ABSTRACT
Diabetes is the result of the insufficiency or dysfunction of pancreatic beta cells alone or in combination with insulin resistance. The replacement or regeneration of beta cells can effectively reverse diabetes in humans and rodents. Therefore, the identification of novel small molecules that promote pancreatic beta-cell proliferation is an attractive approach for diabetic therapy. While numerous hormones, small molecules, and growth factors are able to drive rodent beta cells to replicate, only a few small molecules have demonstrated the ability to stimulate human beta-cell proliferation. Hence, there is an urgent need for therapeutic agents that induce regeneration and expansion of adult human beta cells. Here, we describe a detailed protocol for coating chamber slides, culturing primary islets, performing islet cell disassociation, seeding cells on chamber slides, treating islet cells with compounds or infecting them with adenovirus, immunostaining of proliferation markers and imaging, and data analysis.
Subject(s)
Drug Discovery , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Biomarkers , Cell Culture Techniques , Cell Proliferation/drug effects , Diabetes Mellitus/drug therapy , Diabetes Mellitus/metabolism , Drug Discovery/methods , Fluorescent Antibody Technique , Humans , Hypoglycemic Agents/pharmacology , Immunohistochemistry , Transduction, GeneticABSTRACT
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease, characterized by motor neuron (MN) death, for which there are no truly effective treatments. Here, we describe a new small molecule survival screen carried out using MNs from both wild-type and mutant SOD1 mouse embryonic stem cells. Among the hits we found, kenpaullone had a particularly impressive ability to prolong the healthy survival of both types of MNs that can be attributed to its dual inhibition of GSK-3 and HGK kinases. Furthermore, kenpaullone also strongly improved the survival of human MNs derived from ALS-patient-induced pluripotent stem cells and was more active than either of two compounds, olesoxime and dexpramipexole, that recently failed in ALS clinical trials. Our studies demonstrate the value of a stem cell approach to drug discovery and point to a new paradigm for identification and preclinical testing of future ALS therapeutics.
Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Embryonic Stem Cells/cytology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Induced Pluripotent Stem Cells/cytology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Motor Neurons/cytology , Motor Neurons/drug effects , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/pathology , Animals , Benzazepines/chemistry , Benzazepines/pharmacology , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Cholestenones/chemistry , Cholestenones/pharmacology , Glycogen Synthase Kinase 3/metabolism , Humans , Indoles/chemistry , Indoles/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Motor Neurons/enzymology , Mutation , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/metabolism , Structure-Activity Relationship , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1ABSTRACT
BACKGROUND/AIMS: The steroid hormones, including estradiol (E) and progesterone, act in the brain to regulate female reproductive behavior and physiology. These hormones mediate many of their biological effects by binding to their respective intracellular receptors. The receptors for estrogens (ER) and progestins (PR) interact with nuclear receptor coactivators to initiate transcription of steroid-responsive genes. Work from our laboratory and others reveals that nuclear receptor coactivators, including steroid receptor coactivator-1 (SRC-1) and SRC-2, function in brain to modulate ER-mediated induction of the PR gene and hormone-dependent behaviors. In order for steroid receptors and coactivators to function together, both must be expressed in the same cells. METHODS: Triple-label immunofluorescence was used to determine if E-induced PR cells also express SRC-1 or SRC-2 in reproductively relevant brain regions of the female mouse. RESULTS: The majority of E-induced PR cells in the medial preoptic area (61%), ventromedial nucleus of the hypothalamus (63%) and arcuate nucleus (76%) coexpressed both SRC-1 and SRC-2. A smaller proportion of PR cells expressed either SRC-1 or SRC-2, while a few PR cells expressed neither coactivator. In addition, compared to control animals, 17ß-estradiol benzoate (EB) treatment increased SRC-1 levels in the arcuate nucleus, but not the medial preoptic area or the ventromedial nucleus of the hypothalamus. EB did not alter SRC-2 expression in any of the three brain regions analyzed. CONCLUSIONS: Taken together, the present findings identify a population of cells in which steroid receptors and nuclear receptor coactivators may interact to modulate steroid sensitivity in brain and regulate hormone-dependent behaviors in female mice. Given that cell culture studies reveal that SRC-1 and SRC-2 can mediate distinct steroid-signaling pathways, the present findings suggest that steroids can produce a variety of complex responses in these specialized brain cells.
