PPARgamma agonism inhibits progression of premalignant lesions in a murine lung squamous cell carcinoma model.

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences.

The sorting of RNA molecules to subcellular locations facilitates the activity of spatially restricted processes. We have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these transcripts contain an enrichment of G-quadruplex sequences in their 3' UTRs, suggesting that FMRP recognizes them to promote RNA localization. We observed similar results in neurons derived from Fragile X Syndrome patients. We identified the RGG domain of FMRP as important for binding G-quadruplexes and the transport of G-quadruplex-containing transcripts. Finally, we found that the translation and localization targets of FMRP were distinct and that an FMRP mutant that is unable to bind ribosomes still promoted localization of G-quadruplex-containing messages. This suggests that these two regulatory modes of FMRP may be functionally separated. These results provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

Modeling Hypoxia-Induced Neuropathies Using a Fast and Scalable Human Motor Neuron Differentiation System.

Human motor neuron (MN) diseases encompass a spectrum of disorders. A critical barrier to dissecting disease mechanisms is the lack of appropriate human MN models. Here, we describe a scalable, suspension-based differentiation system to generate functional human MN diseases in 3 weeks. Using this model, we translated recent findings that mRNA mis-localization plays a role in disease development to the human context by establishing a membrane-based system that allows efficient fractionation of MN cell soma and neurites. In response to hypoxia, used to mimic diabetic neuropathies, MNs upregulated mitochondrial transcripts in neurites; however, mitochondria were decreased. These data suggest that hypoxia may disrupt translation of mitochondrial mRNA, potentially leading to neurite damage and development of neuropathies. We report the development of a novel human MN model system to investigate mechanisms of disease affecting soma and/or neurites that facilitates the rapid generation and testing of patient-specific MN diseases.

ß Cell dysfunction during progression of metabolic syndrome to type 2 diabetes.

In a society where physical activity is limited and food supply is abundant, metabolic diseases are becoming a serious epidemic. Metabolic syndrome (MetS) represents a cluster of metabolically related symptoms such as obesity, hypertension, dyslipidemia, and carbohydrate intolerance, and significantly increases type 2 diabetes mellitus risk. Insulin resistance and hyperinsulinemia are consistent characteristics of MetS, but which of these features is the initiating insult is still widely debated. Regardless, both of these conditions trigger adverse responses from the pancreatic ß cell, which is responsible for producing, storing, and releasing insulin to maintain glucose homeostasis. The observation that the degree of ß cell dysfunction correlates with the severity of MetS highlights the need to better understand ß cell dysfunction in the development of MetS. This Review focuses on the current understanding from rodent and human studies of the progression of ß cell responses during the development of MetS, as well as recent findings addressing the complexity of ß cell identity and heterogeneity within the islet during disease progression. The differential responses observed in ß cells together with the heterogeneity in disease phenotypes within the patient population emphasize the need to better understand the mechanisms behind ß cell adaptation, identity, and dysfunction in MetS.

FISHing for ß Cells.

ß cell heterogeneity has emerged as an important contributor to islet function, with potential implications for diabetes. Using an optimized smFISH technique in intact islets, Farack et al. (2018) identify in the islet core an endocrine cell population of "extreme" ß cells with distinct molecular properties.

miR-219 regulates neural progenitors by dampening apical Par protein-dependent Hedgehog signaling.

The transition of dividing neuroepithelial progenitors to differentiated neurons and glia is essential for the formation of a functional nervous system. Sonic hedgehog (Shh) is a mitogen for spinal cord progenitors, but how cells become insensitive to the proliferative effects of Shh is not well understood. Because Shh reception occurs at primary cilia, which are positioned within the apical membrane of neuroepithelial progenitors, we hypothesized that loss of apical characteristics reduces the Shh signaling response, causing cell cycle exit and differentiation. We tested this hypothesis using genetic and pharmacological manipulation, gene expression analysis and time-lapse imaging of zebrafish embryos. Blocking the function of miR-219, a microRNA that downregulates apical Par polarity proteins and promotes progenitor differentiation, elevated Shh signaling. Inhibition of Shh signaling reversed the effects of miR-219 depletion and forced expression of Shh phenocopied miR-219 deficiency. Time-lapse imaging revealed that knockdown of miR-219 function accelerates the growth of primary cilia, revealing a possible mechanistic link between miR-219-mediated regulation of apical Par proteins and Shh signaling. Thus, miR-219 appears to decrease progenitor cell sensitivity to Shh signaling, thereby driving these cells towards differentiation.

microRNA regulation of neural precursor self-renewal and differentiation.

During early stages of development of the vertebrate central nervous system, neural precursors divide symmetrically to produce new precursors, thereby expanding the precursor population. During middle stages of neural development, precursors switch to an asymmetric division pattern whereby each mitosis produces one new precursor and one cell that differentiates as a neuron or glial cell. At late stages of development, most precursors stop dividing and terminally differentiate. Par complex proteins are associated with the apical membrane of neural precursors and promote precursor self-renewal. How Par proteins are down regulated to bring precursor self-renewal to an end has not been known. Our investigations of zebrafish neural development revealed that the microRNA miR-219 negatively regulates apical Par proteins, thereby promoting cessation of neural precursor division and driving terminal differentiation.

miR-219 regulates neural precursor differentiation by direct inhibition of apical par polarity proteins.

Asymmetric self-renewing division of neural precursors is essential for brain development. Partitioning-defective (Par) proteins promote self-renewal, and their asymmetric distribution provides a mechanism for asymmetric division. Near the end of neural development, most asymmetric division ends and precursors differentiate. This correlates with Par protein disappearance, but mechanisms that cause downregulation are unknown. MicroRNAs can promote precursor differentiation but have not been linked to Par protein regulation. We tested a hypothesis that microRNA miR-219 promotes precursor differentiation by inhibiting Par proteins. Neural precursors in zebrafish larvae lacking miR-219 function retained apical proteins, remained in the cell cycle, and failed to differentiate. miR-219 inhibited expression via target sites within the 3' untranslated sequence of pard3 and prkci mRNAs, which encode Par proteins, and blocking miR-219 access to these sites phenocopied loss of miR-219 function. We propose that negative regulation of Par protein expression by miR-219 promotes cell-cycle exit and differentiation.