Christoph Kellendonk

Disruption of CREB function in brain leads to neurodegeneration

Control of cellular survival and proliferation is dependent on extracellular signals and is a prerequisite for ordered tissue development and maintenance. Activation of the cAMP responsive element binding protein (CREB) by phosphorylation has been implicated in the survival of mammalian cells. To define its roles in the mouse central nervous system, we disrupted Creb1 in brain of developing and adult mice using the Cre/loxP system. Mice with a Crem−/− background and lacking Creb in the central nervous system during development show extensive apoptosis of postmitotic neurons. By contrast, mice in which both Creb1 and Crem are disrupted in the postnatal forebrain show progressive neurodegeneration in the hippocampus and in the dorsolateral striatum. The striatal phenotype is reminiscent of Huntington disease and is consistent with the postulated role of CREB-mediated signaling in polyglutamine-triggered diseases.

Genetic dissection of glucocorticoid receptor function in mice

Upon hormone binding, the activated glucocorticoid receptor (GR) functions as a transcription factor via different modes of action to control gene expression. Recent gene-targeting studies in mice provide new insight into the role of GR in vivo and are helping decipher the molecular mechanisms underlying its actions.

Genetic disruption of mineralocorticoid receptor leads to impaired neurogenesis and granule cell degeneration in the hippocampus of adult mice

To dissect the effects of corticosteroids mediated by the mineralocorticoid (MR) and the glucocorticoid receptor (GR) in the central nervous system, we compared MR−/− mice, whose salt loss syndrome was corrected by exogenous NaCl administration, with GR−/− mice having a brain‐specific disruption of the GR gene generated by the Cre/loxP‐recombination system. Neuropathological analyses revealed a decreased density of granule cells in the hippocampus of adult MR−/− mice but not in mice with disruption of GR. Furthermore, adult MR−/− mice exhibited a significant reduction of granule cell neurogenesis to 65% of control levels, possibly mediated by GR due to elevated corticosterone plasma levels. Neurogenesis was unaltered in adult mice with disruption of GR. Thus, we could attribute long‐term trophic effects of adrenal steroids on dentate granule cells to MR. These MR‐related alterations may participate in the pathogenesis of hippocampal changes observed in ageing, chronic stress and affective disorders.

Inactivation of the GR in the Nervous System Affects Energy Accumulation

The homeostatic regulation of body weight protects the organism from the negative consequences of starvation and obesity. Glucocorticoids (GCs) modulate this regulation, although the underlying mechanisms remain unclear. To address the role of central GRs in the regulation of energy balance, we studied mice in which GRs have selectively been inactivated in the nervous system. Mutant mice display marked growth retardation. During suckling age this is associated with normal fat deposition causing a 60% temporary increase of percent body fat, compared with control littermates. After weaning, fat and protein depositions are reduced so that adults are both smaller and leaner than their controls. Decreased food intake and, after weaning, reduced metabolic efficiency account for these developmental disturbances. Plasma levels of leptin and insulin, two important energy balance regulators, are elevated in young mutants but normal in adults. Leptin/body fat ratio is higher at all ages, suggesting disturbed control of circulating leptin as a consequence of chronically elevated GC levels in mutant animals. Adult mutants display increased hypothalamic CRH and NPY levels, but peptide levels of melanin concentrating hormone and Orexin A and B are unchanged. The increased levels of plasma GCs and hypothalamic CRH may act as catabolic signals most likely leading to persistently reduced energy accumulation. (Endocrinology 143: 2333–2340, 2002)

New Insights into Glucocorticoid and Mineralocorticoid Signaling: Lessons from Gene Targeting

Publisher Summary Despite the power of classical gene targeting, this method suffers from the lack of spatial, temporal, and functional selectivity of the introduced mutations. This sometimes renders data interpretation difficult, as distinguishing between direct and indirect effects of gene inactivation is not always possible. Compensatory mechanisms and early lethality of the mutants can prevent the identification of some physiological functions of the receptors. Therefore, it is desirable to generate mouse strains, carrying more refined mutations, in the genes of interest, thereby limiting the effect of the mutation to only certain cell types or functional properties of the receptor. This has been achieved by knock-in approaches, using the Cre-loxP system, leading to somatic mutations, gene replacements, or introduction of point mutations. This chapter discusses mouse mutants for the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) with emphasis on how these mice allow new insights into function and molecular mechanism of these genes. At present, the research is on the analysis of cell-type and function-specific mutations in genes for which null alleles has already been generated. Based on the experience gained, with the analysis of knockout mice, the studies going on can yield even more fascinating results.

Mutagenesis of the glucocorticoid receptor in mice

The glucocorticoid receptor is an ubiquitously expressed transcription factor involved in the regulation of many different physiological processes. Activated by glucocorticoids the receptor regulates transcription positively or negatively either by direct binding to DNA or by protein protein interactions. In order to define the role of the receptor during development and in physiology several mutations have been generated in the mouse. Mice with a disrupted glucocorticoid receptor gene die shortly after birth due to respiratory failure indicating an important role of the receptor in lung function. Transcription of genes encoding gluconeogenic enzymes in the liver is decreased, proliferation of erythroid progenitors is impaired and the HPA axis is strongly upregulated. To analyze molecular mechanisms of glucocorticoid receptor action in vivo a point mutation has been introduced into the mouse genome which allows to separate DNA-binding-dependent from DNA-binding-independent actions of the receptor. Mice homozygous for the point mutation survive indicating that DNA-binding of the receptor is not required for survival. Induction of glucoconegenic enzymes and proliferation of erythroid progenitors however is impaired. Interestingly, repression of corticotropin releasing factor (CRF) synthesis is maintained, whereas proopiomelanocortin (POMC) expression is upregulated. Since mice with a disrupted glucocorticoid receptor gene die shortly after birth attempts using the Cre/loxP-recombination system are made to bypass early lethality and to study the function of the receptor in defined cell types of adult animals.

Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors.

Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post‐translationally inducible Cre‐Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells.