Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models.

microRNA-mRNA Profile of Skeletal Muscle Differentiation and Relevance to Congenital Myotonic Dystrophy.

microRNAs (miRNAs) regulate messenger RNA (mRNA) abundance and translation during key developmental processes including muscle differentiation. Assessment of miRNA targets can provide insight into muscle biology and gene expression profiles altered by disease. mRNA and miRNA libraries were generated from C2C12 myoblasts during differentiation, and predicted miRNA targets were identified based on presence of miRNA binding sites and reciprocal expression.

Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing.

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines, we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls.

HIF2α Is an Essential Molecular Brake for Postprandial Hepatic Glucagon Response Independent of Insulin Signaling.

Glucagon drives hepatic gluconeogenesis and maintains blood glucose levels during fasting. The mechanism that attenuates glucagon action following refeeding is not understood. The present study demonstrates an increase in perivenous liver hypoxia immediately after feeding, which stabilizes hypoxia-inducible factor 2α (HIF2α) in liver.

Transcriptional regulatory events initiated by Ascl1 and Neurog2 during neuronal differentiation of P19 embryonic carcinoma cells.

As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation.

Negative regulation of Yap during neuronal differentiation.

Regulated proliferation and cell cycle exit are essential aspects of neurogenesis. The Yap transcriptional coactivator controls proliferation in a variety of tissues during development, and this activity is negatively regulated by kinases in the Hippo signaling pathway. We find that Yap is expressed in mitotic mouse retinal progenitors and it is downregulated during neuronal differentiation.

Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cyclic AMP-dependent protein kinase.

cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β.

A novel fluorescent transcriptional reporter for cell-based microarray assays.

Cell-based microarrays have been used for a wide variety of assays including gain-of-function, loss-of-function and compound screening. Many of these assays have employed fluorescent proteins as reporters. These fluorescent reporter proteins can be monitored in living cells but have low sensitivity of detection compared to enzymatic reporters.

Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation.

The basic helix-loop-helix transcription factor Ascl1 plays a critical role in the intrinsic genetic program responsible for neuronal differentiation. Here, we describe a novel model system of P19 embryonic carcinoma cells with doxycycline-inducible expression of Ascl1. Microarray hybridization and real-time PCR showed that these cells demonstrated increased expression of many neuronal proteins in a time- and concentration-dependent manner.

Microarray transfection analysis of conserved genomic sequences from three immediate early genes.

In an effort to define novel transcriptional regulatory elements, microarray cotransfection was used to functionally characterize conserved non-coding sequences (CNSs) of three immediate early genes: c-fos, JunB and EGR-1. Cotransfection of fluorescent CNS reporter constructs and expression vectors for constitutively active signaling proteins demonstrated that many of the CNSs alter both the basal and regulated expressions of reporter constructs, but the effects of these CNSs were usually specific for their homologous promoter. One CNS located in the first intron of the c-fos gene conferred regulation by cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and Raf.

Phosphatidylinositol 3-kinase and Akt effectors mediate insulin-like growth factor-I neuroprotection in dorsal root ganglia neurons.

Insulin-like growth factor-I (IGF-I) protects neurons of the peripheral nervous system from apoptosis, but the underlying signaling pathways are not well understood. We studied IGF-I mediated signaling in embryonic dorsal root ganglia (DRG) neurons. DRG neurons express IGF-I receptors (IGF-IR), and IGF-I activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.

Microarray transfection analysis of transcriptional regulation by cAMP-dependent protein kinase.

A wide variety of bioinformatic tools have been described to characterize potential transcriptional regulatory mechanisms based on genomic sequence analysis and microarray hybridization studies. However, these regulatory mechanisms are still experimentally verified using transient transfection methods. Current transfection methods are limited both by their large scale and by the low level of efficiency for certain cell types.

A transforming growth factor beta-induced Smad3/Smad4 complex directly activates protein kinase A.

Transforming growth factor beta (TGFbeta) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFbeta signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA).

Autoinhibition and isoform-specific dominant negative inhibition of the type II cGMP-dependent protein kinase.

In the absence of cyclic nucleotides, the cAMP-dependent protein kinase and cGMP-dependent protein kinases (cGKs) suppress phosphotransfer activity at the catalytic cleft by competitive inhibition of substrate binding with a pseudosubstrate sequence within the holoenzyme. The magnitude of inhibition can be diminished by autophosphorylation near this pseudosubstrate sequence. Activation of type I cGK (cGKI) and type II cGK (cGKII) are differentially regulated by their cyclic nucleotide-binding sites.

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs).