Bettina Holtmann

CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic cytokine as modulator in neuroinflammation

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). So far, immunological mechanisms responsible for demyelination have been the focus of interest. However, mechanisms regulating axon maintenance as well as glial precursor-cell proliferation and oligodendrocyte survival might also influence disease outcome. The cytokine ciliary neurotrophic factor (CNTF), which was originally identified as a survival factor for isolated neurons, promotes differentiation, maturation and survival of oligodendrocytes. To investigate the role of endogenous CNTF in inflammatory demyelinating disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in CNTF-deficient and wild-type C57BL/6 mice. Disease was more severe in CNTF-deficient mice and recovery was poor, with a 60% decrease in the number of proliferating oligodendrocyte precursor cells (OPCs) and a more than 50% increase in the rate of oligodendrocyte apoptosis. In addition, vacuolar dystrophy of myelin and axonal damage were more severe in CNTF-deficient mice. These specific pathological features could be prevented by treatment with an antiserum against tumor necrosis factor-α, suggesting that endogenous CNTF may counterbalance this effect of TNF-α (ref. 7). Here we identify a factor that modulates, in an inflammatory environment, glial cell survival and is an outcome determinant of EAE.

Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues.

Selenoprotein P (SePP), the major selenoprotein in plasma, has been implicated in selenium transport, selenium detoxification or antioxidant defence. We generated SePP-knockout mice that were viable, but exhibited reduced growth and developed ataxia. Selenium content was elevated in liver, but low in plasma and other tissues, and selenoenzyme activities changed accordingly. Our data reveal that SePP plays a pivotal role in delivering hepatic selenium to target tissues.

Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult

Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-β and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L–Cre; Stat3flox/KO mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

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.

Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-κB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Ciliary neurotrophic factor enhances the rate of oligodendrocyte generation

Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.

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.

Early onset of severe familial amyotrophic lateral sclerosis with a SOD-1 mutation: Potential impact of CNTF as a candidate modifier gene

Mutations in the copper/zinc superoxide dismutase 1 (SOD-1) gene are found in approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS), or amyotrophic lateral sclerosis 1. Here we describe a 25-year-old male patient who died from FALS after a rapid disease course of 11 mo. Sequencing of the SOD-1 gene revealed a heterozygous T-->G exchange at position 1513 within exon 5, coding for a V-->G substitution at position 148 of the mature protein. Genetic analysis of this family revealed the same mutation in both his healthy 35-year-old sister and his mother, who did not develop the disease before age 54 years. Screening for candidate modifier genes that might be responsible for the early onset and severe course of the disease in the 25-year-old patient revealed an additional homozygous mutation of the CNTF gene not found in his yet unaffected sister. hSOD-1G93A mice were crossbred with CNTF(-/-) mice and were investigated with respect to disease onset and duration, to test the hypothesis that CNTF acts as a candidate modifier gene in FALS with mutations in the SOD-1 gene. Such hSOD-1G93A/CNTF-deficient mice develop motoneuron disease at a significantly earlier stage than hSOD-1G93A/CNTF-wild-type mice. Linkage analysis revealed that the SOD-1 gene was solely responsible for the disease. However, disease onset as a quantitative trait was regulated by the allelic constitution at the CNTF locus. In addition, patients with sporadic amyotrophic lateral sclerosis who had a homozygous CNTF gene defect showed significantly earlier disease onset but did not show a significant difference in disease duration. Thus, we conclude that CNTF acts as a modifier gene that leads to early onset of disease in patients with FALS who have SOD-1 mutations, in patients with sporadic amyotrophic lateral sclerosis, and in the hSOD-1G93A mouse model.

The role of p75NTR in modulating neurotrophin survival effects in developing motoneurons

Neurotrophins exert their biological functions on neuronal cells through two types of receptors, the trk tyrosine kinases and the low‐affinity neurotrophin receptor (p75NTR), which can bind all neurotrophins with similar affinity. The p75NTR is highly expressed in developing motoneurons and in adult motoneurons after axotomy, suggestive of a physiological role in mediating neurotrophin responses under such conditions. In order to characterize this specific function of p75NTR, we have tested the effects of nerve growth factor (NGF) on embryonic motoneurons from control and p75NTR‐deficient mice. NGF antagonizes brain‐derived neurotrophic factor (BDNF)‐ and neurotrophin‐3 (NT‐3)‐mediated survival in control but not p75NTR‐deficient motoneurons. Survival of cultured motoneurons in the presence of 0.5 ng/mL of either ciliary neurotrophic factor (CNTF) or glial‐derived neurotrophic factor (GDNF) was not reduced by 20 ng/mL NGF. Dose–response investigations revealed that five times higher concentrations of BDNF are required for half‐maximal survival of p75NTR‐deficient motoneurons in comparison to motoneurons from wild‐type controls. After facial nerve lesion in newborn wild‐type mice, local administration of NGF reduced survival of corresponding motoneurons to less than 2% compared to the unlesioned control side. In p75NTR‐deficient mice, the same treatment did not enhance facial motoneuron death on the lesioned side. In the facial nucleus of 1‐week‐old p75NTR–/– mice, a significant reduction of motoneurons was observed at the unlesioned side in comparison to p75NTR +/+ mice. The observation that motoneuron cell numbers are reduced in the facial nucleus of newborn p75NTR‐deficient mice suggests that p75NTR might not function as a physiological cell death receptor in developing motoneurons.

Adenosine receptor A2A-R contributes to motoneuron survival by transactivating the tyrosine kinase receptor TrkB

Neurotrophins are potent survival factors for developing and injured neurons. However, they are not being used to treat neurodegenerative diseases because of difficulties in administration and numerous side effects that have been encountered in previous clinical trials. Their biological activities use Trk (tropomyosin-related kinase) transmembrane tyrosine kinases. Therefore, one alternative approach is to use transactivation pathways such as adenosine 2A receptor agonists, which can activate Trk receptor signaling independent of neurotrophin binding. However, the relevance in vivo and applicability of these transactivation events during neurodegenerative and injury conditions have never been extensively studied. Here we demonstrate that motoneuron survival after facial nerve lesioning is significantly enhanced by transactivation of Trk receptor tyrosine kinases by adenosine agonists. Moreover, survival of motoneurons directly required the activation of the BDNF receptor TrkB and an increase in Akt (AKT8 virus oncogene cellular homolog) activity. The ability of small molecules to activate a trophic response by using Trk signaling provides a unique mechanism to promote survival signals in motoneurons and suggests new strategies for using transactivation in neurodegenerative diseases.