChRs) at the major inputs to MGB and characterize how nAChRs change during aging. In vitro electrophysiological/optogenetic methods were used to examine responses of MGB neurons following activation of nAChRs during a paired-pulse paradigm. Presynaptic nAChR activation increased responses evoked by stimulation of excitatory corticothalamic and inhibitory tectothalamic terminals. Conversely, nAChR activation appeared to have little effect on evoked responses from inhibitory thalamic reticular nucleus and excitatory tectothalamic terminals. In situ hybridization data showed nAChR subunit transcripts in GABAergic inferior colliculus neurons and glutamatergic auditory cortical neurons supporting the present slice findings. Responses to nAChR activation at excitatory corticothalamic and inhibitory tectothalamic inputs were diminished by aging. These findings suggest that cholinergic input to the MGB increases the strength of tectothalamic inhibitory projections, potentially improving signal-to-noise ratio and signal detection while increasing corticothalamic gain, which may facilitate top-down identification of stimulus identity. These mechanisms appear negatively affected by aging, potentially diminishing speech perception in noisy environments. Cholinergic inputs to the MGB appear to maximize sensory processing by adjusting both top-down and bottom-up mechanisms in conditions of attention and arousal.Significance StatementThe pedunculopontine tegmental nucleus (PPTg) is the source of cholinergic innervation for sensory thalamus and is a critical part of an ascending arousal system which controls the firing mode of thalamic cells based on attentional demand. The present study describes the location and impact of aging on presynaptic neuronal nicotinic receptors (nAChRs) within the circuitry of the auditory thalamus (medial geniculate body; MGB). We show that nAChRs are located on ascending inhibitory and descending excitatory presynaptic inputs onto MGB neurons, likely selectively increasing gain and improving temporal clarity. In addition, we show that aging has a deleterious effect on nAChR efficacy. Cholinergic dysfunction at the level of MGB may negatively impact speech understanding in the elderly population.
Rieger, NS;Ng, AJ;Lee, S;Brady, BH;Christianson, JP;
PMID: 36706685 | DOI: 10.1016/j.yhbeh.2023.105313
Prenatal infection increases risk for neurodevelopmental disorders such as autism in offspring. In rodents, prenatal administration of the viral mimic Polyinosinic: polycytidylic acid (Poly I: C) allows for investigation of developmental consequences of gestational sickness on offspring social behavior and neural circuit function. Because maternal immune activation (MIA) disrupts cortical development and sociability, we examined approach and avoidance in a rat social affective preference (SAP) task. Following maternal Poly I:C (0.5 mg/kg) injection on gestational day 12.5, male adult offspring (PN 60-64) exhibited atypical social interactions with stressed conspecifics whereas female SAP behavior was unaffected by maternal Poly I:C. Social responses to stressed conspecifics depend upon the insular cortex where corticotropin releasing factor (CRF) modulates synaptic transmission and SAP behavior. We characterized insular field excitatory postsynaptic potentials (fEPSP) in adult offspring of Poly I:C or control treated dams. Male MIA offspring showed decreased sensitivity to CRF (300 nM) while female MIA offspring showed greater sensitivity to CRF compared to sham offspring. These sex specific effects appear to be behaviorally relevant as CRF injected into the insula of male and female rats prior to social exploration testing had no effect in MIA male offspring but increased social interaction in female MIA offspring. We examined the cellular distribution of CRF receptor mRNA but found no effect of maternal Poly I:C in the insula. Together, these experiments reveal sex specific effects of prenatal infection on offspring responses to social affective stimuli and identify insular CRF signaling as a novel neurobiological substrate for autism risk.
Eyres, M;Bell, JA;Davies, ER;Fabre, A;Alzetani, A;Jogai, S;Marshall, BG;Johnston, DA;Xu, Z;Fletcher, SV;Wang, Y;Marshall, G;Davies, DE;Offer, E;Jones, MG;
PMID: 35977489 | DOI: 10.1016/j.celrep.2022.111230
A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFβ/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.
Cheadle L, Tzeng CP, Kalish BT, Harmin DA, Rivera S, Ling E, Nagy MA, Hrvatin S, Hu L, Stroud H, Burkly LC, Chen C, Greenberg ME.
PMID: 30033152 | DOI: 10.1016/j.neuron.2018.06.036
Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.
Am J Respir Crit Care Med. 2018 Dec 15.
Reyfman PA, Walter JM, Joshi N, Anekalla KR, McQuattie-Pimentel AC, Chiu S, Fernandez R, Akbarpour M, Chen CI, Ren Z, Verma R, Abdala-Valencia H, Nam K, Chi M, Han S, Gonzalez-Gonzalez FJ, Soberanes S, Watanabe S, Williams KJN, Flozak AS, Nicholson TT, Morgan VK, Winter DR, Hinchcliff M, Hrusch CL, Guzy RD, Bonham CA, Sperling AI, Bag R, Hamanaka RB, Mutlu GM, Yeldandi AV, Marshall SA, Shilatifard A, Amaral LAN, Perlman H, Sznajder JI, Argento AC, Gillespie CT, Dematte J, Jain M, Singer BD, Ridge KM, Lam AP, Bharat A, Bhorade SM, Gottardi CJ, Budinger GRS, Misharin AV.
PMID: 30554520 | DOI: 10.1164/rccm.201712-2410OC
Abstract RATIONALE: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. OBJECTIVES: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells or other cell types in lung tissue from subjects with pulmonary fibrosis compared with controls. METHODS: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data in using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. MEASUREMENTS AND MAIN RESULTS: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to non-overlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. CONCLUSIONS: We generated a single cell atlas of pulmonary fibrosis. Using this atlas we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
Gao, F;Li, C;Danopoulos, S;Al Alam, D;Peinado, N;Webster, S;Borok, Z;Kohbodi, GA;Bellusci, S;Minoo, P;
PMID: 35385750 | DOI: 10.1016/j.celrep.2022.110608
The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFβ signaling. Compound inactivation of Alk5 TβR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.
Development (Cambridge, England)
Negretti, NM;Plosa, EJ;Benjamin, JT;Schuler, BA;Habermann, AC;Jetter, CS;Gulleman, P;Bunn, C;Hackett, AN;Ransom, M;Taylor, CJ;Nichols, D;Matlock, BK;Guttentag, SH;Blackwell, TS;Banovich, NE;Kropski, JA;Sucre, JMS;
PMID: 34927678 | DOI: 10.1242/dev.199512
Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages - wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.
YAP regulates alveolar epithelial cell differentiation and AGER via NFIB/KLF5/NKX2-1
Gokey, JJ;Snowball, J;Sridharan, A;Sudha, P;Kitzmiller, JA;Xu, Y;Whitsett, JA;
PMID: 34466790 | DOI: 10.1016/j.isci.2021.102967
Ventilation is dependent upon pulmonary alveoli lined by two major epithelial cell types, alveolar type-1 (AT1) and 2 (AT2) cells. AT1 cells mediate gas exchange while AT2 cells synthesize and secrete pulmonary surfactants and serve as progenitor cells which repair the alveoli. We developed transgenic mice in which YAP was activated or deleted to determine its roles in alveolar epithelial cell differentiation. Postnatal YAP activation increased epithelial cell proliferation, increased AT1 cell numbers, and caused indeterminate differentiation of subsets of alveolar cells expressing atypical genes normally restricted to airway epithelial cells. YAP deletion increased expression of genes associated with mature AT2 cells. YAP activation enhanced DNA accessibility in promoters of transcription factors and motif enrichment analysis predicted target genes associated with alveolar cell differentiation. YAP participated with KLF5, NFIB, and NKX2-1 to regulate AGER. YAP plays a central role in a transcriptional network that regulates alveolar epithelial differentiation.
Proceedings of the National Academy of Sciences of the United States of America
van Doeselaar, L;Stark, T;Mitra, S;Yang, H;Bordes, J;Stolwijk, L;Engelhardt, C;Kovarova, V;Narayan, S;Brix, LM;Springer, M;Deussing, JM;Lopez, JP;Czisch, M;Schmidt, MV;
PMID: 37252963 | DOI: 10.1073/pnas.2300722120
Mental health disorders often arise as a combination of environmental and genetic factors. The FKBP5 gene, encoding the GR co-chaperone FKBP51, has been uncovered as a key genetic risk factor for stress-related illness. However, the exact cell type and region-specific mechanisms by which FKBP51 contributes to stress resilience or susceptibility processes remain to be unravelled. FKBP51 functionality is known to interact with the environmental risk factors age and sex, but so far data on behavioral, structural, and molecular consequences of these interactions are still largely unknown. Here we report the cell type- and sex-specific contribution of FKBP51 to stress susceptibility and resilience mechanisms under the high-risk environmental conditions of an older age, by using two conditional knockout models within glutamatergic (Fkbp5Nex) and GABAergic (Fkbp5Dlx) neurons of the forebrain. Specific manipulation of Fkbp51 in these two cell types led to opposing effects on behavior, brain structure and gene expression profiles in a highly sex-dependent fashion. The results emphasize the role of FKBP51 as a key player in stress-related illness and the need for more targeted and sex-specific treatment strategies.
Hua, SS;Ding, JJ;Sun, TC;Guo, C;Zhang, Y;Yu, ZH;Cao, YQ;Zhong, LH;Wu, Y;Guo, LY;Luo, JH;Cui, YH;Qiu, S;
PMID: 36842495 | DOI: 10.1016/j.biopsych.2023.02.013
The ventromedial prefrontal cortex (vmPFC) has been viewed as a locus to store and recall extinction memory. However, the synaptic and cellular mechanisms underlying this process remain elusive.We combined transgenic mice, electrophysiological recording, activity-dependent cell labeling, and chemogenetic manipulation to analyze the role of adaptor protein APPL1 in the vmPFC for fear extinction retrieval.We found that both constitutive and conditional APPL1 knockout decreases NMDA receptor (NMDAR) function in the vmPFC and impairs fear extinction retrieval. Moreover, APPL1 undergoes nuclear translocation during extinction retrieval. Blocking APPL1 nucleocytoplasmic translocation reduces NMDAR currents and disrupts extinction retrieval. We further identified a prefrontal neuronal ensemble that is both necessary and sufficient for the storage of extinction memory. Inducible APPL1 knockout in this ensemble abolishes NMDAR-dependent synaptic potentiation and disrupts extinction retrieval, while simultaneously chemogenetic activation of this ensemble rescues the impaired behaviors.Therefore, our results indicate that a prefrontal neuronal ensemble stores extinction memory, and APPL1 signaling supports these neurons to retrieve extinction memory via controlling NMDAR-dependent potentiation.
