Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts.

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.

Axotomy-induced motoneuron death is delayed in mice overexpressing PrPc.

The normal function of the cellular prion protein, PrP(c), remains largely unknown. Recently, PrP(c) has been implicated in the regulation of neuronal survival and was shown to confer neuroprotection in the brain. To pursue investigation of the role of PrP(c) in the CNS, we used the facial nerve section, a well-established experimental model of motoneuronal stress. Nerve sections were performed in 2- and 7-day-old newborn mice and in 2 month-old adult mice expressing different levels of PrP(c). We observed no differences in motoneuronal death triggered by facial nerve section between Prnp-/- and wild-type mice, whether in neonatal or adult mice. By contrast, overexpression of PrP(c) in Tga20 newborn mice was correlated with a better survival of motoneurons in the few days following axotomy. The protection was, however transient since motoneuron number in lesioned facial nuclei of Tga20 mice became identical to that of wild-type mice 7 days and 14 days following the lesion when performed in 2- and 7-day-old mice respectively. In Tga20 adult mice, no protection was observed 2 months after the lesion, a time with a significant degree of motoneuron death in adult control mice. These results, while providing further evidence that PrP(c) is endowed with neuroprotective capacity in vivo, also suggest that PrP(c) does not play a physiological role in the regulation of motoneuronal survival.

Transforming growth factor alpha: a promoter of motoneuron survival of potential biological relevance.

Expression of transforming growth factor alpha (TGFalpha), a member of the epidermal growth factor (EGF) family, is a general response of adult murine motoneurons to genetic and experimental lesions, TGFalpha appearing as an inducer of astrogliosis in these situations. Here we address the possibility that TGFalpha expression is not specific to pathological situations but may participate to the embryonic development of motoneurons. mRNA of TGFalpha and its receptor, the EGF receptor (EGFR), were detected by ribonuclease protection assay in the ventral part of the cervical spinal cord from embryonic day 12 (E12) until adult ages. Reverse transcription-PCR amplification of their transcripts from immunopurified E15 motoneurons, associated with in situ double-immunohistological assays, identified embryonic motoneurons as cellular sources of the TGFalpha-EGFR couple. In vitro, TGFalpha promoted the survival of immunopurified E15 motoneurons in a dose-dependent manner, with a magnitude similar to BDNF neuroprotective effects at equivalent concentrations. In a transgenic mouse expressing a human TGFalpha transgene under the control of the metallothionein 1 promoter, axotomy of the facial nerve provoked significantly less degeneration in the relevant motor pool of 1-week-old mice than in wild-type animals. No protection was observed in neonates, when the transgene exhibits only weak expression levels in the brainstem. In conclusion, our results point to TGFalpha as a physiologically relevant candidate for a neurotrophic role on developing motoneurons. Its expression by the embryonic motoneurons, which also synthesize its receptor, suggests that this chemokine is endowed with the capability to promote motoneuron survival in an autocrine-paracrine manner.

Early detection of mouse wobbler mutation: a model of pathological motoneurone death.

The mouse recessive mutation wobbler, carried by the C57BL/6J strain, is a naturally occurring model of motoneurone death. The gene is unknown and in the absence of predictive markers, mutants have to be diagnosed by phenotypic criteria at 4 weeks after birth. We localized the wobbler gene to chromosome 11 at 0.98 +/- 1.1 cM from the glutamine synthetase (Glns) gene. A polymorphic allele of the Glns gene was then introduced into the congenic wobbler strain by intraspecific crossing. One-quarter of the offspring expressed the same phenotypic mutation as true wobbler and were detectable by PCR, as they are homozygous for the wobbler-linked Glns allele. The new mutants exhibit motoneurone degeneration despite the new genetic background.

Differential modulation by glucocorticoids of alternative complement protein secretion in cells of the monocyte/macrophage lineage.

The effect of the synthetic glucocorticoid dexamethasone (DXM) on the secretion by human monocytes of alternative complement proteins C3, factor B and factor H was investigated. Results indicated that DXM modulates this secretion in a direction which would be consistent with its anti-inflammatory properties. DXM, at therapeutic concentrations, had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by monocytes. This differential modulation on C3, factor B and factor H secretion was similar in mature macrophages. Together with previous studies showing that DXM had a suppressive effect on C3 and factor B secretion and a stimulatory effect on factor H secretion by human endothelial cells, our results indicate that DXM appears to have the general property of regulating local production of complement components so as to control complement activation.

Activation of the endothelium by IL-1 alpha and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a.

Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinflammatory cytokine IL-1 alpha has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL-1 alpha and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL-1 alpha, significant potentialization of IL-1 alpha-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10(-7) to 10(-5) M for dexamethasone and 50-200 U for IL-1 alpha. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL-1 alpha on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-1 alpha was not observed for another marker of endothelial activation, IL-1 alpha-induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-1 alpha therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL-1 alpha, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-1 alpha and endogenous glucocorticoids coexist, such as in septic shock.

Bcl-2 sensitivity differentiates two pathways for motoneuronal death in the wobbler mutant mouse.

The molecular events leading to motoneuronal death are still poorly understood. In mammals, the bcl-2 proto-oncogene, which encodes a membrane-associated protein, has been shown to suppress both developmental motoneuronal death and experimental axotomy-induced motoneuronal death. We assessed a potential protective effect of Bcl-2 on pathological motoneuronal death processes in adult rodents. We took advantage of the murine mutant wobbler, which undergoes progressive degeneration of the spinal and brainstem motoneurons. A hybrid carrying both the wobbler mutation and the human bcl-2 transgene under the control of the neuron-specific enolase promoter was produced. Although Bcl-2 protected spinal and brainstem motoneurons from developmental death and the postnatal motoneurons of the facial nucleus from axotomy-induced death, the pathological motoneuronal death was not altered in the adult hybrid. These results demonstrate that Bcl-2 sensitivity distinguishes at least two different motoneuronal death pathways in the wobbler mutant. They support the hypothesis that experimental and pathological motoneuronal death are dependent on different cellular mechanisms.

Transforming growth factor alpha (TGF alpha) expression in degenerating motoneurons of the murine mutant wobbler: a neuronal signal for astrogliosis?

The enhanced expression of the trophic factor transforming growth factor alpha (TGF alpha) in reactive astrocytes following CNS injury suggests that TGF alpha has a role in the development of astrogliosis. We explored this hypothesis in the murine mutant wobbler, which presents a progressive motoneuronal degeneration associated with an astrogliosis. Evolution of astrogliosis, and expression of TGF alpha precursor (pro-TGF alpha) and of its receptor were examined over the course of the disease, using genetically diagnosed animals and immunocytochemical techniques. We report here that degenerating motoneurons of the cervical spinal cord and a subset of astrocytes express pro-TGF alpha, prior to the onset of astrogliosis, when the first clinical manifestations of the disease are observed at 4 weeks of age. TGF alpha expression appeared strongly correlated with motoneuronal degeneration. All pro-TGF alpha-immunoreactive neurons exhibited a degenerative morphology, and the number of pro-TGF alpha-immunoreactive neurons increased with the progression of the disease. At the glial level, we observed that astrogliosis was a transitory phenomenon in the wobbler mice, developing in coordination with the motoneuronal expression of pro-TGF alpha. Astrogliosis became evident in 6-week-old wobbler mice, when the number of pro-TGF alpha-immunoreactive motoneurons was maximal, and regressed in older mutant mice in correlation with the disappearance of pro-TGF alpha-immunoreactive motoneurons. Furthermore, TGF alpha/EGF receptor immunoreactivity was exclusively localized in a subset of reactive astrocytes, its expression following closely the course of the astrogliosis. These data show that TGF alpha synthesis by the affected motoneurons is an early event in the course of the wobbler disease, and suggest a role for TGF alpha as a neuronal inducer of astrocytic reactivity.

Regulation of growth factor gene expression in degenerating motoneurons of the murine mutant wobbler: a cellular patch-sampling/RT-PCR study.

Motoneuronal degenerative diseases are characterized by their progressivity; once affected, the motoneurons remain in altered states during an intermediate phase of degeneration prior to their final disappearance. Whether this survival period coincides with active metabolic rearrangements in the affected neuron remains unknown. As a first step toward the elucidation of this question, we developed cDNA pooled samples obtained from degenerating and control motoneuron mRNA populations through cellular patch sampling and RT-PCR, using the murine wobbler mutant as a model of spinal atrophy. Hybridization of the cDNA pools to various markers of intact or degenerating motoneurons allowed us to verify the cellular specificity of the patch sampling and indicated conservation of the original mRNA population complexity. Exploration of transcriptional alterations of genes encoding growth factors thought to be involved in motoneuronal development revealed that gene expression of the neurotrophin BDNF was induced in affected motoneurons, while expression of neurotrophin-3 was present in both neuronal types. Likewise, expression of a member of the epidermal growth factor (EGF) family, the neuregulin transcript sensory motor neuron-derived factor, was detected in both control and degenerating motoneurons, while transforming growth factor alpha, the functional homolog of EGF, was present only in the affected motoneurons. Immunohistochemical detection of corresponding proteins corroborated these observations. These results demonstrate that, during the course of their degeneration, motoneurons can initiate expression of novel genes which lead to the production of molecules endowed with trophic and/or differentiative properties for the neurons themselves and their glial environment. They also validate the use of the developed cDNA pooled samples for further exploration of transcriptional alterations taking place in degenerating motoneurons.

A role for transforming growth factor alpha as an inducer of astrogliosis.

TGFalpha is a member of the epidermal growth factor (EGF) family with which it shares the same receptor, the EGF receptor (EGFR). Synthesis of TGFalpha and EGFR in reactive astrocytes developing after CNS insults is associated with the differentiative and mitogenic effects of TGFalpha on cultured astrocytes. This suggests a role for TGFalpha in the development of astrogliosis. We evaluated this hypothesis using transgenic mice bearing the human TGFalpha cDNA under the control of the zinc-inducible metallothionein promoter. Expression levels of glial fibrillary acidic protein (GFAP) and vimentin and morphological features of astrocytes were used as indices of astroglial reactivity in adult transgenic versus wild-type mice provided with ZnCl2 in their water for 3 weeks. In the striatum, the hippocampus, and the cervical spinal cord, the three CNS areas monitored, transgenic mice displayed enhanced GFAP mRNA and protein levels and elevated vimentin protein levels. GFAP-immunoreactive astrocytes exhibited numerous thick processes and hypertrophied somata, which are characteristic aspects of reactive astrocytes. Their number increased additionally in the striatum and the spinal cord, but no astrocytic proliferation was observed using bromodeoxyuridine immunohistochemistry. Neither the morphology nor the number of microglial cells appeared modified. A twofold increase in phosphorylated EGFR was detected in the striatum and was associated with the immunohistochemical detection of numerous GFAP-positive astrocytes bearing the EGFR, suggesting a direct action of TGFalpha on astrocytes. Altogether, these results demonstrate that enhanced TGFalpha synthesis is sufficient to trigger astrogliosis throughout the CNS, whereas microglial metabolism is unaffected.

Ciliary neurotrophic factor may activate mature astrocytes via binding with the leukemia inhibitory factor receptor.

Ciliary neurotrophic factor (CNTF) acts on immature astrocytes that express its trimeric receptor. In contrast, mature astrocytes do not significantly express the specific CNTFalpha receptor subunit, yet they respond to CNTF administration in vivo. Here we show that this controversy may be solved by a shift in astroglial sensitivity to CNTF over time, related to a change in the type of receptor bound by the cytokine on mature astrocytes. A convergent set of results supports the hypothesis that the CNTF effect is due to the illegitimate binding on the leukemia inhibitory factor receptor (LIFR): (i) it requires high concentration of recombinant rat CNTF; (ii) it involves the Jak/Stat and Ras-MAPK pathways; (iii) it is preserved in CNTFRalpha-/- cells; (iv) it is potentiated by soluble CNTFRalpha added to the medium; and (v) it is significantly decreased by a partial antagonist of LIFR. On these bases, we propose a mechanistic model in which, in the adult brain, a CNTF/LIFR interglial system may be modulated by neurons that synthesize CNTFRalpha.