CNS-targeted Viral Delivery of G-CSF in an Animal Model for ALS: Improved Efficacy and Preservation of the Neuromuscular Unit
Authors: Alexandre Henriques, Claudia Pitzer, Tanjew Dittgen, Matthias Klugmann, Luc Dupuis, Armin Schneider
Journal: Molecular Therapy (2010)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons. We have recently uncovered a new neurotrophic growth factor, granulocyte-colony stimulating factor (G-CSF), which protects α-motoneurons, improves functional outcome, and increases life expectancy of SOD-1 (G93A) mice when delivered subcutaneously. However, chronic systemic delivery of G-CSF is complicated by elevation of neutrophilic granulocytes. Here, we used adeno-associated virus (AAV) to directly target and confine G-CSF expression to the spinal cord. Whereas intramuscular injection of AAV failed to transduce motoneurons retrogradely, and caused a high systemic load of G-CSF, intraspinal delivery led to a highly specific enrichment of G-CSF in the spinal cord with moderate peripheral effects. Intraspinal delivery improved motor functions, delayed disease progression, and increased survival by 10%, longer than after systemic delivery. Mechanistically, we could show that G-CSF in addition to rescuing motoneurons improved neuromuscular junction (NMJ) integrity and enhanced motor axon regeneration after nerve crush injury. Collectively, our results show that intraspinal delivery improves efficacy of G-CSF treatment in an ALS mouse model while minimizing the systemic load of G-CSF, suggesting a new therapeutic option for ALS treatment. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons. We have recently uncovered a new neurotrophic growth factor, granulocyte-colony stimulating factor (G-CSF), which protects α-motoneurons, improves functional outcome, and increases life expectancy of SOD-1 (G93A) mice when delivered subcutaneously. However, chronic systemic delivery of G-CSF is complicated by elevation of neutrophilic granulocytes. Here, we used adeno-associated virus (AAV) to directly target and confine G-CSF expression to the spinal cord. Whereas intramuscular injection of AAV failed to transduce motoneurons retrogradely, and caused a high systemic load of G-CSF, intraspinal delivery led to a highly specific enrichment of G-CSF in the spinal cord with moderate peripheral effects. Intraspinal delivery improved motor functions, delayed disease progression, and increased survival by 10%, longer than after systemic delivery. Mechanistically, we could show that G-CSF in addition to rescuing motoneurons improved neuromuscular junction (NMJ) integrity and enhanced motor axon regeneration after nerve crush injury. Collectively, our results show that intraspinal delivery improves efficacy of G-CSF treatment in an ALS mouse model while minimizing the systemic load of G-CSF, suggesting a new therapeutic option for ALS treatment. IntroductionAmyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease, characterized by progressive weakness, muscle wasting and death ensuing 3–5 years after diagnosis.1Mitchell JD Borasio GD Amyotrophic lateral sclerosis.Lancet. 2007; 369: 2031-2041Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar The etiopathogenesis and pathophysiology of ALS is complex with many players involved that lead to the functional decline of the motor pathway.2Gonzalez de Aguilar JL Echaniz-Laguna A Fergani A René F Meininger V Loeffler JP et al.Amyotrophic lateral sclerosis: all roads lead to Rome.J Neurochem. 2007; 101: 1153-1160Crossref PubMed Scopus (67) Google Scholar,3Pasinelli P Brown RH Molecular biology of amyotrophic lateral sclerosis: insights from genetics.Nat Rev Neurosci. 2006; 7: 710-723Crossref PubMed Scopus (894) Google Scholar,4Rothstein JD Current hypotheses for the underlying biology of amyotrophic lateral sclerosis.Ann Neurol. 2009; 65: S3-S9Crossref PubMed Google Scholar Due to insufficient insights into the molecular pathway(s) crucial to ALS pathogenesis the most rational strategy at present remains to rescue and strengthen motor units with neurotrophic factors.5Henriques A Pitzer C Schneider A Neurotrophic growth factors for the treatment of amyotrophic lateral sclerosis: where do we stand?.Front Neurosci. 2010; 4: 32PubMed Google Scholar A number of growth factors have been clinically tested in ALS without success so far, but major pharmacokinetic problems and unexpected peripheral effects preclude any conclusion as to the true therapeutic potential of this concept.5Henriques A Pitzer C Schneider A Neurotrophic growth factors for the treatment of amyotrophic lateral sclerosis: where do we stand?.Front Neurosci. 2010; 4: 32PubMed Google ScholarGranulocyte-colony stimulating factor (G-CSF) is a 19.6 kd cytokine first described as a hematopoietic growth factor that stimulates proliferation and differentiation of myeloid precursors.6Welte K Platzer E Gabrilove JL Lu L Levi E Polivka A et al.Purification to apparent homogeneity and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor.Haematol Blood Transfus. 1985; 29: 398-401PubMed Google Scholar We and others have uncovered neurotrophic properties of G-CSF. G-CSF and its receptor are expressed by neurons in many regions of the adult brain and spinal cord.7Schneider A Krüger C Steigleder T Weber D Pitzer C Laage R et al.The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.J Clin Invest. 2005; 115: 2083-2098Crossref PubMed Scopus (594) Google Scholar,8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar,9Pitzer C Klussmann S Krüger C Letellier E Plaas C Dittgen T et al.The hematopoietic factor granulocyte-colony stimulating factor improves outcome in experimental spinal cord injury.J Neurochem. 2010; 113: 930-942Crossref PubMed Scopus (39) Google Scholar G-CSF appears clinically attractive, because it is generally well-tolerated, crosses the intact blood–brain barrier,7Schneider A Krüger C Steigleder T Weber D Pitzer C Laage R et al.The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.J Clin Invest. 2005; 115: 2083-2098Crossref PubMed Scopus (594) Google Scholar and its pharmacokinetic properties are well established. A large number of studies in various animal models of neurodegenerative diseases demonstrated that G-CSF is neuroprotective and proregenerative in models of stroke,7Schneider A Krüger C Steigleder T Weber D Pitzer C Laage R et al.The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.J Clin Invest. 2005; 115: 2083-2098Crossref PubMed Scopus (594) Google Scholar Parkinson's disease,10Meuer K Pitzer C Teismann P Krüger C Göricke B Laage R et al.Granulocyte-colony stimulating factor is neuroprotective in a model of Parkinson's disease.J Neurochem. 2006; 97: 675-686Crossref PubMed Scopus (105) Google Scholar and of spinal cord injury.9Pitzer C Klussmann S Krüger C Letellier E Plaas C Dittgen T et al.The hematopoietic factor granulocyte-colony stimulating factor improves outcome in experimental spinal cord injury.J Neurochem. 2010; 113: 930-942Crossref PubMed Scopus (39) Google ScholarIn 2008, we reported that G-CSF had beneficial effects in SOD-1 (G93A) transgenic mice, an animal model for ALS, after systemic delivery using subcutaneous pump delivery or transgenic expression of G-CSF.8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar One key mechanism of G-CSF beneficial action is its direct antiapoptotic effect on motoneurons, as supported by G-CSF-mediated protection of motoneurons after neonatal sciatic nerve axotomy.11Henriques A Pitzer C Dupuis L Schneider A G-CSF protects motoneurons against axotomy-induced apoptotic death in neonatal mice.BMC Neurosci. 2010; 11: 25Crossref PubMed Scopus (26) Google ScholarOne challenge for growth factor treatment in ALS is the likely need for a very chronic treatment with those proteins. In the case of G-CSF, any form of systemic delivery, such as subcutaneous delivery, has the inherent consequence of a chronic rise in white blood cells (WBC). The effects of such chronic elevation of WBCs have not been explored extensively in humans. In addition, the limited plasma half-life (~4 hours) would require repeated dosing or some sort of pump delivery of the protein.To circumvent these problems, direct delivery of G-CSF to the central nervous system (CNS) using viral vectors might be advantageous. Recombinant adeno-associated virus12Atchison RW Casto BC Hammon WM Adenovirus-associated defective virus particles.Science. 1965; 149: 754-756Crossref PubMed Scopus (540) Google Scholar is replication-deficient, derived from a nonpathogenic virus, and able to infect numerous cell types, including neurons, resulting in its presence in the nucleus as episomal concatamers.13Bouard D Alazard-Dany D Cosset FL Viral vectors: from virology to transgene expression.Br J Pharmacol. 2009; 157: 153-165Crossref PubMed Scopus (227) Google Scholar In nondividing neuronal cells, the virus may persist in that form for the lifetime of the cell. AAV serotype 2 was described to be able to transduce spinal motoneurons of SOD-1 (G93A) mice after intramuscular and intraspinal injections,14Dodge JC Haidet AM Yang W Passini MA Hester M Clarke J et al.Delivery of AAV-IGF-1 to the CNS extends survival in ALS mice through modification of aberrant glial cell activity.Mol Ther. 2008; 16: 1056-1064Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar,15Kaspar BK Lladó J Sherkat N Rothstein JD Gage FH Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model.Science. 2003; 301: 839-842Crossref PubMed Scopus (741) Google Scholar and lead to the production of neurotrophic factors.We used a chimeric AAV1/2, known to have enhanced transduction efficiency over AAV2 in the CNS to deliver G-CSF to spinal motor neurons by either intramuscular or direct intraspinal injection of AAV. We show here that intraspinal delivery improved efficacy of G-CSF treatment and decreased peripheral load and elevation of leukocyte count.ResultsMuscular injection fails to transduce motoneurons in SOD-1 (G93A) transgenic miceAn elegant and clinically feasible way to bring G-CSF to motoneurons would be to exploit the retrograde transport ability of AAV and inject the virus into skeletal muscles where it would be taken up by presynaptic neuromuscular junctions (NMJs). Indeed, successful targeting of motoneurons with AAV using this route has been described.15Kaspar BK Lladó J Sherkat N Rothstein JD Gage FH Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model.Science. 2003; 301: 839-842Crossref PubMed Scopus (741) Google Scholar Motoneurons are then expected to synthesize and secrete G-CSF that will bind to its neuronally expressed receptor and induce antiapoptotic pathways. Such a strategy would have the additional advantage of mimicking the endogenous autocrine behavior of the ligand in neurons.7Schneider A Krüger C Steigleder T Weber D Pitzer C Laage R et al.The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis.J Clin Invest. 2005; 115: 2083-2098Crossref PubMed Scopus (594) Google ScholarWe injected a total of 0.9 × 1010 particles of G-CSF-expressing and control virus into the gastrocnemius and longissimus thoracis muscles of SOD-1 (G93A) transgenic mice. We next studied virus-mediated enhanced green fluorescent protein (eGFP) expression 4 weeks after injection, i.e., the reported time for the maximal expression of AAV-encoded proteins.16Palomeque J Chemaly ER Colosi P Wellman JA Zhou S Del Monte F et al.Efficiency of eight different AAV serotypes in transducing rat myocardium in vivo.Gene Ther. 2007; 14: 989-997Crossref PubMed Scopus (91) Google Scholar Surprisingly, we were unable to detect any fluorescent signal in the spinal cord of intramuscularly (i.m.) injected mice (n = 10; Figure 1a,c), while there was strong eGFP expression in all injected muscles (Figure 1e), suggesting either that AAV particles were not retrogradely transported or that transported particles did not lead to detectable amounts of eGFP expression. For a more sensitive detection of virus presence in the spinal cord after i.m. delivery, we employed quantitative PCR analysis of DNA extracted from the thoracic and lumbar spinal cord. However, we were unable also with this method to detect viral DNA in the spinal cord of any of the animals studied (n = 4; Supplementary Figure S1).We therefore decided to directly inject viral particles into the spinal cord to transduce motoneurons.17Lepore AC Haenggeli C Gasmi M Bishop KM Bartus RT Maragakis NJ et al.Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS.Brain Res. 2007; 1185: 256-265Crossref PubMed Scopus (98) Google Scholar This mode of injection led to strong and mainly neuronal expression of the virus-derived eGFP in the ventral spinal cord (Figure 1d)18Klugmann M Leichtlein CB Symes CW Serikawa T Young D During MJ Restoration of aspartoacylase activity in CNS neurons does not ameliorate motor deficits and demyelination in a model of Canavan disease.Mol Ther. 2005; 11: 745-753Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar over a length of at least 2 mm (Figure 1b,d), but not in the musculature (Figure 1f). Thus, intramuscular injection of AAV particles in our hands did not result in detectable retrograde transport of the virus, whereas intraspinal delivery appears as a very efficient way to deliver AAV particles to the spinal cord.Intraspinal injection of AAV leads to a highly CNS-specific expression of G-CSFAfter intramuscular injection at week 15, we observed a high level of G-CSF in the serum accompanied by a moderate increase of G-CSF in spinal cord extracts. In contrast to this, intraspinal injection of AAV G-CSF strongly increased spinal G-CSF levels, but only moderately elevated serum G-CSF (serum: i.m. G-CSF: 1.890 pg/ml; intraspinal G-CSF: 420.1 pg/ml; eGFP: 92.5 pg/ml; P < 0.05 for both injections; spinal cord: i.m. G-CSF: 1.25 pg/mg; intraspinal G-CSF: 229.2 pg/mg; eGFP: 0.3 pg/mg; P < 0.05 for both injections Figure 2a,b).Figure 2Distribution of G-CSF after intramuscular and intraspinal injection of AAV vector. (a) The level of G-CSF in the serum is increased in mice injected with AAV G-CSF compared to mice injected with the control vector (n = 10, *P < 0.05). Muscular injection of AAV G-CSF leads to a higher level of circulating G-CSF compared to the spinal injection (P < 0.05). (b) Level of G-CSF in the total spinal cord is increased in mice injected with AAV G-CSF compared to mice injected with the control vector (n = 6, P < 0.05). The increase following intraspinal injection is 150-fold higher than after intramuscular injection (P < 0.05). (c) Neutrophil numbers following intramuscular and intraspinal injections of AAV G-CSF. The number of neutrophils increases after both muscular and spinal injections. Neutrophil counting was performed using an automatic counting system (n = 7, *P < 0.05). AAV, adeno-associated virus; G-CSF, granulocyte-colony stimulating factor; i.m., intramuscular; i.sp., intraspinal.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serum G-CSF is able to stimulate the proliferation of neutrophil precursors and their differentiation into mature neutrophilic granulocytes.19Neidhart J Mangalik A Kohler W Stidley C Saiki J Duncan P et al.Granulocyte colony-stimulating factor stimulates recovery of granulocytes in patients receiving dose-intensive chemotherapy without bone marrow transplantation.J Clin Oncol. 1989; 7: 1685-1692Crossref PubMed Scopus (127) Google Scholar Indeed, we noted a significant elevation of neutrophils after both intramuscular and intraspinal injections when compared to the control SOD-1 (G93A) mice. Whereas neutrophil count was eightfold elevated with i.m. injection (4.04 neutrophils/nl), intraspinal delivery resulted only in a threefold elevation (1.68 neutrophils/nl), still within the normal value range for mice20Hedrich H Bullock G The Laboratory Mouse (The Handbook of Experimental Animals). Elsevier Academic Press, London2004: 278Google Scholar (Figure 2c). Thus, muscular injection of AAV predominantly led to a systemic delivery of G-CSF produced in the injected muscles, along with its expected consequences in terms of neutrophil elevation and did not pose any advantage over systemic subcutaneous delivery. In contrast, intraspinal injection led to a highly CNS-specific delivery with low systemic levels, and a moderate increase of neutrophils, suggesting that this mode of delivery could maximize the neuroprotective effects, and minimize the peripheral effects. We thus focused our efforts on this mode of delivery.Spinal delivery of G-CSF is beneficial for SOD-1 (G93A) miceSince intraspinal injection in contrast to i.m. injection requires a relatively lengthy surgery, we studied postoperative behavior, weight, and motor performance of the mice and found no obvious difference between mice before and 1 week after surgery (Supplementary Figure S2), suggesting that the surgery did not have a major impact on general mouse health and motor functions.We monitored Rotarod and grip strength performance as indicators of muscular endurance and strength weekly in AAV G-CSF or control injected animals (n = 12 female mice per group, litter-matched). Our experimental settings comply with the guidelines for preclinical studies in ALS established by the European ALS/MND group.21Ludolph AC Bendotti C Blaugrund E Chio A Greensmith L Loeffler JP et al.Guidelines for preclinical animal research in ALS/MND: A consensus meeting.Amyotroph Lateral Scler. 2010; 11: 38-45Crossref PubMed Scopus (244) Google Scholar From week 20 on we noted a relative improvement in muscular strength in the G-CSF versus the control group (AAV- eGFP: 151 mN; AAV G-CSF: 285 mN; P < 0.05 by repeated measures analysis of variance and Fisher's LSD) and a better performance on the Rotarod (AAV-eGFP: 34 seconds; AAV G-CSF: 135 seconds, P < 0.05 by repeated measures analysis of variance and Fisher's LSD) (Figure 3).Figure 3AAV G-CSF improves motor functions of SOD-1 (G93A) mice. (a) Muscular strength measured by the grip strength test. AAV G-CSF treatment leads to a relative preservation of the muscular strength of SOD-1 (G93A) mice at the mid- to end-point of the disease (n = 12). (b) Performance on Rotarod. AAV G-CSF treatment leads to an improvement of endurance/coordination performance at the mid- to end-point of the disease (n = 12). AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; G-CSF, granulocyte-colony stimulating factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The disease progression in SOD-1 (G93A) mice is well described. The disease at its onset is characterized by a slight hind limb tremor, the midpoint of disease is defined by gait impairment and weight loss, and the end stage of disease is marked by paresis. Onset of body mass decrease, defined as a drop of 5% of the mouse maximal weight (around 1 g), was significantly delayed by G-CSF treatment by >2 weeks (P < 0.05; Figure 4a). The onset of gait impairment, defined as abnormal limb movement in at least one hind limb, was not significantly different between the two groups despite a trend (P < 0.17), but the onset of paresis, defined as the inability to use one limb in the coordinated stride, was significantly delayed after G-CSF treatment (P < 0.05; Figure 4b). Most importantly, the clinical end point of the disease was delayed by 15 days, increasing the survival by 10% (Figure 4c). This gain in survival was higher than the increases observed after subcutaneous delivery (7% increased survival8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar). An increase of 7% in survival was also seen in the mice treated by i.m. injection, which was essentially a systemic delivery (Supplementary Figure S3).Figure 4AAV G-CSF delays symptoms progression and enhances survival of SOD-1 (G93A) mice. Kaplan–Meyer graphs showing time to (a) body mass decrease, (b) onset of paresis, and (c) clinical endstage of the disease. (a) Body mass decrease, associated with muscular atrophy, is delayed after AAV G-CSF treatment (P < 0.05). (b) First manifestation of paresis of the hind limbs is delayed after AAV G-CSF treatment (P < 0.05). (c) Survival of SOD-1 (G93A) mice is increased by 10% after AAV G-CSF treatment (P < 0.05). AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; G-CSF, granulocyte-colony stimulating factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Thus, G-CSF delivered by AAV to the spinal cord is able to delay disease progression and improve survival in SOD-1 (G93A) mice.Spinal delivery of G-CSF maintains motor-unit integrity in SOD-1 (G93A) tg miceMotoneuron survival. G-CSF is known to protect α-motoneurons under proapoptotic conditions.8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar,11Henriques A Pitzer C Dupuis L Schneider A G-CSF protects motoneurons against axotomy-induced apoptotic death in neonatal mice.BMC Neurosci. 2010; 11: 25Crossref PubMed Scopus (26) Google Scholar We have previously shown an increase by 30% in the total number of α-motoneurons at midpoint of the disease (week 15) after systemic delivery of G-CSF.8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar Here, we sought to determine the effect of a direct delivery of G-CSF to motor neurons at two different spinal segments: the cervical (C3–C4) and lumbar level (L3–L4) of 15-week-old SOD-1 (G93A) mice. We used the previously described criteria based on localization, size, and ChAT positivity.8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar We noted a loss of α-motoneurons at both lumbar and cervical spinal segments for the SOD-1 (G93A) mice when compared to the littermate wild types (P < 0.05; Figure 5). After G-CSF treatment, we noted a rescue of α-motoneurons at both the cervical (+50% motoneurons; P < 0.05; Figure 5a) and lumbar level (+35% motoneurons; P < 0.05; Figure 5c). The analysis of the size distribution of the remaining motoneurons indicates that large α-motoneurons are particularly protected by G-CSF treatment at both levels (Figure 5b,d; P < 0.05). Supplementary Figure S4 shows a histogram size distribution of cervical and lumbar motoneurons. In addition, we assayed microglial numbers in the spinal cord as a possible cellular element contributing to disease pathophysiology.22Boillée 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: 1389-1392Crossref PubMed Scopus (1263) Google Scholar While microglial numbers were increased in the SOD-1 (G93A) model at week 15 in contrast to wild-type littermates, we could not detect any influence of G-CSF on this elevation, a result in concordance with our previous study8Pitzer C Krüger C Plaas C Kirsch F Dittgen T Müller R et al.Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis.Brain. 2008; 131: 3335-3347Crossref PubMed Scopus (104) Google Scholar (Supplementary Figure S5).Figure 5AAV G-CSF improves α-motoneuron survival in SOD-1 (G93A) mice. (a,c) Quantification of surviving α-motoneurons after AAV G-CSF treatment, in SOD-1 (G93A) mice at 15 weeks of age. Motoneuron survival is increased after spinal injection of AAV G-CSF at both the (a) cervical (+50%; P < 0.05; n = 9) and (c) lumbar (+35%; P < 0.05; n = 9) level of the spinal cord when compared to control. (b,d) Size evaluation of all ChAT+ cells in SOD-1 (G93A) mice at 15 weeks of age. There is an upward shift in mean size distribution by AAV G-CSF treatment at both the (b) cervical and (d) lumbar level (P < 0.05). AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; G-CSF, granulocyte-colony stimulating factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Preservation of NMJs. In ALS, the disruption of the NMJs occurs long before the degeneration of the cell body of motoneurons.23Fischer LR Culver DG Tennant P Davis AA Wang M Castellano-Sanchez A et al.Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man.Exp Neurol. 2004; 185: 232-240Crossref PubMed Scopus (988) Google Scholar Therefore, the rescue of α-motoneuron cell bodies is not sufficient to explain a therapeutic effect that inherently implies preserved muscle innervation.24Dupuis L Loeffler JP Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models.Curr Opin Pharmacol. 2009; 9: 341-346Crossref PubMed Scopus (153) Google Scholar To determine whether G-CSF treatment can preserve muscular innervation, we investigated the state of the NMJs in the gastrocnemius muscle of 15-week-old mice. At this age, mice present clear gait impairment and decreased performance in both Rotarod and grip strength analyses. At first, we determined the innervation fraction of the gastrocnemius muscle, defined as the number of innervated NMJs per total NMJs (innervated and denervated). We found that the gastrocnemius muscle in SOD1 (G93A) mice showed clear denervation when compared to the littermate control wild-type mice where virtually all muscular endplates were found innervated (Figure 6a). The severity of this denervation is reduced by G-CSF treatment (AAV-eGFP: 57.7% innervation ratio; AAV G-CSF: 75.7%; wild type: 90.2%; P < 0.05; Figure 6b). This effect is also seen when comparing the total number of innervated NMJs per muscle volume (40% increase in the total number of NMJs after AAV G-CSF transduction; AAV-eGFP: 40.6 innervated NMJs/mm3; AAV G-CSF: 57.4; P < 0.01; Figure 6c).Figure 6AAV G-CSF enhances muscular innervation in SOD-1 (G93A) mice. (a) Double fluorescence immunostaining of nicotinic acetylcholine receptors (AChr, red) and axons (neurofilament-L, green) in the gastrocnemius muscle of 15-week-old mice (AAV-eGFP, AAV G-CSF, and wild-type mice). (b) Innervated NMJs in percentage of total NMJs, in the gastrocnemius muscle of 15-week-old mice. AAV G-CSF treatment increases the fraction of innervated sites (P < 0.0005; n = 5). (c) Number of innervated NMJs normalized to muscle volume. AAV G-CSF treatment increases the number of innervated NMJs per volume (P < 0.0005; n = 5). AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; G-CSF, granulocyte-colony stimulating factor; NMJ, neuromuscular junction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Motor axon regeneration. An important intrinsic compensatory mechanism in ALS is that surviving motoneurons partially reinnervate postsynaptic NMJ sites that belonged to the motor unit of a damaged neighboring motoneuron.25Schaefer AM Sanes JR Lichtman JW A compensatory subpopulation of motor neurons in a mouse model of amyotrophic lateral sclerosis.J Comp Neurol. 2005; 490: 209-219Crossref PubMed Scopus (200) Google Scholar A G-CSF-induced higher propensity for motor axon outgrowth may therefore be an additional mechanism that leads to a higher number of innervated NMJs, especially since G-CSF enhances neurite outgrowth in vitro.9Pitzer C Klussmann S Krüger C Letellier E Plaas C Dittgen T et al.The hematopoietic factor granulocyte-colony stimulating factor improves outcome in experimental spinal cord injury.J Neurochem. 2010; 113: 930-942Crossref PubMed Scopus (39) Google Scholar,26Pan HC Wu HT Cheng FC Chen CH Sheu ML Chen CJ Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury.Biochem Biophys Res Commun. 2009; 382: 177-182Crossref PubMed Scopus (31) Google Scholar To approach this question, we performed sciatic nerve crush injury on SOD-1 (G93A) mice at 15 weeks of age. Sciatic nerve crush in adult mice results in axonal degeneration and in muscular denervation. Due to the small length of the sciatic nerve and a high rege