Age- and sex- divergent translatomic responses of the mouse retinal pigmented epithelium.

Aging is the main risk factor for age-related macular degeneration (AMD), a retinal neurodegenerative disease that leads to irreversible blindness, particularly in people over 60 years old. Retinal pigmented epithelium (RPE) atrophy is an AMD hallmark. Genome-wide chromatin accessibility, DNA methylation, and gene expression studies of AMD and control RPE demonstrate epigenomic/transcriptomic changes occur during AMD onset and progression. However, mechanisms by which molecular alterations of normal aging impair RPE function and contribute to AMD pathogenesis are unclear. Here, we specifically interrogate the RPE translatome with advanced age and across sexes in a novel RPE reporter mouse model. We find differential age- and sex- associated transcript expression with overrepresentation of pathways related to inflammation in the RPE. Concordant with impaired RPE function, the phenotypic changes in the aged translatome suggest that aged RPE becomes immunologically active, in both males and females, with some sex-specific signatures, which supports the need for sex representation for in vivo studies.

Translatomic response of retinal Müller glia to acute and chronic stress.

Analysis of retina cell type-specific epigenetic and transcriptomic signatures is crucial to understanding the pathophysiology of retinal degenerations such as age-related macular degeneration (AMD) and delineating cell autonomous and cell-non-autonomous mechanisms. We have discovered that Aldh1l1 is specifically expressed in the major macroglia of the retina, Müller glia, and, unlike the brain, is not expressed in retinal astrocytes. This allows use of Aldh1l1 cre drivers and Nuclear Tagging and Translating Ribosome Affinity Purification (NuTRAP) constructs for temporally controlled labeling and paired analysis of Müller glia epigenomes and translatomes. As validated through a variety of approaches, the Aldh1l1cre/ERT2-NuTRAP model provides Müller glia specific translatomic and epigenomic profiles without the need to isolate whole cells. Application of this approach to models of acute injury (optic nerve crush) and chronic stress (aging) uncovered few common Müller glia-specific transcriptome changes in inflammatory pathways, and mostly differential signatures for each stimulus. The expression of members of the IL-6 and integrin-linked kinase signaling pathways was enhanced in Müller glia in response to optic nerve crush but not aging. Unique changes in neuroinflammation and fibrosis signaling pathways were observed in response to aging but not with optic nerve crush. The Aldh1l1cre/ERT2-NuTRAP model allows focused molecular analyses of a single, minority cell type within the retina, providing more substantial effect sizes than whole tissue analyses. The NuTRAP model, nucleic acid isolation, and validation approaches presented here can be applied to any retina cell type for which a cell type-specific cre is available.

Repeated cocaine or methamphetamine treatment alters astrocytic CRF2 and GLAST expression in the ventral midbrain.

Dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) play a central role in the reinforcing properties of abused drugs including methamphetamine and cocaine. Chronic effects of psychostimulants in the SN/VTA also involve non-dopaminergic transmitters, including glutamate and the stress-related peptide corticotropin-releasing factor (CRF). In the SN/VTA, astrocytes express a variety of membrane-bound neurotransmitter receptors and transporters that influence neurotransmission. CRF receptor type 2 (CRF2) activity in the VTA is important for stress-induced relapse and drug-seeking behaviour, but the localization of its effects is incompletely understood. Here, we first identified CRF2 transcript in astrocytes of the SN/VTA using RNA-Seq in Aldh1l1;NuTRAP mice and confirmed it using in situ hybridization (RNAscope) in wild-type mice. We then used immunofluorescence to quantify the astrocytic marker protein S100ß, glial-specific glutamate/aspartate transporter GLAST, and CRF2 in the SN/VTA following 12 days of treatment (i.p.) with methamphetamine (3 mg/kg), cocaine (10 mg/kg), or saline. We observed a significant decrease in GLAST immunofluorescence in brains of psychostimulant treated mice compared with saline controls. In addition, we observed increased labelling of CRF2 in drug treated groups, a decrease in the number of S100ß positive cells, and an increase of co-staining of CRF2 with both S100ß and tyrosine hydroxylase (dopamine neurons). Our results suggest a significant interaction between CRF2, GLAST, and astrocytes in the midbrain that emerges with repeated exposure to psychostimulants. These findings provide rationale for future investigation of astrocyte-based strategies for altering cellular and circuit function in response to stress and drug exposure.

Adipocytes fail to maintain cellular identity during obesity due to reduced PPARγ activity and elevated TGFß-SMAD signaling.

OBJECTIVE: Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. METHODS: We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), 3) and PPARγ cistrome (ChIP-seq) profiles in mice fed chow or a high-fat diet (HFD) for 10 weeks. We also assessed the impact of the PPARγ agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from the HFD-fed mice. We integrated these data to determine the transcription factors underlying adipocyte responses to HFD and conducted functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. RESULTS: Adipocytes from the HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes and enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARγ binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGFß1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and associated with myofibroblast signature genes. TGFß1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFß1. CONCLUSIONS: Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPARγ activity and elevated TGFß-SMAD signaling. This cellular identity crisis may be a fundamental mechanism that drives functional decline of adipose tissues during obesity.

Simultaneous Transcriptional and Epigenomic Profiling from Specific Cell Types within Heterogeneous Tissues In Vivo.

Epigenomic mechanisms direct distinct gene expression programs for different cell types. Various in vivo tissues have been subjected to epigenomic analysis; however, these studies have been limited by cellular heterogeneity, resulting in composite gene expression and epigenomic profiles. Here, we introduce "NuTRAP," a transgenic mouse that allows simultaneous isolation of cell-type-specific translating mRNA and chromatin from complex tissues. Using NuTRAP, we successfully characterize gene expression and epigenomic states of various adipocyte populations in vivo, revealing significant differences compared to either whole adipose tissue or in vitro adipocyte cell lines. We find that chromatin immunoprecipitation sequencing (ChIP-seq) using NuTRAP is highly efficient, scalable, and robust with even limited cell input. We further demonstrate the general utility of NuTRAP by analyzing hepatocyte-specific epigenomic states. The NuTRAP mouse is a resource that provides a powerful system for cell-type-specific gene expression and epigenomic profiling.