ABSTRACT Signalling by receptor tyrosine kinases (RTKs) coordinates basic cellular processes during development and in adulthood. Whereas aberrant RTK signalling can lead to cancer,

reactivation of RTKs is often found following stress or cell damage. This has led to the common belief that RTKs can counteract degenerative processes and so strategies to exploit them for

therapy have been extensively explored. An understanding of how RTK stimuli act at cellular levels is needed, however, to evaluate their mechanism of therapeutic action. In this study, we

genetically explored the biological and functional significance of enhanced signalling by the Met RTK in neurons, in the context of a neurodegenerative disease. Conditional _met_-transgenic

ameliorates motor performance, by selectively delaying disease onset. Thus, our studies highlight the properties of RTKs to counteract toxic signals in a disease characterized by dysfunction

of multiple cell types by acting in MNs. Moreover, they emphasize the relevance of genetically assessing the effectiveness of agents targeting neurons during ALS evolution. SIMILAR CONTENT

BEING VIEWED BY OTHERS THE MOTOR NEURON M6A REPERTOIRE GOVERNS NEURONAL HOMEOSTASIS AND FTO INHIBITION MITIGATES ALS SYMPTOM MANIFESTATION Article Open access 30 April 2025 GENETIC

INACTIVATION OF RIP1 KINASE DOES NOT AMELIORATE DISEASE IN A MOUSE MODEL OF ALS Article 29 September 2020 ATAXIN-2 INTERMEDIATE-LENGTH POLYGLUTAMINE EXPANSIONS ELICIT ALS-ASSOCIATED

METABOLIC AND IMMUNE PHENOTYPES Article Open access 29 August 2024 MAIN Signalling by receptor tyrosine kinases (RTKs) is involved in cell communication events regulating tissue

morphogenesis during development and tissue homeostasis during adulthood.1 _In vivo_, RTK signalling levels vary according to a number of parameters, such as RTK expression levels, ligand

availability, action of positive/negative signalling regulators, and components of the signalling cascade. The levels of RTK signalling determine qualitatively different biological

outcomes.2 Given the multiple roles of RTKs in coordinating basic biological processes, modulating their activation levels is a means of achieving different cellular responses in normal

processes and in pathological conditions. Notably, RTK activation is tightly regulated in healthy adult tissues as aberrant signalling in susceptible cells can cause pathologies, such as

cancer.1, 3 Conversely, studies on degenerative diseases have shown that following stress or cell damage there is nearly always a reactivation of RTK signalling, coinciding with periods of

active fight for survival/repair.4, 5 For example, genetic analysis of RTK functions in neurodegenerative processes have demonstrated their requirement for repair of damaged tissues.4 In

contrast, studies on cultured cells and on animal models have shown that symptoms linked to degenerative diseases can be ameliorated when activation of appropriate RTKs is achieved through

exogenous ligand administration.4, 6 However, it is still not clear to which extent levels of remobilized endogenous RTKs are limiting for effective neuronal repair in disease CNS. Over the

last years, a regional and temporal map of RTK-dependency has emerged, suggesting that an appropriate enhancement of RTK signalling might be beneficial to efficiently counteract disease

onset and progression. This issue is particularly relevant for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), an adult onset motor neuron (MN) disease caused by

pathological processes occurring in both neuronal and non-neuronal cells.7 ALS involves progressive degeneration of upper and lower MNs, culminating in muscle wasting, and death mostly due

to respiratory failure. Although the aetiology of most cases remains unknown, 10–20% of familial ALS is caused by mutations in the _superoxide dismutase1_ (_SOD1)_ gene. Consistently,

transgenic mice expressing the mutant forms of human _SOD1_ recapitulate a number of ALS symptoms and have been instrumental in evaluating the molecular and cellular events underlying ALS

pathology.7 Moreover, genetic and cell biological studies based on the differential expression of mutant SOD1 in distinct cell types have demonstrated that although death of MNs causes ALS

symptoms, the disease also renders other cells, such as astrocytes and microglia, dysfunctional. Thus, MN loss, in addition to a cell-autonomous origin, is also triggered by

non-cell-autonomous defects involving toxicity of other unhealthy cells.8, 9 Notably, these different cell types play distinct roles in ALS pathogenesis. Damage within MNs is primarily

associated with disease onset and its early progression phase, whereas damage within microglia and astrocytes accelerates MN degeneration and ALS progression.8, 9, 10, 11, 12 The recognition

that multiple cell types determine ALS evolution has boosted the need to understand the relative contribution of providing beneficial signals to different cell types involved in the

disease. Among several strategies to alleviate ALS symptoms, a major hope has been placed on the ability of trophic factors, acting on MN in culture or during development,4, 7 to activate

endogenous RTKs. Initial results were disappointing as infusion of specific trophic factors had little or no beneficial effects. The weakness of these approaches appeared to be inadequate

delivery of these factors to the right cells. This possibility was further supported by genetic studies assessing growth factor efficacy depending on the delivery site.13, 14 Therefore,

understanding the relative contribution of enhancing RTK signalling in distinct cell types is needed to further clarify the ALS biology and to evaluate how beneficial signals should be

delivered for therapy. In this study, we assessed the biological and functional significance of enhanced signalling, above endogenous levels, downstream of the Met RTK specifically in

neurons during neuro-degenerative diseases, such as ALS. A number of features makes the Met receptor of special interest to address RTK signalling levels during neuro-degeneration.15, 16

During development, activation of the Met receptor by its ligand HGF regulates MN fate at multiple levels, including identity acquisition, axonal growth, and survival.2, 17, 18, 19, 20

During injury, HGF is able to enhance regeneration of the lesioned spinal cord and of crushed peripheral nerves.21, 22 Intracerebral delivery or transgenic-mediated neuronal expression of

HGF in mutant _SOD1_ animal models acts simultaneously on different dysfunctional cell types.23, 24 However, the contribution of enhancing Met signalling uniquely in neurons and its relative

impact on ALS onset and progression remains to be established. To enhance Met signalling above a threshold level in a temporal and tissue-specific manner, we generated conditional _met_

transgenic mice using the cre-loxP system. Here, we show that transgene-mediated neuronal expression of the Met RTK in _SOD1__G93A_ mice selectively delays disease onset, without slowing

down its progression. Our findings show that boosting RTK signalling in a cell-type-restricted manner can have a distinct beneficial impact in counteracting the processes underlying the

evolution of neurodegenerative diseases. RESULTS GENERATION OF CONDITIONAL _ROSA26__LACZ−STOP−MET_ TRANSGENIC MICE To enhance Met signalling levels in a temporally and spatially regulated

manner, we generated mice carrying a conditional mouse–human chimeric _met_ transgene (_met__tg_). In particular, we engineered a mouse strain in which a cytomegalovirus

(CMV)-enhancer/_β-_actin-promoter controls the expression of a floxed-_β-geo_-reporter gene followed by a _met__tg_. Such a strategy was chosen to keep the _met__tg_ silent, unless the stop

cassette is excised by cre-mediated recombination. To avoid integration site effects and favour loxP-site accessibility, the construct was inserted into the _Rosa26_ locus

(_Rosa26__LacZ−stop−Met_, referred to _R26__LacZ−stop−Met_; Figures 1a–c). To specifically gain expression in neural cells, we crossed the _R26__LacZ−stop−Met_ mice with the _nestin-cre_

transgenics25 (_nestin-cre_;_Rosa26__LacZ−stop−Met_, referred to as _Nes-R26__Met_), leading to the excision of the _lacZ_-floxed cassette and consequently expression of the Met chimeric

protein (Mettg) in neural tissues (Figures 1c and d). Western blot analysis of E15.5 embryos and P7 mice revealed the presence of Mettg in dissected brains and spinal cords only after

recombination (Figure 1d). To estimate Mettg levels _versus_ endogenous Met, we performed western blot analysis of brain and spinal cord protein extracts at different developmental stages

using antibodies recognizing the kinase domain of both endogenous and Mettg. Quantification analyses indicated that Mettg levels were at least 5/7-fold increased in brains and spinal cords

of heterozygous _Nes-R26__Met_, when compared with endogenous mouse Met (Figures 1e and f). Consistently, Mettg levels were twofold higher in homozygous mice compared to heterozygous

littermates (Figures 1d–f). We next characterized the _R26__LacZ−stop−Met_ mice before and after recombination by following the expression of the _lacZ_-stop cassette and _met__tg_

transcript in adult brains and spinal cords. We found a decrease in _β_-galactosidase activity and _lacZ_ transcripts in _Nes-R26__Met_ compared with _R26__LacZ−stop−Met_ transgenics,

indicating that recombination in several brain regions occurred as expected (Figures 2a–d, Supplementary Figures 1 and 2). Conversely, _met__tg_ was expressed in brains only after

recombination in a pattern complementary to _lacZ_ distribution (Figures 2e and f). High levels were found in the hippocampus, cerebellum, cerebral cortex, and cervical spinal cord (Figures

2g–i). Expression studies performed on adult lumbar spinal cord sections revealed also a complementary distribution pattern. In particular, _lacZ_ or _met__tg_ were found in cells with large

nuclei resembling MNs of _R26__LacZ−stop−Met_ and _Nes-R26__Met_, respectively (Figure 3). Quantification analysis revealed that cre-mediated excision occurred in approximately 56% of these

cells (56.3±1.1; _P_<0.0001; Figure 3g). MOLECULAR AND CELLULAR CHARACTERIZATION OF _NES-R26__MET_ MICE Although the _Rosa26_ locus drives gene expression ubiquitously,26 we observed

that the _lacZ_ distribution in brains and spinal cords of adult _R26__LacZ−stop−Met_ mice appeared restricted to distinct cell types (Figures 2a–d and 3a–f, Supplementary Figures 1 and 2).

Colocalization studies revealed _β_-galactosidase activity predominantly in NeuN-positive neurons, but not in glial fibrillary acidic protein (GFAP)-positive astrocytes (Figures 4a–h). The

restricted neuronal _lacZ_ expression was also observed in cultured cells (Figures 4i–n). Thus, the genetic setting we adopted (CMV-enhancer/_β-_actin-promoter in _Rosa26_) results in an

animal model with a restricted expression of the transgene, indicating that _met__tg_ should be predominantly confined to neurons after _nestin-cre_-mediated recombination. Consistently,

_met__tg_ transcripts colocalized with Smi32-positive neurons, but not with GFAP-positive astrocytes (Supplementary Figure 3). The restricted expression of _met__tg_ in neurons was also

observed in _Nes-R26__Met_ adult spinal cords, where it was found in dorsal horn neurons, intermediate lateral neurons, and MNs, but not in GFAP-positive astrocytes (Figure 5). Thus,

_met__tg_ is predominantly restricted to neurons in _Nes-R26__Met_ mice. We next examined whether Mettg protein was active by following its phosphorylation state using anti-phospho-Met

antibodies. High levels of phosphorylated Mettg were found in the pons, medulla, lateral ventricles, rostral-migratory stream, olfactory bulbs, cerebral and lumbar spinal cords (Figure 6 and

data not shown). These results show that Mettg is predominantly functional in spatially restricted domains, which possibly correlate to a map of cellular competence for Mettg activation

influenced by a combination of parameters, such as environmental contexts (e.g., endogenous HGF levels) or permissive intracellular mechanisms (e.g., signalling modulators). Altogether these

data show that _Nes-R26__Met_ mice can be a useful animal model to investigate the functional consequences of enhancing Met levels in distinct cell types, using available tissue-specific

_cre_-lines. PHENOTYPICAL CHARACTERIZATION OF _NES-R26__MET_ MICE As previously discussed, Met regulates specification, axonal growth, and survival of MN subtypes during development.17, 18,

19, 20 We therefore evaluated whether enhanced Met levels influence MN numbers in _Nes-R26__Met_ mice. By staining spinal cord sections either with cresyl violet or with

vesicular-acetylcholie-transporter (VAChT) antibodies, similar MN numbers were found at thoracic (data not shown) or lumbar spinal cord levels in _Nes-R26__Met_ and control animals

(_P_>0.05; Figures 7a–d and g). As expected by the colocalization studies, no differences in GFAP-fluorescence intensity were observed (_P_>0.05; Figures 7e, f and h). As body weight

is a generic indicator of animal physiology influenced by body metabolism, activity, and feeding behaviour, the weight of _Nes-R26__Met_ mice was followed over-time and no significant

differences were found _versus_ controls (_P_>0.05; Figure 7j). We next performed the rotarod test to evaluate the overall motor function and no significant differences were found in

_Nes-R26__Met_ _versus_ controls (_P_>0.05; Figure 7i). Altogether, these studies show that enhancing Met levels in neurons does not cause gross physiological abnormalities.

NEURONAL-ENHANCED MET LEVELS COUNTERACT ALS SYMPTOMS IN SOD TRANSGENIC MICE We next investigated the functional relevance of genetically enhancing Met signalling levels in the context of a

neurodegenerative disorder, such as the ALS. The _Nes-R26__Met_ mice were therefore crossed with a strain carrying high copy numbers of the _SOD1__G93A_ transgene to generate an ALS animal

model with increased Met levels in neurons. Five groups of mice were generated: (a) wild-type; (b) _Rosa26__LacZ−stop−Met_, (c) _Nes-R26__Met_, (d) _SOD1__G93A_ (referred to as _SOD_), and

(e) _Nes-R26__Met__;SOD1__G93A_ (referred to as _Nes-R26__Met__-SOD_). As no significant changes were observed between wild-type, _Rosa26__LacZ−stop−Met_, and _Nes-R26__Met_ for all

parameters examined, results of control animals included these three groups. By evaluating the life span of _SOD_ and _Nes-R26__Met__-SOD_ mice, we found that neuronal-enhanced Met levels

prolonged the survival by 13 days (mean age, _SOD_: 136±2 days; _Nes-R26__Met__-SOD_: 149±3 days; _P_=0.0021; Figure 8a). As reduction of body weight is an objective measure of ALS disease,

we examined its evolution in _SOD_ and _Nes-R26__Met__-SOD_ mice, and found that neuronal-enhanced Met levels delayed body weight loss. In particular, loss of 10% body weight was retarded

for 13 days (_SOD_: 123±3 days; _Nes-R26__Met__-SOD_: 136±5 days; _P_=0.0094; Figure 8b), whereas no significant differences were observed during disease progression (_SOD_: 12.9±2.5 days;

_Nes-R26__Met__-SOD_: 13±2.4 days; _P_>0.05; Figure 8c). Together, these results indicate that enhanced Met levels in neurons counteract ALS symptoms in _SOD_ transgenic mice. IMPROVED

MOTOR PERFORMANCE AND DELAYED ONSET OF PARALYSIS IN _SOD_ MICE WITH NEURONAL-ENHANCED MET LEVELS The neuro-degeneration defects causing ALS disease lead to progressive muscle weakness,

atrophy, and paralysis. Screwed hindlimbs and locomotor defects are among the first symptoms affecting transgenic ALS mice. We monitored the appearance and progression of motor defects in

_SOD versus Nes-R26__Met__-SOD_ compared with controls by employing swimming tank and footprint assays.27 Onset of swimming defects was delayed by 14 days, as estimated by the increased time

that mice needed to execute this motor task (_SOD_: 113±3 days; _Nes-R26__Met__-SOD_: 127±3 days; _P_=0.0015; Figure 8d), whereas disease progression was unchanged (_SOD_: 30±3.9 days;

_Nes-R26__Met__-SOD_: 22±2.7 days; _P_>0.05; Figure 8e). Therefore, neuronal-enhanced Met improved motor strength and swimming performance of _SOD_ mice by acting specifically on disease

onset. The motor capability of _Nes-R26__Met__-SOD versus SOD_ mice was further evaluated by performing footprint studies (Supplementary Figure 4). The forepaw/hindpaw overlap analysis

revealed a delay of 19 days in the locomotor gait dysfunction (_SOD_: 108±3 days; _Nes-R26__Met__-SOD_: 127±3 days; _P_=0.0009; Figure 8f), accompanied by a delay of 9 days in the appearance

of step-length defects (_SOD_: 106±2 days; _Nes-R26__Met__-SOD_: 115±2 days; _P_=0.0136; Figure 8h). The motor performance decline rate was not appreciably different between _SOD_ and

_Nes-R26__Met__-SOD_ mice, showing that when the disease has started, it progresses at the same rate (overlap, _SOD_: 30±3.9 days; _Nes-R26__Met__-SOD_: 22±22.7 days; _P_>0.05;

step-length, _SOD_: 32±3.1 days; _Nes-R26__Met__-SOD_: 34±1.7 days; _P_>0.05; Figures 8g and i). NEURONAL-ENHANCED MET LEVELS ATTENUATE MN LOSS IN SPINAL CORDS OF _SOD_ MICE As motor

performances were transiently improved in _Nes-R26__Met__-SOD_ mice by delaying disease onset, we evaluated the neuroprotective effects of increased Met levels by quantifying lumbar spinal

cord MNs. For these studies, we selected three animals among the _Nes-R26__Met__-SOD_, _SOD_, and controls at the symptomatic disease phase (120 days). This stage was chosen because all

behavioural studies showed significant differences between groups. Lumbar spinal cord sections were stained with VAChT antibodies and MN numbers were determined (Figures 9a–c and m). As

expected, we observed a significant 60% MN loss in _SOD_ mice compared with controls (_P_=0.0065). By contrast, neuronal-enhanced Met in _Nes-R26__Met__-SOD_ mice led to an improvement of MN

maintenance as the surviving MN numbers increased by 32% compared with _SOD_ (_P_=0.0002; Figure 9m). We next assessed to what extent enhancing Met function in neurons influenced

astrogliosis and microglia activation, which appear at disease onset and become more prominent during progression.9 In contrast to controls, activated GFAP-positive astrocytes (changes in

fluorescence intensity) were detected in lumbar spinal cords, although reduced in _Nes-R26__Met__-SOD_ compared with _SOD_ (_SOD_: 15.5±1.9 × 108; _Nes-R26__Met__-SOD_: 9.9±1.4 × 108;

_P_=0.0164; Figures 9d–f and n). Similarly, the number of microglial cells was reduced in _Nes-R26__Met__-SOD_ mice compared with _SOD_ (_SOD_: 281.7±14.6; _Nes-R26__Met__-SOD_: 222.1±15.5;

_P_=0.0016; Figures 9g–i and o). Analysis of muscle innervation revealed that the increased MN numbers in _Nes-R26__Met__-SOD_ was accompanied by an enhanced integrity of neuro-muscular

junctions (NMJs; _SOD_: 16±1.8%; _Nes-R26__Met__-SOD_: 41.7±11.4%; _P_=0.041; controls: 84.4±7.9%; Figure 9p). Thus, increased neuronal Met levels elicit a combination of protective effects

in different cell types: (1) cell-autonomous protective effects on spinal cord MNs and for NMJ maintenance; (2) non-cell-autonomous delay of astrocyte activation and increased microglia cell

numbers. DISCUSSION Most of neurodegenerative diseases result from a combinatorial action of pathological signals produced by neurons themselves and by neighbouring cells acting in a

non-cell-autonomous manner.7 A number of molecules including trophic factors and their receptors can elicit beneficial effects on disease-related cells when applied _in vitro_ and/or when

delivered in disease animal models. Understanding how these molecules act on dysfunctional cells remains a key topic to clarify disease mechanisms and to evaluate their use for therapies. We

show here that enhanced signalling by Met has an impact on a specific stage of ALS pathology when it is selectively upregulated in neurons. Indeed, transgene-mediated neuronal expression of

Met elicits a beneficial effect in _SOD1__G93A_ by delaying disease onset, but not progression. Our results are based on a genetic approach involving the generation of conditional _met__tg_

mice, in which Met signalling levels is modulated in a temporally and spatially regulated manner. Such an approach offers the possibility of exploring how enhanced Met signalling above

endogenous levels influences cell fate in developmental events, in adult physiology, and in pathological conditions. Loss of Met function during development interferes with identity

acquisition, axonal growth, and survival of MN subsets.2, 17, 18, 19, 20 We show here that neuronal-enhanced Met signalling levels do not affect either MN development or function in

adulthood, whereas it influences MN maintenance in ALS pathological conditions. Thus, it is likely that excessive Met functions are restrained by mechanisms such as tissue homeostasis or by

limiting amounts of ligand. Importantly, the dispensable function of Met in several adult tissues is in contrast to its requirement in counteracting degenerative processes following

injuries, such as axotomy,21, 22 hepatectomy,28 and skin-wound.29 These regenerative studies together with our findings indicate that in a pathological context, cell types like neurons

become sensitive to the beneficial effects provided by Met. The generation of compound transgenics by crossing the _R26__LacZ−stop−Met_ mice with available _cre-_lines will offer a unique

genetic setting for determining tissue-specific sensitiveness to enhanced Met signalling either during development, in adulthood, or in pathologies. The pleiotropic functions elicited by the

HGF/Met system in neurons have boosted the interest in exploring its potential for ALS therapy through several strategies. HGF intrathecal administration at disease onset provided evidence

that, when present at high doses and accessible to different disease cells, HGF/Met attenuates MN degeneration and retards disease progression by 11 days.30 However, these studies did not

clarify whether, and to what extent, the HGF/Met system exerts support on MNs in a cell-autonomous manner. Insights to this issue come from studies based on genetic neuronal delivery of HGF

in the low copy number _SOD1__G93A_ mice. In particular, _hgf_ expression driven by the neuron-specific _enolase_-promoter delayed disease onset by approximately 28 days, rather than

influencing its progression.23 However, as exogenous HGF is expressed and secreted by neurons, these _hgf_ transgenic mice did not allow discriminating between the HGF effects on MNs

_versus_ those elicited on astrocytes, which in turn influence MNs and microglia function. Consistently, a decrease in the number of microglia, reactive astrocytes, and MN loss was observed

in _hgf_ transgenics. Our mouse model allowed discrimination between the cell-autonomous effects elicited by enhanced Met signalling in MNs and those influenced by HGF on other dysfunctional

cells. Moreover, the _Nes-R26__Met_ mice established that Met signalling in MNs selectively counteracts ALS disease onset. It remains to be investigated what contribution enhanced Met

signalling, above endogenous levels in astrocytes and microglia, could have with respect to disease evolution. The severity of ALS and the lack of effective therapeutic strategies are

driving efforts to explore agents, applied either separately or in combination, to counteract disease symptoms. Concerning trophic factors, HGF could offer therapeutic advantages at multiple

levels. It is noteworthy that endogenous Met is upregulated starting from ALS onset in ventral spinal cords of _SOD1__G93A_ mice (data not shown and see Sun _et al._23). These observations

indicate that endogenous Met is turned on to counteract ALS symptoms, but endogenous HGF levels may not be sufficient to reverse the damage, as was found studying regeneration after

optic-nerve axotomy (unpublished results). Moreover, as HGF/Met elicits functions in muscle cells,31, 32 it is possible that ectopic HGF in muscles would also counteract

neuro-muscular-junction denervation and muscle atrophy. Indeed, HGF favours the formation of NMJs during development.33 Although enhanced Met activation is often associated with tumour

formation and metastasis, we did not observe side effects such as neoplasia in _Nes-R26__Met_ mice despite Mettg expression levels in regions of the nervous system. Thus, the beneficial

effects of HGF/Met on ALS degenerative processes appear to be well tolerated in healthy nervous system tissues. The use of the conditional ALS mouse model, in which the mutant _SOD1_ gene

can be deleted according to the _cre_-transgenic line used, has been instrumental in clarifying the influence of distinct cell types during ALS evolution. In particular, selective reduction

of mutant _SOD1_ in MNs predominantly impacts on disease onset and its early phase. In contrast, reduced mutant _SOD1_ either in astrocytes or in microglia mainly influences the late stage

of disease progression.8, 9 Thus, although recombination in _Nes-R26__Met_ mice did not occur in 44% of lumbar spinal cord MNs, rebalancing the levels of stress and survival signals and/or

protecting presynaptic terminals in ALS MNs, either by providing RTK support (our studies), or by removing signals such as BAX influencing NMJ integrity,12 or by depleting mutant SOD,8, 9

ameliorates ALS by selectively delaying disease onset. It is known that the initial MN damage is followed by a progressive increase in cytotoxic and inflammatory mediator levels, which

further affect MN themselves and neighbouring cells. Astrocytes and microglia are the predominant cell types driving disease progression towards death. Although disease progression is

unchanged in _Nes-R26__Met_ mice, our genetic analysis show that enhanced-neuronal Met in _SOD_ mice impacts on distinct cell types involved in the ALS disease: MNs, in a cell-autonomous

manner, possibly by providing trophic support and/or maintenance of NMJ integrity and astrocytes and microglia, in a non-cell-autonomous manner, by delaying their activation. Future studies

will establish the relative contribution of each individual event in delaying the onset of the disease by neuronal Met signalling and uncover the underlying mechanisms. Understanding how

therapeutic reagents act at cellular levels is needed to accelerate the progress of promising treatments towards the clinic. Our findings emphasize the relevance of genetically assessing the

effects of agents on distinct cell types implicated in a disease and during its evolution. Although successful therapies for ALS will possibly require concomitant actions on distinct

dysfunctional cells, our results highlight the considerable therapeutic potential of modulating RTK signalling in MNs to combat degenerative signals. MATERIALS AND METHODS GENERATION OF

_R26__LACZ−STOP−MET_ MICE The _R26__LacZ−stop−Met_ mice were generated by taking advantage of the loxP-flanked (‘floxed’)-stop cassette system. To generate the chimeric mouse–human gene, the

human Met cDNA encoding the transmembrane and cytoplasmic portion was fused in-frame with the mouse extracellular cDNA sequence using the _Pvu_II site present in both sequences. We chose

such a strategy to ensure that the Met chimeric protein interacts efficiently with the endogenous mouse HGF, but can still be identified from the endogenous mouse Met protein. The fusion

product was subsequently subcloned into the pCALL2 vector downstream of the insert containing the CMV enhancer-chicken _β_-actin promoter followed by the loxP-flanked _β-_geo/3xpA cassette.

This vector is referred to as pCALL-Met. To generate the Rosa26 targeting construct, the insert containing the CMV-enhancer/chicken-_β_-actin-promoter-loxP-flanked _β-_geo/3xpA-Met was

subcloned into the _Xho_I site of pRosa-1 vector previously modified using a poly-linker. The targeting vector was electroporated into R1 ES cell lines. Cell culture, electroporation,

selection, and Southern blot analyses were performed as previously described.32 To identify recombined clones, genomic DNA was digested with _Eco_RI or _Eco_RV and probed with an external or

internal probe, respectively. Two selected ES cell lines carrying the homologous recombination were used to generate the _R26__LacZ−stop−Met_ mice through blastocyst injections. TRANSGENIC

MICE The mouse line expressing cre recombinase under the nestin promoter was previously described.25 The B6SJL-Tg(SOD1*G93A)1Gur(SOD1G93A (SOD1G93A) mouse line was used as ALS disease

model.34 Both _R26__LacZ−stop−Met_ and nestin-cre mice were backcrossed into the B6SJL genetic background before breeding with the SOD1G93A transgenics. The number of mice used for

behavioural and immuno-histochemical studies are indicated in figure legends. Each genetic group consisted of a mixed population of equal numbers of males and females. The presence of a

vaginal plug in the morning was considered as 0.5 embryonic day (E0.5). All procedures involving the use of animals were performed in accordance with the European Community Council Directive

of 24 November 1986 on the protection of animals used for experimental purposes (86/609/EEC). The experimental protocols were carried out in compliance with institutional ethical committee

guidelines for animal research. All efforts were made to minimize the number of animals used and their suffering. When paralysis started, food and water were placed directly into the cage.

To reduce animal pain, mice were killed when they were unable to right themselves within 30 s when placed on their back. ANTIBODIES Antibodies used were anti-tubulin and anti-GFAP and

anti-VAChT (1 : 1000; Sigma-Aldrich, St. Louis, MO, USA), anti-MetKD (1 : 1000) and anti-phospho Y1234-1235-Met (1 : 50; Cell Signaling, Danvers, MA, USA), anti-human Met (1 : 500;

Santa-Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-Smi32 (1 : 500; Sternberger monoclonals, Covance, Dallas, TX, USA), anti-NeuN (1 : 200; Chemicon, Millipore, Billerica, MA, USA),

and anti-neurofilament-145 (1 : 1000; AB1987; Millipore), anti-mouse or rabbit fluorescent-coupled secondary antibodies (1 : 400; Jackson, West Grove, PA, USA), anti-mouse or rabbit

biotin-coupled secondary antibodies (1 : 500; Jackson). For western blot analyses, the following secondary antibodies were used: anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase (1 : 

4000, Jackson). HISTOLOGICAL ANALYSIS Anesthetized mice were intra-cardiacally perfused first with PBS then with 4% para-formaldehyde (PFA, Sigma) in PBS. Brains, spinal cords, and muscles

were dissected, postfixed in 4% PFA and embedded. For brain and spinal cords, 16 or 30-_μ_m thick cryo-sections were performed (Leica, Wetzlar, Germany). _In situ_ hybridization,

immuno-histochemistry, and X-Gal staining were performed as previously described.27, 35, 36 MN numbers were determined on 16-_μ_m thick lumbar spinal cord sections stained with VAChT

antibodies or cresyl violet. A total of 10 sections per mouse were analyzed. Astrogliosis was monitored by measuring fluorescence-levels of sections stained with anti-GFAP antibodies (Image

J software, ImageJ 1.41, NIH, Bethesda, MD, USA). For NMJ staining, 35-_μ_m thick longitudinal sections were collected on Superfrost Plus Slides (CML, Thermo Scientific, Braunscheweig,

Germany). Tissue sections were incubated in blocking solution (0.5% Triton X-100, 5% BSA in PBS) at 37 °C for 2 h. Rabbit polyclonal anti neurofilament-145 antibodies were diluted in the

same blocking solution and incubated overnight at 4 °C. Anti-rabbit-alexa 488-conjugated secondary antibody (Invitrogen, Life Technologies, Carlsbad, CA, USA) and

_α_-bungarotoxin-tetramethylrhodamine-conjugate (1 : 1000; Invitrogen) were incubated for 2 h at room temperature (1% BSA in PBS), before washing and mounting. Stained end-plates on sections

were examined under an Axio microscope (Zeiss, Oberkochen, Germany). Innervated (yellow) or denervated (red) end-plates were counted on apoptome (Zeiss) 35-_μ_m Z-stacks. BIOCHEMICAL

STUDIES Protein extracts were prepared from freshly dissected brains and spinal cords at the appropriate stages and western blot analyses were performed as previously described.37, 38, 39

Quantifications were done by measuring band intensities with the Image J software. CULTURES Cell culture procedures were previously described.40 Briefly, E12.5 spinal cords from

_R26__LacZ−stop−Met_ transgenic embryos were dissected in Hank's Balanced Salt Solution containing 7 mM HEPES pH7.4 and 4.5 g/l glucose. Cells were dissociated in Ham-F10 medium

(Invitrogen) with 0.025% Trypsin (Sigma) and centrifuged over a 4% (w/v) BSA cushion at 800 × _g_ for 5 min. Cells were plated on poly-ornithine/laminin-treated coverslips in supplemented

Neurobasal medium (Invitrogen) containing neurotrophic factors (0.1 ng/ml GDNF, 1 ng/ml BDNF, and 10 ng/ml CNTF) and maintained at 37 °C in 7.5% CO2 atmosphere for 3 days before being

processed for immunochemistry. Astrocytes were prepared from P2 _R26__LacZ−stop−Met_ spinal cords. When confluent, cells were trypsinized and plated onto coverslips in Dulbecco's

modified Eagle's (DMEM, Invitrogen) medium supplemented with 10% fetal bovine serum and penicillin–streptomycin. Cells were then cultured at 37 °C in 5% CO2 atmosphere for 3 days before

immunostaining. Cultured cells were processed for immunocytochemistry as above described. We used the following primary antibodies diluted in PBS containing 4% BSA, 2% donkey serum:

anti-_β-_Gal (1 : 4000; Cappel, MP Biomedicals, Ullkirch, France), anti-neurofilament 160 (1 : 600; NN18, Sigma), anti-GFAP (1 : 500; MAB360, Millipore). Alexa Fluor 488 (1 : 500; A21206;

Invitrogen) and 555 (1 : 1000; A31570; Invitrogen)-conjugated donkey anti-rabbit and anti-mouse were used as secondary antibodies (Invitrogen). BEHAVIOURAL TESTS Body weight measurements and

all behavioural tests began when mice reached the age of 70 days and recording was performed weekly. To analyze motor functions, locomotor tests included the rotarod, the 1 m swimming tank

and the footprint, which have been done as previously described.27 We performed four trials at each time point for each animal and recorded the three best performances for statistical

analysis. Briefly, the rotarod test was performed by placing mice on an accelerating rod (3 cm diameter) and by recording the time each animal took to fall from the rod. The speed of the

rotarod accelerated from 4 to 40 r.p.m. over a 5-min period. The swimming tank device allows evaluation of the hindlimb strength and performance; it is suitable for assessing onset and

progression of ALS symptoms. For the swimming tank, each mouse performed four trials and the time needed to reach the platform was recorded. A mouse was considered at the onset of motor

defect when it needed 6.8 s to perform the swimming tank test. The 6.8 s were chosen as reference as this value corresponds to the first significant difference _versus_ control mice. For the

footprint test, the fore-toes and the hind-toes were labelled in blue and red, respectively. Weekly monitoring of walking patterns allows assessment of motor coordination and synchrony

through the evaluation of several parameters, including toe spread, forepaw/hindpaw overlap, and step-length. Two parameters were measured: (1) the overlap distance between forepaw and

hindpaw on the same side; (2) the stride length as the distance of the hindpaw on the same side between each step. For the overlap, the Kaplan–Meier curve indicates the mice when the

distance between forepaw and hindpaw increased by 45%. For the step length, the Kaplan–Meier curve indicates when the distance of the hindpaw for each step decreased by 40%. To reduce mouse

stress and fatigue, the swimming and the footprint behavioural tests were carried out on different days. STATISTICAL ANALYSIS Results were expressed as the mean±S.E.M. Statistically

significant differences on cell counts were assessed by the Student's _t-_test. Statistically significant differences among the groups of mice were assessed by two-way ANOVA. _Post hoc_

Bonferroni's correction was also used to test all pair-wise comparisons between groups and time points per group. A log-rank test was used to calculate the statistical differences in

the onset and survival of the different mouse cohorts. Statistical significance was defined as ns: _P_>0.05; *_P_<0.05; **_P_<0.01; ***_P_<0.001. ABBREVIATIONS * RTK: receptor

tyrosine kinase * MN: motor neuron * ALS: amyotrophic lateral sclerosis * CMV: cytomegalovirus * GFAP: glial fibrillary acidic protein * VAChT: vesicular-acetylcholie-transporter * SOD:

superoxide dismutase * NMJ: neuro-muscular junction REFERENCES * Lemmon MA, Schlessinger J . Cell signaling by receptor tyrosine kinases. _Cell_ 2010; 141: 1117–1134. Article  CAS  Google

Scholar  * Maina F, Pante G, Helmbacher F, Andres R, Porthin A, Davies AM _et al_. Coupling Met to specific pathways results in distinct developmental outcomes. _Mol Cell_ 2001; 7:

1293–1306. Article  CAS  Google Scholar  * Furlan A, Stagni V, Hussain A, Richelme S, Conti F, Prodosmo A _et al_. Abl interconnects oncogenic Met and p53 core pathways in cancer cells.

_Cell Death Differ_ 2011 in press. * Connor B, Dragunow M . The role of neuronal growth factors in neurodegenerative disorders of the human brain. _Brain Res Brain Res Rev_ 1998; 27: 1–39.

Article  CAS  Google Scholar  * Snider WD, Zhou FQ, Zhong J, Markus A . Signaling the pathway to regeneration. _Neuron_ 2002; 35: 13–16. Article  CAS  Google Scholar  * Siegel GJ, Chauhan NB

. Neurotrophic factors in Alzheimer's and Parkinson's disease brain. _Brain Res Brain Res Rev_ 2000; 33: 199–227. Article  CAS  Google Scholar  * Kanning KC, Kaplan A, Henderson

CE . Motor neuron diversity in development and disease. _Annu Rev Neurosci_ 2010; 33: 409–440. Article  CAS  Google Scholar  * Boillee S, Yamanaka K, Lobsiger CS, Copeland NG, Jenkins NA,

Kassiotis G _et al_. Onset and progression in inherited ALS determined by motor neurons and microglia. _Science_ 2006; 312: 1389–1392. Article  CAS  Google Scholar  * Yamanaka K, Chun SJ,

Boillee S, Fujimori-Tonou N, Yamashita H, Gutmann DH _et al_. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. _Nat Neurosci_ 2008; 11: 251–253.

Article  CAS  Google Scholar  * Miller TM, Kaspar BK, Kops GJ, Yamanaka K, Christian LJ, Gage FH _et al_. Virus-delivered small RNA silencing sustains strength in amyotrophic lateral

sclerosis. _Ann Neurol_ 2005; 57: 773–776. Article  CAS  Google Scholar  * Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA, Lee DC _et al_. Silencing mutant SOD1 using RNAi protects

against neurodegeneration and extends survival in an ALS model. _Nat Med_ 2005; 11: 429–433. Article  CAS  Google Scholar  * Gould TW, Buss RR, Vinsant S, Prevette D, Sun W, Knudson CM _et

al_. Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS. _J Neurosci_ 2006; 26: 8774–8786. Article  CAS  Google Scholar  * Guillot S,

Azzouz M, Deglon N, Zurn A, Aebischer P . Local GDNF expression mediated by lentiviral vector protects facial nerve motoneurons but not spinal motoneurons in SOD1(G93A) transgenic mice.

_Neurobiol Dis_ 2004; 16: 139–149. Article  CAS  Google Scholar  * Li W, Brakefield D, Pan Y, Hunter D, Myckatyn TM, Parsadanian A . Muscle-derived but not centrally derived transgene GDNF

is neuroprotective in G93A-SOD1 mouse model of ALS. _Exp Neurol_ 2007; 203: 457–471. Article  CAS  Google Scholar  * Maina F, Hilton MC, Andres R, Wyatt S, Klein R, Davies AM . Multiple

roles for hepatocyte growth factor in sympathetic neuron development. _Neuron_ 1998; 20: 835–846. Article  CAS  Google Scholar  * Maina F, Klein R . Hepatocyte growth factor—a versatile

signal for developing neurons. _Nat Neurosci_ 1999; 2: 213–217. Article  CAS  Google Scholar  * Ebens A, Brose K, Leonardo ED, Hanson MG, Bladt F, Birchmeier C _et al_. Hepatocyte growth

factor/Scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons. _Neuron_ 1996; 17: 1157–1172. Article  CAS  Google Scholar  * Maina F, Hilton MC,

Ponzetto C, Davies AM, Klein R . Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons. _Genes Dev_ 1997; 11:

3341–3350. Article  CAS  Google Scholar  * Yamamoto Y, Livet J, Pollock RA, Garces A, Arce V, deLapeyriere O _et al_. Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor

for a subpopulation of embryonic motoneurons. _Development_ 1997; 124: 2903–2913. CAS  PubMed  Google Scholar  * Helmbacher F, Dessaud E, Arber S, deLapeyriere O, Henderson CE, Klein R _et

al_. Met signaling is required for recruitment of motor neurons to PEA3-positive motor pools. _Neuron_ 2003; 39: 767–777. Article  CAS  Google Scholar  * Kato N, Nemoto K, Nakanishi K,

Morishita R, Kaneda Y, Uenoyama M _et al_. Nonviral HVJ (hemagglutinating virus of Japan) liposome-mediated retrograde gene transfer of human hepatocyte growth factor into rat nervous system

promotes functional and histological recovery of the crushed nerve. _Neurosci Res_ 2005; 52: 299–310. Article  CAS  Google Scholar  * Kitamura K, Iwanami A, Nakamura M, Yamane J, Watanabe

K, Suzuki Y _et al_. Hepatocyte growth factor promotes endogenous repair and functional recovery after spinal cord injury. _J Neurosci Res_ 2007; 85: 2332–2342. Article  CAS  Google Scholar

  * Sun W, Funakoshi H, Nakamura T . Overexpression of HGF retards disease progression and prolongs life span in a transgenic mouse model of ALS. _J Neurosci_ 2002; 22: 6537–6548. Article 

CAS  Google Scholar  * Kadoyama K, Funakoshi H, Ohya W, Nakamura T . Hepatocyte growth factor (HGF) attenuates gliosis and motoneuronal degeneration in the brainstem motor nuclei of a

transgenic mouse model of ALS. _Neurosci Res_ 2007; 59: 446–456. Article  CAS  Google Scholar  * Tronche F, Kellendonk C, Kretz O, Gass P, Anlag K, Orban PC _et al_. Disruption of the

glucocorticoid receptor gene in the nervous system results in reduced anxiety. _Nat Genet_ 1999; 23: 99–103. Article  CAS  Google Scholar  * Soriano P . Generalized lacZ expression with the

ROSA26 Cre reporter strain. _Nat Genet_ 1999; 21: 70–71. Article  CAS  Google Scholar  * Raoul C, Abbas-Terki T, Bensadoun JC, Guillot S, Haase G, Szulc J _et al_. Lentiviral-mediated

silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. _Nat Med_ 2005; 11: 423–428. Article  CAS  Google Scholar  * Huh CG, Factor VM,

Sanchez A, Uchida K, Conner EA, Thorgeirsson SS . Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair. _Proc Natl Acad Sci USA_ 2004;

101: 4477–4482. Article  CAS  Google Scholar  * Chmielowiec J, Borowiak M, Morkel M, Stradal T, Munz B, Werner S _et al_. c-Met is essential for wound healing in the skin. _J Cell Biol_

2007; 177: 151–162. Article  CAS  Google Scholar  * Ishigaki A, Aoki M, Nagai M, Warita H, Kato S, Kato M _et al_. Intrathecal delivery of hepatocyte growth factor from amyotrophic lateral

sclerosis onset suppresses disease progression in rat amyotrophic lateral sclerosis model. _J Neuropathol Exp Neurol_ 2007; 66: 1037–1044. Article  CAS  Google Scholar  * Bladt F,

Riethmacher D, Isenmann S, Aguzzi A, Birchmeier C . Essential role for the c-_met_ receptor in the migration of myogenic precursor cells into the limb bud. _Nature_ 1995; 376: 768–771.

Article  CAS  Google Scholar  * Maina F, Casagranda F, Audero E, Simeone A, Comoglio P, Klein R _et al_. Uncoupling of Grb2 from the Met receptor _in vivo_ reveals complex roles in muscle

development. _Cell_ 1996; 87: 531–542. Article  CAS  Google Scholar  * Madhavan R, Peng HB . HGF induction of postsynaptic specializations at the neuromuscular junction. _J Neurobiol_ 2006;

66: 134–147. Article  CAS  Google Scholar  * Chiu AY, Zhai P, Dal Canto MC, Peters TM, Kwon YW, Prattis SM _et al_. Age-dependent penetrance of disease in a transgenic mouse model of

familial amyotrophic lateral sclerosis. _Mol Cell Neurosci_ 1995; 6: 349–362. Article  CAS  Google Scholar  * Dono R . Fibroblast growth factors as regulators of central nervous system

development and function. _Am J Physiol Regul Integr Comp Physiol_ 2003; 284: R867–R881. Article  CAS  Google Scholar  * Zuniga A, Michos O, Spitz F, Haramis AP, Panman L, Galli A _et al_.

Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression. _Genes Dev_ 2004; 18: 1553–1564. Article  CAS  Google

Scholar  * Segarra J, Balenci L, Drenth T, Maina F, Lamballe F . Combined Signaling through ERK, PI3K/AKT, and RAC1/p38 Is Required for Met-triggered Cortical Neuron Migration. _J Biol Chem_

2006; 281: 4771–4778. Article  CAS  Google Scholar  * Moumen A, Ieraci A, Patane S, Sole C, Comella JX, Dono R _et al_. Met signals hepatocyte survival by preventing Fas-triggered FLIP

degradation in a PI3k-Akt-dependent manner. _Hepatology_ 2007; 45: 1210–1217. Article  CAS  Google Scholar  * Moumen A, Patane S, Porras A, Dono R, Maina F . Met acts on Mdm2 via mTOR to

signal cell survival during development. _Development_ 2007; 134: 1443–1451. Article  CAS  Google Scholar  * Aebischer J, Cassina P, Otsmane B, Moumen A, Seilhean D, Meininger V _et al_.

IFNgamma triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1. _Cell Death Differ_, e-pub ahead of print 12 November 2010.

Download references ACKNOWLEDGEMENTS We are particularly grateful to C Henderson, A Moqrich, K Dudley, and all lab members for discussions and comments. We thank: V Girod-David, L Jullien,

staff members at IBDML and CIML animal house and transgenic facilities for help with mouse husbandry; IBDML imaging platform; CG Lobe for the pCALL2 vector. This work was supported by funds

from INCa, ARC, FRM, AFM, FdF, Fondation Bettencourt-Schueller, Marie Curie Host Fellowship for the Transfer-of-Knowledge to FM and RD. MG was supported by University-Franco-Italy

fellowship, EC by AFM, AF by FRM, HH by FdF, ARC. AUTHOR INFORMATION Author notes * M Genestine & E Caricati Present address: 4Current address: Departments of Anatomy, Pharmacology, and

Forensic Medicine, c.so M.D’Azeglio 2, 10126 Torino, Italy., * E Caricati and A Fico: These authors equally contributed to the work. * F Maina and R Dono: Shared last authors. AUTHORS AND

AFFILIATIONS * Developmental Biology Institute of Marseille-Luminy (IBDML), UMR 6216, CNRS – Inserm – Université de la Méditerranée, Campus de Luminy-Case 907, Marseille Cedex 09, France M

Genestine, E Caricati, A Fico, S Richelme, H Hassani, F Lamballe, F Helmbacher, F Maina & R Dono * Inserm-Avenir Team, The Mediterranean Institute of Neurobiology, Marseille, France C

Sunyach, B Pettmann & C Raoul * Departments of Anatomy, Pharmacology, and Forensic Medicine, Torino, Italy G C Panzica Authors * M Genestine View author publications You can also search

for this author inPubMed Google Scholar * E Caricati View author publications You can also search for this author inPubMed Google Scholar * A Fico View author publications You can also

search for this author inPubMed Google Scholar * S Richelme View author publications You can also search for this author inPubMed Google Scholar * H Hassani View author publications You can

also search for this author inPubMed Google Scholar * C Sunyach View author publications You can also search for this author inPubMed Google Scholar * F Lamballe View author publications You

can also search for this author inPubMed Google Scholar * G C Panzica View author publications You can also search for this author inPubMed Google Scholar * B Pettmann View author

publications You can also search for this author inPubMed Google Scholar * F Helmbacher View author publications You can also search for this author inPubMed Google Scholar * C Raoul View

author publications You can also search for this author inPubMed Google Scholar * F Maina View author publications You can also search for this author inPubMed Google Scholar * R Dono View

author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to F Maina or R Dono. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no conflict of interest. ADDITIONAL INFORMATION Edited by A Verkhraski Supplementary Information accompanies the paper on Cell Death and Disease website SUPPLEMENTARY INFORMATION

SUPPLEMENTARY FIGURE 1 (JPG 458 KB) SUPPLEMENTARY FIGURE 2 (JPG 367 KB) SUPPLEMENTARY FIGURE 3 (JPG 369 KB) SUPPLEMENTARY FIGURE 4 (JPG 156 KB) SUPPLEMENTARY INFORMATION (DOC 35 KB) RIGHTS

AND PERMISSIONS This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit

http://creativecommons.org/licenses/by-nc-nd/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Genestine, M., Caricati, E., Fico, A. _et al._ Enhanced neuronal Met

signalling levels in ALS mice delay disease onset. _Cell Death Dis_ 2, e130 (2011). https://doi.org/10.1038/cddis.2011.11 Download citation * Received: 09 September 2010 * Revised: 11

January 2011 * Accepted: 01 February 2011 * Published: 17 March 2011 * Issue Date: March 2011 * DOI: https://doi.org/10.1038/cddis.2011.11 SHARE THIS ARTICLE Anyone you share the following

link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature

SharedIt content-sharing initiative KEYWORDS * RTK signalling * conditional transgenesis * neuro-degenerative disease * HGF/Met * ALS