Hsp27 upregulation and phosphorylation is required for injured sensory and motor neuron survival.

Peripheral nerve transection results in the rapid death by apoptosis of neonatal but not adult sensory and motor neurons. We show that this is due to induction and phosphorylation in all adult axotomized neurons of the small heat shock protein Hsp27 and the failure of such induction in most neonatal neurons. In vivo delivery of human Hsp27 but not a nonphosphorylatable mutant prevents neonatal rat motor neurons from nerve injury-induced death, while knockdown in vitro and in vivo of Hsp27 in adult injured sensory neurons results in apoptosis. Hsp27's neuroprotective action is downstream of cytochrome c release from mitochondria and upstream of caspase-3 activation. Transcriptional and posttranslational regulation of Hsp27 is necessary for sensory and motor neuron survival following peripheral nerve injury.

An inhibitor of serine proteases, neuroserpin, acts as a neuroprotective agent in a mouse model of neurodegenerative disease.

Various studies suggest that proteolytic activity may be involved in a number of neurodegenerative disorders, including stroke and seizure. In this report, we examined the role of tryptic serine proteases, plasminogen activators (PAs), in the evolution of a neurodegenerative disease. Transgenic mice overexpressing an axonally secreted inhibitor of serine proteases (neuroserpin) were crossed with mice characterized by a "dying-back" motor neuron disease [progressive motor neuronopathy (pmn/pmn)]. Compared with pmn/pmn mice that showed an increase in PA activity, double mutant mice had decreased PA activity in sciatic nerves and spinal cord; their lifespan was increased by 50%, their motor behavior was stabilized, and histological analysis revealed increased numbers of myelinated axons and rescue of motoneuron number and size. This is the first report showing that a class of serine proteases (PAs) may be involved in the pathogenesis of a motor neuron disease and more specifically in axonal degeneration. Inhibiting serine proteases could offer a new strategy for delaying these disorders.

Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease.

Apoptosis is a hallmark of motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) [1]. In a widely used mouse model of motoneuron disease (progressive motor neuronopathy or pmn) [2-4], transgenic expression of the anti-apoptotic bcl-2 gene [5] or treatment with glial cell-derived neurotrophic factor [6] prevents the apoptosis of the motoneuron soma; however, they were unable to affect the life span of the animals. The goal of the present work was to determine whether the pmn phenotype could be rescued by means of a gene that inhibits axon degeneration. For this reason, the pmn mice were crossed with mice bearing the dominant Wlds ("slow Wallerian degeneration") mutation, which slows axon degeneration and synapse loss [7-9]. We show here that the Wlds gene product attenuates symptoms, extends life span, prevents axon degeneration, rescues motoneuron number and size, and delays retrograde transport deficits in pmn/pmn mice. These results suggest new pathogenic mechanisms and therapeutic avenues for motoneuron diseases.

Grafted neural stem cells increase the life span and protect motoneurons in pmn mice.

In this study, we have grafted neural stem cells (NSCs) into the lumbar spinal cord of a mouse mutant that has a specific loss of motoneurons (progressive motor neuronopathy/pmn). A small number of grafted cells ( approximately 3000) increased the life span of the mice by 56%. The improved survival was accompanied by a rescue of host motoneurons, a stabilization in the weight and an increase in the size of the muscle fibers. The grafted NSCs were small and round and exhibited no neural markers, suggesting that they remained in an undifferentiated state. Thus grafting of NSCs in a mouse model with motoneuron degeneration exerts a neuroprotective effect.

Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain.

Nociceptors are a particular subtype of dorsal root ganglion (DRG) neurons that detect noxious stimuli and elicit pain. Although recent efforts have been made to reveal the molecular profile of nociceptors in normal conditions, little is known about how this profile changes in pathological conditions. In this study we exploited laser capture microdissection to specifically collect individual injured and non-injured nociceptive DRG neurons and to define their gene profiling in rat spared nerve injury (SNI) model of neuropathic pain. We found minimal transcriptional changes in non-injured neurons at 7 days after SNI. In contrast, several novel transcripts were altered in injured nociceptors, and the global signature of these LCM-captured neurons differed markedly from that the gene expression patterns found previously using whole DRG tissue following SNI. Pathway analysis of the transcriptomic profile of the injured nociceptors revealed oxidative stress as a key biological process. We validated the increase of caspase-6 (CASP6) in small-sized DRG neurons and its functional role in SNI- and paclitaxel-induced neuropathic pain. Our results demonstrate that the identification of gene regulation in a specific population of DRG neurons (e.g., nociceptors) is an effective strategy to reveal new mechanisms and therapeutic targets for neuropathic pain from different origins.

Motoneuron resistance to apoptotic cell death in vivo correlates with the ratio between X-linked inhibitor of apoptosis proteins (XIAPs) and its inhibitor, XIAP-associated factor 1.

Apoptotic cell death occurs in motoneurons in the neonate but not in the adult after a lesion of a peripheral nerve. To investigate the molecular basis for this difference, we have analyzed the expression and localization of inhibitors of apoptosis proteins (IAPs) and their inhibitors X-linked IAP (XIAP)-associated factor 1 (XAF1), Smac/DIABLO, and Omi/HtrA2 in motoneurons at both ages. Quantitative immunohistochemical and immunoblotting analysis of these proteins in motoneurons revealed an increase in IAP expression [XIAP, neuronal apoptosis inhibitory protein, human IAP1 (HIAP1), and HIAP2] during postnatal development as opposed to XAF1, which decreased during the same period; there was no significant alteration in either Smac/DIABO or Omi/HtrA2. The regulation of IAPs and XAF1 varied after axotomy of the sciatic nerve; in the neonate, there was a significant loss of IAP in the injured motoneurons as opposed to the adult, in which there was only a moderate decrease. By overexpressing exogenous IAPs in neonatal axotomized motoneurons, it was possible to delay motoneuron cell death (Perrelet et al., 2000, 2002). In opposition, the overexpression of exogenous XAF1 in adult motoneurons totally abrogated the natural resistance of these cells to axotomy. The degradation in the adult, induced by XAF1, could be overcome by simultaneously expressing high levels of exogenous XIAP in adult motoneurons. These experiments suggest that it may be the ratio between XAF1 and XIAP that confers the resistance of adult motoneurons to axotomy. In addition, the regulation in the levels of IAPs and XAF1 may be essential in the cell death mechanism of injured motoneurons.

Fas/tumor necrosis factor receptor death signaling is required for axotomy-induced death of motoneurons in vivo.

Activation of the Fas death receptor leads to the death of motoneurons in culture. To investigate the role of Fas in programmed cell death and pathological situations, we used several mutant mice deficient for Fas signaling and made a novel transgenic FADD-DN (FAS-associated death domain-dominant-negative) strain. In vitro, motoneurons from all of these mice were found to be resistant to Fas activation and to show a delay in trophic deprivation-induced death. During normal development in vivo, no changes in motoneuron survival were observed. However, the number of surviving motoneurons was twofold higher in animals deficient for Fas signaling after facial nerve transection in neonatal mice. These results reveal a novel role for Fas as a trigger of axotomy-induced death and suggest that the Fas pathway may be activated in pathological degeneration of motoneurons.

Rapid and reproducible methods using fluorogold for labelling a subpopulation of cervical motoneurons: application in the wobbler mouse.

A murine model of motoneuron disease, the wobbler mouse, is characterized by a selective loss of cervical spinal cord motoneurons. To determine the number of motoneurons that degenerate in mice with ongoing disease, we have developed two rapid and reproducible methods for labelling specific pools of cervical motoneurons using the retrograde tracer fluorogold. The motoneurons can be labelled either by capsule application of the tracer onto the sectioned musculo-cutaneous, median and ulnar nerves or by intramuscular (i.m.) injection of the tracer into the biceps brachii muscle and flexor muscles of the forelimb. In wild-type animals, the largest number of retrogradely labelled motoneurons was found 4 days following capsule application ( approximately equal 1900 motoneurons labelled) and 6 days after i.m. injection ( approximately equal1500 motoneurons labelled). Application of these techniques in 5 week-old wobbler mice showed a 36% loss of motoneurons 4 days following tracer application to the cut nerves and a 16% loss 6 days after i.m. injections as compared to values obtained in age-matched wild-type animals in the same conditions. Our results indicate that these procedures can be applied to any rodent model to analyse quantitatively the loss of specific subpopulations of cervical motoneurons and are valuable tools for evaluating novel therapeutics.

Differential vulnerability of cranial motoneurons in mouse models with motor neuron degeneration.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of selective motoneuron populations, yet it remains unclear why some groups of motoneurons are more vulnerable than others. Our aim was to compare the motoneuron loss in five cranial nuclei at different stages of the disease in three mouse models of ALS: two naturally occurring murine models (progressive motor neuronopathy (pmn) and wobbler) and a transgenic mouse model with a human G93A mutation in the superoxide dismutase-1 (SOD1) gene. By quantifying these different motoneuron populations we report that the degree of degeneration in the various cranial motoneuron nuclei depends on the mouse model and the stage of the disease. The biologically most significant difference between the mutations occurs in the oculomotor/trochlear nucleus which is affected in the pmn mouse but not in the wobbler and SOD G93A mice. These results suggest that there is a selective degeneration of cranial motoneurons in these mouse models as in ALS patients.

Axonal involvement in the Wlds neuroprotective effect: analysis of pure motoneurons in a mouse model protected from motor neuron disease at a pre-symptomatic age.

The identification of the Wlds gene that delays axonal degeneration in several models of neurodegenerative disease provides an interesting tool to study mechanisms of axonal loss. We showed that crossing a mouse mutant with a motoneuron disease (pmn for progressive motor neuronopathy) with mice that express the Wlds gene delayed axonal loss, increased the life span, partially rescued axonal transport deficit and prolonged the survival of the motoneuron cell bodies. To determine factors involved in the neuroprotective effect of Wlds, we combined laser capture microdissection and microarray analysis to identify genes that are differentially regulated at a pre-symptomatic age in motoneuron cell bodies in pmn/pmn,Wlds/Wlds mice as compared with pmn/pmn mice. Only 56 genes were de-regulated; none of the 'classical' genes implicated in apoptosis were de-regulated. Interestingly, a large proportion of these genes are related to axonal function and to retrograde and anterograde transport (i.e. members of the dynactin complex and kinesin family). These results were confirmed by real-time PCR, in situ hybridization and at protein level in sciatic nerves. Thus, genes related to axonal function and in particular to axonal transport may be involved at an early stage in the neuroprotective property of the Wlds gene and confirm the importance of axonal involvement in this model of motor neuron disease.

The neuroprotective effects of the WldS gene are correlated with proteasome expression rather than apoptosis.

The Wld(s) gene (slow Wallerian degeneration) specifically delays axonal degeneration following injury and in several models of neurodegenerative diseases. It thus provides an interesting tool to study mechanisms of neurodegeneration. We previously crossed the Wld(s) mice with a mouse mutant that has a motoneuron disease (pmn for progressive motor neuronopathy) and showed that the Wld(s) gene prevented axonal loss, increased the life-span and prolonged the survival of the motoneuron cell bodies. In this study we show that spinal motoneurons of pmn/pmn mice, as opposed to axons, die by apoptosis that cannot be prevented by the Wld(s) gene. However, this same gene could partially rescue the proteasome impairment observed in motoneuron cell bodies and axons of pmn/pmn mice. We conclude that the neuroprotective effect of the Wld(s) gene is not related to an inhibition of apoptosis but could possibly be linked to a regulation in proteasome expression.

Cell death pathways differ in several mouse models with motoneurone disease: analysis of pure motoneurone populations at a presymptomatic age.

To identify candidate genes that are responsible for motoneurone degeneration, we combined laser capture microdissection with microarray technology. We analysed gene expression in pure motoneurones from two mouse mutants that develop motoneurone degeneration, progressive motor neuronopathy and wobbler. At a presymptomatic age, there was a significant differential expression of a restricted number of genes (25 and 72 in progressive motor neuronopathy and wobbler respectively, of 22 600 transcripts screened). We compared these results to our previous analyses in the copper-zinc superoxide dismutase mutant mouse (SOD1(G93A)) in which we observed a de-regulation of 27 genes. Some of these genes were de-regulated uniquely in one mouse mutant and some have already been identified in cell death pathways implicated in amyotrophic lateral sclerosis and animal models of motoneurone degeneration (i.e. de-regulation of intermediate filaments, axonal transport, the ubiquitin-proteasome system and excitotoxicity). One gene, vimentin, was differentially up-regulated in all mouse mutants; this main candidate gene has been confirmed by in situ hybridization and immunohistochemistry to be expressed in motoneurones in all mouse mutants. Furthermore, vimentin expression correlated with the state of motoneurone degeneration. These results identify early molecular changes that may be involved in the pathogenesis of motoneurones leading to cell death and favour a complex multipathway induction of the disease; surprisingly, there was no important modification in cell death-associated genes. This is the first study to show a clear difference in the genes that are de-regulated at an early stage in three different mouse models of motoneurone disease.

No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model.

To identify candidate genes that may be involved in motoneuron degeneration, we combined laser capture microdissection with microarray technology. Gene expression in motoneurons was analyzed during the progression of the disease in transgenic SOD1(G93A) mice that develop motoneuron loss. Three major observations were made: first, there was only a small number of genes that were differentially expressed in motoneurons at a pre-symptomatic age (27 out of 34 000 transcripts). Secondly, there is an early specific up-regulation of the gene coding for the intermediate filament vimentin that is increased even further during disease progression. Using in situ hybridization and immunohistochemical analysis, we show that vimentin expression was not only elevated in motoneurons but that the protein formed inclusions in the motoneuron cytoplasm. Thirdly, a time-course analysis of the motoneurons at a symptomatic age (90 and 120 days) showed a modest de-regulation of only a few genes associated with cell death pathways; however, a massive up-regulation of genes involved in cell growth and/or maintenance was observed. This is the first description of the gene profile of SOD1(G93A) motoneurons during disease progression and unexpectedly, no widespread induction of cell death-associated genes was detected in motoneurons of SOD1(G93A) mice.