Androgen deprivation induces neuroendocrine phenotypes in prostate cancer cells through CREB1/EZH2-mediated downregulation of REST.

Although effective initially, prolonged androgen deprivation therapy (ADT) promotes neuroendocrine differentiation (NED) and prostate cancer (PCa) progression. It is incompletely understood how ADT transcriptionally induces NE genes in PCa cells. CREB1 and REST are known to positively and negatively regulate neuronal gene expression in the brain, respectively. No direct link between these two master neuronal regulators has been elucidated in the NED of PCa. We show that REST mRNA is downregulated in NEPC cell and mouse models, as well as in patient samples. Phenotypically, REST overexpression increases ADT sensitivity, represses NE genes, inhibits colony formation in culture, and xenograft tumor growth of PCa cells. As expected, ADT downregulates REST in PCa cells in culture and in mouse xenografts. Interestingly, CREB1 signaling represses REST expression. In studying the largely unclear mechanism underlying transcriptional repression of REST by ADT, we found that REST is a direct target of EZH2 epigenetic repression. Finally, genetic rescue experiments demonstrated that ADT induces NED through EZH2's repression of REST, which is enhanced by ADT-activated CREB1 signaling. In summary, our study has revealed a key pathway underlying NE gene upregulation by ADT, as well as established novel relationships between CREB1 and REST, and between EZH2 and REST, which may also have implications in other cancer types and in neurobiology.

The identification of a Distinct Astrocyte Subtype that Diminishes in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of two hallmark pathologies: the accumulation of Amyloid beta (Aß) and tau proteins in the brain. There is a growing body of evidence suggesting that astrocytes, a type of glial cell in the brain, play crucial roles in clearing Aß and binding to tau proteins. However, due to the heterogeneity of astrocytes, the specific roles of different astrocyte subpopulations in response to Aß and tau remain unclear. To enhance the understanding of astrocyte subpopulations in AD, we investigated astrocyte lineage cells based on single-nuclei transcriptomic data obtained from both human and mouse samples. We characterized the diversity of astrocytes and identified global and subpopulation-specific transcriptomic changes between control and AD samples. Our findings revealed the existence of a specific astrocyte subpopulation marked by low levels of GFAP and the presence of AQP4 and CD63 expression, which showed functional enrichment in Aß clearance and tau protein binding, and diminished in AD. We verified this type of astrocytes in mouse models and in AD patient brain samples. Furthermore, our research also unveiled significant alterations of the ligand-receptor interactions between astrocytes and other cell types. These changes underscore the complex interplay between astrocytes and neighboring cells in the context of AD. Overall, our work gives insights into astrocyte heterogeneity in the context of AD and reveals a distinct astrocyte subpopulation that holds potential for therapeutic interventions in AD. Targeting specific astrocyte subpopulations may offer new avenues for the development of novel treatments for AD.

Androgen deprivation induces neuroendocrine phenotypes in prostate cancer cells through CREB1/EZH2-mediated downregulation of REST.

Although effective initially, prolonged androgen deprivation therapy (ADT) promotes neuroendocrine differentiation (NED) and prostate cancer (PCa) progression. It is incompletely understood how ADT transcriptionally induces NE genes in PCa cells. CREB1 and REST are known to positively and negatively regulate neuronal gene expression in the brain, respectively. No direct link between these two master neuronal regulators has been elucidated in the NED of PCa. We show that REST mRNA is downregulated in NEPC cell and mouse models, as well as in patient samples. Phenotypically, REST overexpression increases ADT sensitivity, represses NE genes, inhibits colony formation in culture, and xenograft tumor growth of PCa cells. As expected, ADT downregulates REST in PCa cells in culture and in mouse xenografts. Interestingly, CREB1 signaling represses REST expression. In studying the largely unclear mechanism underlying transcriptional repression of REST by ADT, we found that REST is a direct target of EZH2 epigenetic repression. Finally, genetic rescue experiments demonstrated that ADT induces NED through EZH2's repression of REST, which is enhanced by ADT-activated CREB signaling. In summary, our study has revealed a key pathway underlying NE gene upregulation by ADT, as well as established novel relationships between CREB1 and REST, and between EZH2 and REST, which may also have implications in other cancer types and in neurobiology.

The melanocortin action is biased toward protection from weight loss in mice.

The melanocortin action is well perceived for its ability to regulate body weight bidirectionally with its gain of function reducing body weight and loss of function promoting obesity. However, this notion cannot explain the difficulty in identifying effective therapeutics toward treating general obesity via activation of the melanocortin action. Here, we provide evidence that altered melanocortin action is only able to cause one-directional obesity development. We demonstrate that chronic inhibition of arcuate neurons expressing proopiomelanocortin (POMC) or paraventricular hypothalamic neurons expressing melanocortin receptor 4 (MC4R) causes massive obesity. However, chronic activation of these neuronal populations failed to reduce body weight. Furthermore, gain of function of the melanocortin action through overexpression of MC4R, POMC or its derived peptides had little effect on obesity prevention or reversal. These results reveal a bias of the melanocortin action towards protection of weight loss and provide a neural basis behind the well-known, but mechanistically ill-defined, predisposition to obesity development.

Novel role of dynamin-related-protein 1 in dynamics of ER-lipid droplets in adipose tissue.

Dynamin-Related-Protein 1 (DRP1) critically regulates mitochondrial and peroxisomal fission in multicellular organisms. However, the impact of DRP1 on other organelles, especially its direct influence on ER functions remains largely unclear. Here, we report that DRP1 translocates to endoplasmic reticulum (ER) in response to ß-adrenergic stimulation. To further investigate the function of DRP1 on ER-lipid droplet (LD) dynamics and the metabolic subsequences, we generated an adipose tissue-specific DRP1 knockout model (Adipo-Drp1flx/flx ). We found that the LDs in adipose tissues of Adipo-Drp1flx/flx mice exhibited more unilocular morphology with larger sizes, and formed less multilocular structures upon cold exposure. Mechanistically, we discovered that abnormal LD morphology occurs because newly generated micro-LDs fail to dissociate from the ER due to DRP1 ablation. Conversely, the ER retention of LDs can be rescued by the overexpressed DRP1 in the adipocytes. The alteration of LD dynamics, combined with abnormal mitochondrial and autophagy functions in adipose tissue, ultimately lead to abnormalities in lipid metabolism in Adipo-Drp1flx/flx mice.

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

Isolation and characterization of alveolar epithelial type II cells derived from mouse embryonic stem cells.

The use of embryonic stem cells (ESCs) to regenerate distal lung epithelia damaged by injuries or diseases requires development of safe and efficient methodologies that direct ESC differentiation into transplantable distal lung epithelial progenitors. Time-consuming culture procedure and low differentiation efficiency are major problems that are associated with conventional differentiation approaches via embryoid body formation. The use of a growth factor cocktail or a lung-specific cell-conditioned medium to enrich definitive endoderm for efficient differentiation of mouse ESCs (mESC) into alveolar epithelial progenitor type II cells (ATIICs) has been reported, but not yet successful for generating a homogenous population of ATIICs for tissue regeneration purpose, and it remains unclear whether or not those mESC-derived ATIICs possess normal biological functions. Here, we report a novel method using a genetically modified mESC line harboring an ATIIC-specific neomycin(R) transgene in Rosa 26 locus. We showed that ATIICs can be efficiently differentiated from mESCs as early as day 7 by culturing them directly on Matrigel-coated plates in DMEM containing 15% knockout serum replacement. With this culture condition, the genetically modified mESCs can be selectively differentiated into a homogenous population (>99%) of ATIICs. Importantly, the mESC-derived ATIICs (mESC-ATIICs) exhibited typical lamellar bodies and expressed surfactant protein A, B, and C as normal control ATIICs. When cultured with an air-liquid-interface culture system in Small Airway Epithelial Cell Growth Medium, the mESC-ATIICs can be induced to secrete surfactant proteins after being treated with dibutyryl cAMP+dexamethasone. These mESC-ATIICs can synthesize and secrete surfactant lipid in response to secretagogue, demonstrating active surfactant metabolism in mESC-ATIICs as that seen in normal control ATIICs. In addition, we demonstrated that the selected mESC-ATIICs can be maintained on Matrigel-coated plates for at least 4 days with robust proliferative capacity. When cultured in DMEM medium containing 10% FBS, mESC-ATIICs spontaneously differentiated into alveolar epithelial type I cells. Collectively, these data demonstrate that the genetically modified mESCs can be selectively differentiated into a homogenous population of functional ATIICs, providing a reliable cell source to explore their therapeutic potential in lung tissue regeneration.

Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity.

We examined tyrosine hydroxylase (TH-GAL4) expression and anti-TH immunoreactivity in the Drosophila protocerebrum and characterized single cell clones of the TH-GAL4 neurons. Eight clusters of putative dopaminergic neurons were characterized. Neurons in three of the clusters project to the mushroom body neuropil: PAM neurons project to the medial portion of the horizontal lobes; PPL1 neurons project to the vertical lobes, the junction area, the heel and distal peduncle; and PPL2ab neurons project to the calyx. Five types of PPL1 neurons were discovered that innervate different zones of the mushroom body lobes. Functional imaging experiments showed that the dopaminergic processes in four of the zones differ in response properties to odor, electric shock, or following the pairing of odor and electric shock. These results indicate that distinct dopaminergic neurons define separate zones of the mushroom body lobes and are probably involved in different functions. Differences in functional response properties of these neurons suggest that they are involved in different behavioral processes.

Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila.

Targeted gene expression has become a standard technique for the study of biological questions in Drosophila. Until recently, transgene expression could be targeted in the dimension of either time or space, but not both. Several new systems have recently been developed to direct transgene expression simultaneously in both time and space. We describe here two such systems that we developed in our laboratory. The first system provides a general method for temporal and regional gene expression targeting (TARGET) with the conventional GAL4-upstream activator sequence (UAS) system and a temperature-sensitive GAL80 molecule, which represses GAL4 transcriptional activity at permissive temperatures. The second system, termed Gene-Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. We have used both systems for simultaneous spatial and temporal rescue of memory dysfunction in the rutabaga (rut) memory mutant of Drosophila. In this protocol, we provide guidelines for the use of these two novel systems, which should have general utility in studying Drosophila biology and in using the fly as a model for human disease.

Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch.

The GAL4-based Gene-Switch system has been engineered to regulate transgene expression in Drosophila in both time and space. We constructed a Gene-Switch transgene in which Gene-Switch expression is restricted spatially by a defined mushroom body enhancer. This system allows Gene-Switch to be active only in the mushroom bodies and only on administration of the pharmacological Gene-Switch ligand RU486. This line was used to drive the expression of a rutabaga cDNA in otherwise rutabaga mutant flies. Induction of the rutabaga cDNA in the mushroom bodies only during adulthood, or during adulthood along with the larval and pupal developmental stages, corrects the olfactory memory impairment found in rutabaga mutants. Induction of the cDNA only during the larval and pupal stages was inconsequential to performance in olfactory memory tasks. These data indicate that normal rutabaga function must be expressed in adulthood for normal memory and conclusively delimit the time and space expression requirements for correcting the rutabaga memory impairment. Such combined pharmacogenetic regulation of transgene expression now allows this time and space dissection to be made for other behavioral mutants.