ABSTRACT
During embryonic development, and in certain neurodegenerative diseases, neurons die by apoptosis. A new family of anti-apoptotic proteins, termed inhibitors of apoptosis (IAP), suppresses apoptosis through the direct inhibition of caspases. The anti-apoptotic activity of IAPs is inhibited by second mitochondria-derived activator of caspase (Smac)/DIABLO and XAF1 (ref. 8). IAPs, as well as neurotrophic factors, can protect degenerating neurons both in vivo and in vitro. However, the downstream targets of neurotrophic factors have not yet been identified. Here, we demonstrate that XIAP and NAIP, but not HIAP2, are directly involved in the intracellular response to glial cell-derived neurotrophic factor (GDNF). In newborn rats, GDNF regulates endogenous levels of XIAP and NAIP in motor neurons after sciatic nerve axotomy. The inhibition of XIAP or NAIP activity prevents GDNF-mediated neuroprotective effects. These results suggest that XIAP and NAIP are essential for intracellular signalling of GDNF in motor neuron survival.
Subject(s)
Motor Neurons/drug effects , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/pharmacology , Neuroprotective Agents/pharmacology , Proteins/metabolism , Animals , Apoptosis/physiology , Axotomy , Brain-Derived Neurotrophic Factor/pharmacology , Ciliary Neurotrophic Factor/pharmacology , Enzyme Inhibitors/metabolism , Glial Cell Line-Derived Neurotrophic Factor , Humans , Inhibitor of Apoptosis Proteins , Lumbar Vertebrae , Motor Neurons/cytology , Motor Neurons/metabolism , Motor Neurons/pathology , Nerve Growth Factors/pharmacology , Nerve Tissue Proteins/genetics , Neuronal Apoptosis-Inhibitory Protein , Proteins/genetics , Rats , Rats, Sprague-Dawley , Sciatic Nerve/cytology , Sciatic Nerve/drug effects , Sciatic Nerve/surgery , Spinal Cord/cytology , Spinal Cord/metabolism , X-Linked Inhibitor of Apoptosis ProteinABSTRACT
A murine model of motoneuron disease, the pmn/pmn mouse, shows a reduction in the retrograde transport of fluorescent probes applied directly onto the cut end of sciatic nerve. Brain-derived neurotrophic factor (BDNF), when co-applied with fluorescent tracers, increases the number of retrograde labelled motoneurons. We demonstrate here that spinal cord tissue from pmn/pmn mice had significantly reduced phosphatidylinositol 3-kinase activity and expression in the particulate fraction compared to controls, without changes in the activities or expression of the downstream kinases, protein kinase B/Akt or Erk1. Systemic administration of BDNF augmented phosphatidylinositol 3-kinase specific activity in spinal cord tissue from pmn/pmn and control mice, with a greater elevation in the particulate fractions of pmn/pmn mice than in controls. We examined the effect of inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase on the retrograde labelling of motoneurons, 24h following the direct application of inhibitors and Fluorogold to the cut end of sciatic nerve in control and pmn/pmn mice (labelling index). The mitogen-activated protein kinase kinase inhibitor PD 98059 had no effect on the labelling index in control or pmn/pmn mice. In the absence of exogenous BDNF, phosphatidylinositol 3-kinase inhibitors reduced the number of labelled motoneurons in control mice, without changing the labelling index in pmn/pmn. Co-application of phosphatidylinositol 3-kinase inhibitors with BDNF to the cut end of sciatic nerve blocked the action of BDNF on retrograde labelling in pmn/pmn mice. These results indicate that the retrograde labelling of motoneurons is mediated by phosphatidylinositol 3-kinase-dependent and -independent pathways. In pmn/pmn mice, phosphatidylinositol 3-kinase activity in spinal neurons is below the level required for optimal retrograde labelling of motoneurons and labelling can be augmented by the administration of growth factors stimulating phosphatidylinositol 3-kinase activity. The data indicate that phosphatidylinositol 3-kinase activity is important in the uptake and/or retrograde transport of substances by motoneurons and is altered in this model of motoneuron diseases.
Subject(s)
Motor Neuron Disease/enzymology , Motor Neurons/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases , Animals , Brain-Derived Neurotrophic Factor/pharmacology , Mice , Mice, Mutant Strains , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Ribosomal Protein S6 Kinases/metabolism , Spinal Cord/enzymologyABSTRACT
Apoptosis and mitochondrial dysfunction are thought to be involved in the aetiology of neurodegenerative diseases. We have tested an orally active anti-apoptotic molecule (CGP 3466B) that binds to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an animal model with motoneuron degeneration, i.e. a mouse mutant with progressive motor neuronopathy (pmn). In pmn/pmn mice, CGP 3466B was administered orally (10 - 100 nmol kg(-1)) at the onset of the clinical symptoms (2 weeks after birth). CGP 3466B slowed disease progression as determined by a 57% increase in life-span, preservation of body weight and motor performance. This improvement was accompanied by a decreased loss of motoneurons and motoneuron fibres as well as an increase in retrograde transport. Electron microscopic analysis showed that CGP 3466B protects mitochondria which appear to be selectively disrupted in the motoneurons of pmn/pmn mice. The data support evaluation of CGP 3466B as a potential treatment for motor neuron disease.
Subject(s)
Apoptosis/drug effects , Disease Models, Animal , Mitochondria/drug effects , Motor Neuron Disease/drug therapy , Nerve Growth Factors , Oxepins/pharmacology , Administration, Oral , Animals , Glial Cell Line-Derived Neurotrophic Factor , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Mice , Motor Neuron Disease/mortality , Motor Neurons/drug effects , Nerve Tissue Proteins/pharmacology , Weight Loss/drug effectsABSTRACT
There is accumulating evidence that cytokines are involved in the functioning of the brain and the spinal cord. However, it has been controversial whether they exert a neurotoxic or a neuroprotective effect. To address this question in vivo, we have examined the survival of injured motoneurons in a line of transgenic mice that overexpress the soluble form of tumour necrosis factor receptor-1 (sTNFR1). In these animals, all of the circulating TNF and lymphotoxin-alpha are neutralized by the continuous expression of the soluble receptor. Following axotomy of the facial nerve in 7-day-old control mice, we observed a loss of approximately 90% of the motoneurons at two weeks survival. In the transgenic mice under the same conditions, the percentage of motoneuron survival was increased two-fold (515 vs. 224) and varied as a function of the level of the circulating receptor. These results indicate that neutralization of endogenous TNF and lymphotoxin-alpha by means of overexpression of the soluble receptor can decrease cell death of injured motoneurons and suggest that these cytokines may play an important role in neuronal degeneration in the CNS following a lesion.
Subject(s)
Motor Neurons/cytology , Motor Neurons/physiology , Receptors, Tumor Necrosis Factor/physiology , Spinal Cord/cytology , Age Factors , Animals , Axotomy , Cell Survival/physiology , Facial Nerve/chemistry , Facial Nerve/physiology , Lymphotoxin-alpha/analysis , Mice , Mice, Inbred BALB C , Mice, Transgenic , Nerve Degeneration/physiopathology , Solubility , Transgenes/physiology , Tumor Necrosis Factor-alpha/analysisABSTRACT
Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.
Subject(s)
Cell Death/physiology , Insect Proteins/genetics , Motor Neurons/cytology , Proteins , Adenoviridae/genetics , Animals , Axotomy , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/pharmacology , Ciliary Neurotrophic Factor/genetics , Ciliary Neurotrophic Factor/pharmacology , Gene Expression/physiology , Humans , Inhibitor of Apoptosis Proteins , Motor Neurons/physiology , Nerve Degeneration/drug therapy , Nerve Degeneration/physiopathology , Neuroprotective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Sciatic Nerve/cytology , Sciatic Nerve/physiology , Spinal Cord/cytology , Transfection/methods , Transgenes/physiology , Ubiquitin-Protein LigasesABSTRACT
Nerve growth factor (NGF) promotes the survival of several neuronal populations, but recently it has also been shown to induce neuronal cell death. Here we report the effects of NGF on lesioned motoneurons. We have analyzed facial and sciatic motoneurons in newborn and adult BALB/c and C57BL/6 mice, in addition to mice deficient in the low-affinity p75 receptor for the neurotrophins (p75NTR). NGF application did not alter survival of lesioned facial motoneurons in any of the strains examined independent of the age of the animals. Only in the adult C57BL/6 mouse strain where the sciatic nerve had been crushed prior to factor application did NGF induce cell death of axotomized sciatic motoneurons. Our results illustrate the importance of the genetic background and the motoneuron sub-type in studies related to cell death and survival of motoneurons in relation to NGF and p75NTR.
Subject(s)
Cell Death/drug effects , Facial Nerve/cytology , Motor Neurons/cytology , Nerve Growth Factor/pharmacology , Sciatic Nerve/cytology , Animals , Blotting, Western , Cell Survival/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Motor Neurons/chemistry , Motor Neurons/drug effects , Nerve Crush , Receptor, Nerve Growth Factor/analysis , Receptor, Nerve Growth Factor/metabolism , Species SpecificityABSTRACT
It has been shown that abnormalities in axonal transport occur in several mouse models with motoneuron degeneration and also in the human disease amyotrophic lateral sclerosis. In this report, we have examined the potential of neurotrophic factors to act on axonal transport properties in a mouse mutant, progressive motor neuronopathy (pmn). This mouse mutant has been characterized as a "dying-back" motoneuronopathy, with a loss of motoneuron cell bodies and motor fibers. Retrograde transport to the spinal cord motoneurons was determined using fluorescent tracers either injected into the gastrocnemius muscle or applied directly onto the cut sciatic nerve. Because the rate of retrograde labeling was significantly reduced in the pmn, we examined the potential of neurotrophic factors to compensate for the impairment. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) but not glial-derived neurotrophic factor (GDNF) or nerve growth factor (NGF) were capable of significantly improving the rate of labeling. The differential effects of these factors agree with previous studies showing that molecules that promote cell survival do not necessarily compensate for axonal deficiency. Because impairment of axonal properties appears as an early event in motoneuron pathology, our results may have important clinical implications in the treatment of motoneuron diseases.