Klotho is a transmembrane protein expressed in the renal tubules where it acts as a permissive coreceptor for fibroblast growth factor 23 (FGF23). FGF23 signaling reduces the abundance of CYP27b1 and phosphate cotransporters NPT2a and NPT2c, leading to a decrease in 1,25(OH)2D3synthesis and a rise in urinary phosphate excretion, respectively. Systemic or whole-nephron deletion of Klotho in mice results in renal FGF23 resistance characterized by high 1,25(OH)2D3 and phosphate levels and premature aging. Expression of Klotho is highest in the distal tubules, whereas 25OH vitamin D 1α hydroxylation and phosphate reabsorption predominantly occur in the proximal tubules. Currently, the segment-specific roles of Klotho in renal tubules are not fully understood. Here we have generated mice with Klotho specifically ablated from the proximal tubules using 3 different Cre mouse strains. All 3 models displayed impaired urinary phosphate excretion and increased abundance of NPT2a in the brush border membrane. Notably, hyperphosphatemia in knockout mice was mild or nonexistent under basal conditions but occurred upon high phosphate loading, indicating the presence of compensatory mechanisms. Effects on 1,25(OH)2D3 varied between mouse strains but were modest overall. Thus, Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo.

Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration.

Although the Ventana immunohistochemistry (IHC) platform for detecting anaplastic lymphoma kinase gene (ALK) (D5F3) expression was recently approved by the US Food and Drugs Administration (FDA), fluorescence in situ hybridization (FISH) is still the "gold-standard" method recommended by the US National Comprehensive Cancer Network (NCCN) guideline for NSCLC. We evaluated 6 ALK-positive lung adenocarcinoma patients who tested Ventana IHC-positive and FISH-negative and assessed their clinical responses to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Histologic and cytologic specimens from the 6 patients were stained with Ventana anti-ALK(D5F3) rabbit monoclonal primary antibody using the OptiView™ DAB IHC detection kit and OptiView™ amplification kit on a Ventana BenchMark XT processor. In addition, they were also tested by FISH, qRT-PCR, next-generation sequencing (NGS), and RNAscope ISH analysis. All patients received crizotinib treatment and their follow-up clinical data were recorded. The objective response rate achieved with crizotinib therapy was 66.7% (4/6 partial responses and 2/6 stable disease). One patient in whom a new fusion type (EML4->EXOC6B->ALK fusion) was identified obtained a partial response. These findings indicate that patients with ALK-positive lung adenocarcinoma who test Ventana IHC-positive and FISH-negative may still respond to crizotinib therapy.

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signalingpathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required forMerkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel celldifferentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures.

Abstract

Abstract

Diabetic Nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in: 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.

Phosphaturic mesenchymal tumors typically cause paraneoplastic osteomalacia, chiefly as a result of FGF23 secretion. In a prior study, we identified FN1-FGFR1 fusion in 9 of 15 phosphaturic mesenchymal tumors. In this study, a total of 66 phosphaturic mesenchymal tumors and 7 tumors resembling phosphaturic mesenchymal tumor but without known phosphaturia were studied. A novel FN1-FGF1 fusion gene was identified in two cases without FN1-FGFR1 fusion by RNA sequencing and cross-validated with direct sequencing and western blot. Fluorescence in situ hybridization analyses revealed FN1-FGFR1 fusion in 16 of 39 (41%) phosphaturic mesenchymal tumors and identified an additional case with FN1-FGF1 fusion. The two fusion genes were mutually exclusive. Combined with previous data, the overall prevalence of FN1-FGFR1 and FN1-FGF1 fusions was 42% (21/50) and 6% (3/50), respectively. FGFR1 immunohistochemistry was positive in 82% (45/55) of phosphaturic mesenchymal tumors regardless of fusion status. By contrast, 121 cases of potential morphologic mimics (belonging to 13 tumor types) rarely expressed FGFR1, the main exceptions being solitary fibrous tumors (positive in 40%), chondroblastomas (40%), and giant cell tumors of bone (38%), suggesting a possible role for FGFR1 immunohistochemistry in the diagnosis of phosphaturic mesenchymal tumor. With the exception of one case reported in our prior study, none of the remaining tumors resembling phosphaturic mesenchymal tumor had either fusion type or expressed significant FGFR1. Our findings provide insight into possible mechanisms underlying the pathogenesis of phosphaturic mesenchymal tumor and imply a central role of the FGF1-FGFR1 signaling pathway. The novel FN1-FGF1 protein is expected to be secreted and serves as a ligand that binds and activates FGFR1 to achieve an autocrine loop. Further study is required to determine the functions of these fusion proteins.

Understanding the neural basis of behaviour requires studying brain activity in behaving subjects using complementary techniques that measure neural responses at multiple spatial scales, and developing computational tools for understanding the mapping between these measurements. Here we report the first results of widefield imaging of genetically encoded calcium indicator (GCaMP6f) signals from V1 of behaving macaques. This technique provides a robust readout of visual population responses at the columnar scale over multiple mm(2) and over several months. To determine the quantitative relation between the widefield GCaMP signals and the locally pooled spiking activity, we developed a computational model that sums the responses of V1 neurons characterized by prior single unit measurements. The measured tuning properties of the GCaMP signals to stimulus contrast, orientation and spatial position closely match the predictions of the model, suggesting that widefield GCaMP signals are linearly related to the summed local spiking activity.

Abstract

ABSTRACT

GPR88 is an orphan G protein-coupled receptor highly expressed in striatal D1R and D2R-expressing medium spiny neurons. This receptor is involved in activity and motor responses, and we previously showed that this receptor also regulates anxiety-like behaviors. To determine whether GPR88 in D2R-expressing neurons contributes to this emotional phenotype, we generated conditional Gpr88 knockout mice using A2AR-Cre-driven recombination, and compared anxiety-related responses in both total and A2AR-Gpr88 KO mice. A2AR-Gpr88 KO mice showed a selective reduction of Gpr88 mRNA in D2R- but not D1R-expressing neurons. These mutant mice showed increased locomotor activity and decreased anxiety-like behaviors in light dark and elevated plus maze tests. These phenotypes were superimposable to those observed in totalGpr88 KO mice, demonstrating that the previously reported anxiogenic activity of GPR88 operates at the level of A2AR-expressing neurons. Further, A2AR-Gpr88 KO mice showed no change in novelty preference and novelty-suppressed feeding, while these responses were increased and decreased, respectively, in the total Gpr88 KO mice. Also, A2AR-Gpr88 KO mice showed intact fear conditioning, while the fear responses were decreased in total Gpr88 KO. We therefore also show for the first time that GPR88 activity regulates approach behaviors and conditional fear, however these behaviors do not seem mediated by receptors in A2AR-neurons. We conclude thatGpr88 expressed in A2AR neurons enhances ethological anxiety-like behaviors without affecting conflict anxiety and fear responses.

Significance Statement: GPR88, a striatal enriched orphan G protein-coupled receptor has been implicated in regulation of anxiety-like behaviors. In the striatum, GPR88 is most abundant in both medium spiny neurons expressing dopamine D1 receptors and dopamine D2 receptors. To evaluate the contribution of GPR88 in D2R-neurons, we compared anxiety-like and fear-related behavioral responses of newly generated conditional A2AR-Gpr88 mice, with those of total Gpr88knockout animals. Our data show that GPR88 expressed in A2AR -neurons increases ethological anxiety-like behaviors without affecting conflict anxiety and fear responses. These results represent a first step towards understanding circuit mechanisms underlying GPR88 function in the brain. Future studies will evaluate the role of GPR88 in D1R-neurons.

Tumor suppression that is mediated by oncogene-induced senescence (OIS) is considered to function as a safeguard during development ofpancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that regulate OIS in PDAC are poorly understood. Here, we have determined that nuclear RelA reinforces OIS to inhibit carcinogenesis in the Kras mouse model of PDAC. Inactivation of RelA acceleratedpancreatic lesion formation in Kras mice by abrogating the senescence-associated secretory phenotype (SASP) gene transcription signature. Using genetic and pharmacological tools, we determined that RelA activation promotes OIS via elevation of the SASP factor CXCL1 (also known as KC), which activates CXCR2, during pancreatic carcinogenesis. In Kras mice, pancreas-specific inactivation of CXCR2 prevented OIS and was correlated with increased tumor proliferation and decreased survival. Moreover, reductions in CXCR2 levels were associated with advanced neoplastic lesions in tissue from human pancreatic specimens. Genetically disabling OIS in Kras mice caused RelA to promote tumor proliferation, suggesting a dual role for RelA signaling in pancreatic carcinogenesis. Taken together, our data suggest a pivotal role for RelA in regulating OIS in preneoplastic lesions and implicate the RelA/CXCL1/CXCR2 axis as an essential mechanism of tumor surveillance in PDAC.