Rudolf Götz

Cryptic physiological trophic support of motoneurons by LIF revealed by double gene targeting of CNTF and LIF

BACKGROUND The survival and differentiation of motoneurons during embryonic development, and the maintenance of their function in the postnatal phase, are regulated by a great variety of neurotrophic molecules which mediate their effects through different receptor systems. The multifactorial support of motoneurons represents a system of high security, because the inactivation of individual ligands has either no detectable, or relatively small, atrophic or degenerative effect on motoneurons. RESULTS Leukaemia inhibitory factor (LIF) has been demonstrated to support motoneuron survival in vitro and in vivo under different experimental conditions. However, when LIF was inactivated by gene targeting, there were no apparent changes in the number and structure of motoneurons and no impairment of their function. The slowly appearing, relatively mild degenerating effects in motoneurons that resulted from ciliary neurotrophic factor (CNTF) gene targeting were substantially potentiated by simultaneous inactivation of the LIF gene, however. Thus, in mice deficient in LIF and CNTF, the degenerative changes in motoneurons were more extensive and appeared earlier. These changes were also functionally reflected by a marked reduction in grip strength. CONCLUSIONS Degenerative disorders of the nervous system, in particular those of motoneurons, may be based on multifactorial inherited and/or acquired defects which individually do not result in degenerative disorders, but which become apparent when additional (cryptic) inherited disturbances or sub-threshold concentrations of noxious factors come into play. Accordingly, the inherited inactivation of the CNTF gene in a high proportion of the Japanese population may represent a predisposing factor for degenerative disorders of motoneurons.

Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells

Bag1 is a cochaperone for the heat-shock protein Hsp70 that interacts with C-Raf, B-Raf, Akt, Bcl-2, steroid hormone receptors and other proteins. Here we use targeted gene disruption in mice to show that Bag1 has an essential role in the survival of differentiating neurons and hematopoietic cells. Cells of the fetal liver and developing nervous system in Bag1−/− mice underwent massive apoptosis. Lack of Bag1 did not disturb the primary function of Akt or Raf, as phosphorylation of the forkhead transcription factor FKHR and activation of extracellular signal–regulated kinase (Erk)-1/2 were not affected. However, the defect was associated with the disturbance of a tripartite complex formed by Akt, B-Raf and Bag1, in addition to the absence of Bad phosphorylation at Ser136. We also observed reduced expression of members of the inhibitor of apoptosis (IAP) family. Our data show that Bag1 is a physiological mediator of extracellular survival signals linked to the cellular mechanisms that prevent apoptosis in hematopoietic and neuronal progenitor cells.

The anti-apoptotic protein ITA is essential for NGF-mediated survivalof embryonic chick neurons

The avian ITA is homologous to the baculoviral and mammalian inhibitor of apoptosis (IAP) proteins, which can prevent apoptosis by inhibition of specific caspases. We investigated the role of ITA in embryonic chick sympathetic and dorsal root ganglionic neurons, which depend on nerve growth factor (NGF) for their survival. Within 6 hours, NGF upregulated ITA protein production more than 25-fold in sensory and sympathetic neurons. Overexpression of ITA in primary neurons supported survival of these cells in the absence of NGF, and ita antisense constructs inhibited NGF-mediated survival. Thus the induction of ITA expression seems to be an essential signaling event for survival of sympathetic and dorsal root ganglionic sensory neurons in response to NGF.

Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure

BackgroundSignaling networks promoting cell growth and proliferation are frequently deregulated in cancer. Tumors often are highly dependent on such signaling pathways and may become hypersensitive to downregulation of key components within these signaling cascades. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus and provides attractive molecular targets for cancer treatment. For example, Ras and Raf kinase inhibitors are already in a number of ongoing phase II and phase III clinical trials. In this study the effect of the Raf kinase inhibitor BAY 43-9006 and of the MEK inhibitor CI-1040 (PD184352) on a Raf dependent lung tumor mouse model was analyzed in detail.MethodsWe have generated a lung cancer mouse model by targeting constitutively active C-Raf kinase to the lung. These mice develop adenomas within 4 months of life. At this time-point they received daily intraperitoneal injections of either 100 mg/kg BAY 43-9006 or CI-1040 for additional 21 days. Thereafter, lungs were isolated and the following parameters were analyzed using histology and immunohistochemistry: overall lung structure, frequency of adenoma foci, proliferation rate, ERK activity, caspase-3 activation, and lung differentiation.ResultsBoth inhibitors were equally effective in vitro using a sensitive Raf/MEK/ERK ELISA. In vivo, the systemic administration of the MEK inhibitor CI-1040 reduced adenoma formation to a third and significantly restored lung structure. The proliferation rate of lung cells of mice treated with CL-1040 was decreased without any obvious effects on differentiation of pneumocytes. In contrast, the Raf inhibitor BAY 43-9006 did not influence adenoma formation in vivo.ConclusionThe MEK inhibitor CI-1040 may be used for the treatment of Ras and/or Raf-dependent human malignancies.

Lin9, a Subunit of the Mammalian DREAM Complex, Is Essential for Embryonic Development, for Survival of Adult Mice, and for Tumor Suppression

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) and related p107 and p130 “pocket proteins” function together with the E2F transcription factors to repress gene expression during the cell cycle and development. Recent biochemical studies have identified the multisubunit DREAM pocket protein complexes in Drosophila melanogaster and Caenorhabditis elegans in regulating developmental gene repression. Although a conserved DREAM complex has also been identified in mammalian cells, its physiological function in vivo has not been determined. Here we addressed this question by targeting Lin9, a conserved core subunit of DREAM. We found that LIN9 is essential for early embryonic development and for viability of adult mice. Loss of Lin9 abolishes proliferation and leads to multiple defects in mitosis and cytokinesis because of its requirement for the expression of a large set of mitotic genes, such as Plk1, Aurora A, and Kif20a. While Lin9 heterozygous mice are healthy and normal, they are more susceptible to lung tumorigenesis induced by oncogenic c-Raf than wild-type mice. Together these experiments provide the first direct genetic evidence for the role of LIN9 in development and mitotic gene regulation and they suggest that it may function as a haploinsufficient tumor suppressor.

BAG-1 haplo-insufficiency impairs lung tumorigenesis

BackgroundBAG-1 is a multifunctional co-chaperone of heat shock proteins (Hsc70/Hsp70) that is expressed in most cells. It interacts with Bcl-2 and Raf indicating that it might connect protein folding with other signaling pathways. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial.MethodsWe have evaluated the effect of BAG-1 heterozygosity in mice in a model of non-small-cell lung tumorigenesis with histological and molecular methods. We have generated mice heterozygous for BAG-1, carrying a BAG-1 null allele, that in addition express oncogenic, constitutively active C-Raf kinase (SP-C C-Raf BxB) in type II pneumocytes. SP-C C-Raf BxB mice develop multifocal adenomas early in adulthood.ResultsWe show that BAG-1 heterozygosity in mice impairs C-Raf oncogene-induced lung adenoma growth. Lung tumor initiation was reduced by half in BAG-1 heterozygous SP-C C-Raf BxB mice compared to their littermates. Tumor area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies. Whereas BAG-1 heterozygosity did not affect the rate of cell proliferation or signaling through the mitogenic cascade in adenoma cells, it increased the rate of apoptosis.ConclusionReduced BAG-1 expression specifically targets tumor cells to apoptosis and impairs tumorigenesis. Our data implicate BAG-1 as a key player in oncogenic transformation by Raf and identify it as a potential molecular target for cancer treatment.

Cortical migration defects in mice expressing A-RAF from the B-RAF locus

ABSTRACT We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAFKIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAFKIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.

Raf kinases in lung tumor development.

The specific response of a cell to an extracellular signal is mediated by signaling cascades that relay the excitation of one or more receptor(s) to the co-ordinated activity of a set of effector molecules which regulate cell division and growth and prevent an intrinsic cell death program. The small G protein Ras is an essential component in the transduction of extracellular signals. Raf is a serine/threonine protein kinase and a direct downstream effector of Ras, which is activated to its GTP-bound state in response to various ligands binding to their cognate receptors. Raf kinases are located at the head of one of the best characterized signaling cascades, the Raf-MEK-ERK kinase cascade which is involved in many signaling processes (Fig. 1). In multicellular organisms, the family of Raf kinases consists of three members, ARaf, BRaf and CRaf. Although all of these Raf kinases phosphorylate MEK, they differ in the way they are activated (Hagemann and Rapp, 1999). For example, cAMP potentiates BRaf kinase activity but inhibits CRaf (Erhardt et al., 1995; Cook and McCormick, 1993; Dhillon et al., 2002). Genetic and biochemical data also indicate that isoform specific functions exist for Raf kinases which correlate with specific protein interactions (Hagemann and Rapp, 1999). Recent findings regarding interaction of Raf with lipid membrane microdomains (Hekman et al., 2002; Raabe and Rapp, 2002) and the discovery of Rab binding scaffold proteins that may be regulated by MAP kinases in their ability to activate actin comet formation for vesicle transport (Kerkhoff et al., 2001; Eden et al., 2002) have suggested to us a revised model for membrane translocation of Raf (Fig. 2). The model incorporates suggestions on the role of rafts in protein sorting (Simons and Ikonen, 1997) and anticipates a self organizing element in the maturation of signaling complexes. As pictured in Fig. 2, joint sorting of Raf, Ras and upstream signal regulators, e.g., growth factor receptors together with elements of the Ras cycle as well as effectors of Ras/Raf signaling such as MEK and ERK (perhaps

Regulation of neuronal cell death and differentiation by NGF and IAP family members

Nerve growth factor (NGF) and other neurotrophins were identified because of their trophic role for distinct populations of neurons in the peripheral nervous system. We know that neuronal cell death is regulated by a genetically encoded programme, called apoptosis, that is conserved from worms to humans. Dysregulation of this programme is thought to contribute to neurodegenerative diseases which are characterized by the loss of neurons. This article will review recent findings about the motoneuron disease spinal muscular atrophy (SMA). Two closely linked candidate genes for SMA, the SMN (survival motor neuron) gene and the NAIP (neuronal apoptosis inhibitory protein) gene have been reported. The SMN protein forms a complex with several other proteins and this complex containing SMN plays a critical role in the assembly of spliceosomes and in pre-mRNA splicing. NAIP, c-IAP1 (inhibitor of apoptosis-1), c-IAP2, X-IAP and survivin comprise the mammalian inhibitor of apoptosis family. Its members can protect mammalian cells from apoptosis induced by a variety of stimuli. Some of the IAP molecules have been shown to interact both with cell signalling molecules and with specific caspases but details concerning their cellular role are only incompletely characterized.

Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum

This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.