I. Nuclear receptor function
Genetic approaches to define the role of the estradiol receptor a in the regulation of the reproductive axis.
To define the role of the G protein-coupled receptor GPR54 in the estrogen-mediated feedback regulation of GnRH synthesis we follow two approaches. GPR54 expression is controlled by the decapeptide Kiss1 synthesized in the AVPV neurons expressing the estradiol receptor a. Since germ line mutants of GPRR54 are infertile and have a similar phenopype as the ERa forebrain-specific mutants we reason that ERa leads to increased synthesis of Kiss1 which binds and activates the GPR54 receptor in GnRH neurons. To test this hypothesis we use GPR54-/- mice (obtained from Schering Plough) in which we express GPR54 under control of the GnRH gene. We also generate mice with a floxed allele of the GPR54 gene which we will cross with forebrainspecific Cre-expressing mice. With these experiments we hope to provide conclusive evidence that the critical positive feedback action of estrogens upon GnRH neurons are mediated via the Kiss1/GPR54 system and that the action of the receptor is indirect.
Evaluation of corticosteroid receptor function in the brain
Corticosteroid hormones regulate a variety of developmental, physiological and pathological processes by interacting with specific receptors, the glucocorticoid and mineralocorticoid receptor. Using genetic technologies we have generated a panel of tissue-specific and function-selective mutations of the genes for these two corticosteroid hormone receptors in the mouse. These mouse models have allowed us to gain important insights in corticosteroid hormone function in the animal. Using these technologies we have been able to investigate steroid hormone receptor function, both in a tissue- or cell type-selective manner since germ line mutations for the glucocorticoid and mineralocorticoid receptor lead to lethality. We therefore have had a strong interest to develop conditional alleles for these two receptors. For example, using hepatocyte-specific mutations in the mouse we could show that the glucocorticoid receptor and Stat5 in hepatocytes are essential for normal postnatal growth. Surprisingly the glucocorticoid receptor does not bind to DNA but its activity is mediated through interaction with the Stat5 protein. To identify the function of the glucocorticoid receptor in allergic skin conditions we have analyzed contact hypersensitivity in various specific glucocorticoid receptor mutant mouse strains. We could establish that macrophages and neutrophils are the targets for immune suppression of glucocorticoids in contact allergy responses. To generate mutations of the gluco- and mineralocorticoid receptor in the adult brain we have developed mice which express an inducible Cre recombinase in neurons of the forebrain (Fig. 1).
|Fig. 1: Ablation of GR protein expression in neurons of the adult hippocampus upon tamoxifen treatment Vibratome sections of mice homozygous for a conditional GR allele (GRflox) and heterozygous for the inducible CaMKCreERT2 transgene (GRCaMKCreERT2 mice) have been analyzed for GR protein expression by immunohistochemistry.|
These mice with inducible mutations of the gluco- as well as the mineralocorticoid receptor, alone or in combination, are presently investigated to define the role of these receptors in functions like learning and memory, hypothalamic feedback control, and the process of addiction. Function-selective mutations combined with gene expression profiling will help to clarify the transcription mechanism involved in the action of corticosteroid hormones in vivo in the brain.
The nuclear receptor tailless (Tlx) is required for generation and maintenance of adult neural stem cells and participates in brain tumor formation
The tailless (Tlx) gene encodes an orphan nuclear receptor which is expressed in the periventricular neurogenic zone during mouse embryonic development. Mutant mice survive but display specific anatomical defects in the cortex and the limbic system. In particular late developing structures such as the upper cortical layers and the dendate gyrus are reduced in size due to depletion of progenitor cells in the subventricular zone. To determine the cell types which express the Tlx gene we have chosen an approach where a Cre fusion protein is used as a reporter for the Tlx gene. We have shown that the Tlx gene is expressed exclusively in adult neural stem cells, the B cells, but not any more in progenitors. Cell fate mapping experiments showed that Tlx-expressing cells are multipotent which can differentiate into neurons, astrocytes as well as into oligodentrocytes. Using BrdU-incorporation, GFAP and Cre staining we could show that these cells are able to selfrenew. Thus, Tlx is specifically expressed by long-term selfrenewing neural stem cells. Tlx-expressing cells also express CD133, a marker of brain tumor stem cells. Furthermore, we could show that loss of Tlx leads to upregulation of PTEN in the subventricular zone compatible with its function in control of neural stem cell proliferation. These findings establish Tlx as an indicator for and regulator of adult neural stem cells
|Fig. 2: Cre antibody staining of Tlx-CreERT2 in the subventricular zone (SVZ) and rostral migratory stream (RMS) of Tlx-CreERT2-expressing mice after tamoxifen induction.|
II. A new model for Parkinson's disease
Disruption of nucleolar function leads to compromised mitochondrial activity
We have reported that disruption of nucleolar integrity based on the ablation of the gene for the transcription initiator factor TIF-IA results in stabilization of p53 and p53-mediated apoptosis. These findings are extremely interesting since a crucial role for p53 has been debated in several neurodegenerative diseases. Disruption of nucleolar integrity in dopaminergic neurons following ablation of the transcription initiation factor TIF-IA generated mice with the major hallmarks of Parkinson´s disease: progressive and differential loss of dopaminergic neurons in the Substantia nigra and Ventral Tegmental Area, depletion of striatal dopamine content and responsiveness to L-DOPA treatment. The observation that constitutive and inducible mutations in the TIF-IA gene mimic so closely Parkinson´s disease symptoms, stimulated us to ask whether nucleolar impairment affects mitochondrial activity and induces oxidative stress. Ablation of TIF-IA in dopaminergic neurons leads to disruption of nucleolar organization as reflected by altered distribution of the protein nucleophosmin, which is specifically found in the nucleolus of wild type mice but distributed throughout the entire nucleus in the mutants. Further, disruption of nucleolar integrity due to loss of TIF-IA is associated with the deficiency in the activity of cytochrome c oxidase (COX), the terminal enzyme complex of the mitochondrial respiratory chain. We therefore concluded that nucleolar disruption following TIF-IA ablation leads to impairment of mitochondrial functions. As signs of oxidative damage we find a strong increase in the number of dopaminergic neurons positive for markers of nitrosylated proteins, neuroketals, which are products of oxydised membrane fatty acid components and oxydised DNA. We reason that p53 overexpression mediates the effects of nucleolar disruption on mitochondria.
|Fig. 3: The Cre recombinase fusion protein is expressed exclusively in B cells expressing GFAP but not in C or A cells expressing EGFR or doublecortin (DCX), resp.|