C52 HEPATOCYTE GROWTH FACTOR (HGF) AS A NOVEL NEUROTROPHIC FACTOR FOR AMYOTROPHIC LATERAL SCLEROSIS (ALS)
Funakoshi H, Nakamura T
Osaka University Graduate School of Medicine, Osaka, Japan
E‐mail address for correspondence: [email protected]
Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes and was molecularly cloned in 1989 (Nakamura et al., BBRC, 1984; Nakamura et al., Nature, 1989). HGF prevents endotoxin induced lethal hepatic failure in mice with fulminant hepatitis via its anti‐apoptotic activity, and HGF gene therapy is capable of improving the survival rate of rats with lethal liver cirrhosis (Kosai et al., Hepatology, 1999; Ueki et al., Nat Med, 1999). In addition to its role as a hepatotrophic factor, extensive expression and functional studies, including knock‐out/knock‐in mouse strategies, revealed HGF to be a novel neurotrophic factor for a variety of neurons (Honda et al., Mol Brain Res, 1995; Maina et al., Nat Neurosci, 1999). Furthermore, HGF is one of the most potent survival‐promoting factors for motor neurons. We show that overexpression of HGF in the nervous system attenuates motor neuron death and axonal degeneration, improves motor function and prolongs the life span of transgenic mice overexpressing mutated Cu2+/Zn2+ SOD1. HGF prevented induction of proapoptotic proteins (various types of activated caspases) in both spinal and brainstem motor neurons and increased X‐linked inhibitor of apoptosis protein (XIAP) in these motor neurons. In addition, HGF retained the levels of the glial‐specific glutamate transporter (EAAT2/GLT1) in reactive astrocytes, presumably favoring a reduction in glutamatergic neurotoxicity. We also present the efficient activation of c‐Met/HGF receptor by HGF in an ALS‐dependent fashion. Taken together with the evidence that HGF and c‐Met are regulated in the spinal cord of both familiar and sporadic patients with ALS in a manner similar to the transgenic ALS model, we propose that local application of HGF may be a safe and effective therapeutic for patients with ALS.
C53 INTRATHECAL DELIVERY OF HEPATOCYTE GROWTH FACTOR AT THE ONSET OF PARALYSIS SLOWS DISEASE PROGRESSION IN A RAT MODEL OF ALS
Aoki M1, Ishigaki A1, Nagai M1, Warita H1, Kato S3, Kato M4, Nakamura T5, Funakoshi H5, Itoyama Y1
1Department of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan, 2Tohoku University Hospital ALS Center, Sendai, Japan, 3Department of Neuropathology, Institute of Neurological Sciences, Faculty of Medicine Tottori University, Yonago, Japan, 4Division of Pathology, Tottori University Hospital,, Yonago, Japan, 5Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
E‐mail address for correspondence: [email protected]
Background and objectives: We developed a rat model of ALS expressing a human SOD1 transgene with two ALS‐associated mutations, G93A and H46R. Some experimental manipulations are difficult in Tg mice because of size limitations. However, this Tg rat model allows routine implantation of infusion pumps for intrathecal drug delivery.
Hepatocyte growth factor (HGF) is one of the most potent survival‐promoting factors for motor neurons. We reported that introduction of the HGF gene into neurons of G93A Tg mice attenuates motor neuron degeneration and increases the lifespan of these mice. Thus, HGF is a good candidate agent for treatment of ALS. Currently, treatment using recombinant protein is closer to clinical application than gene therapy. Therefore, we examined continuous intrathecal delivery of human recombinant HGF (hrHGF) into Tg rats using implanted infusion pumps for selective and less invasive supply of HGF to the spinal cord.
Methods: To examine the motor neuron protective effect and therapeutic potential of hrHGF, we administered 40 and 200 µg of hrHGF or vehicle alone to 100‐day‐old G93A Tg rats around the age that pathological changes of the spinal cord appear but the rats do not show weakness clinically. 200 µg of hrHGF or vehicle alone was administered to 115‐day‐old G93A Tg rats at the time of onset of paralysis. HrHGF was administered for four weeks in each case.
Results:Administration of hrHGF to 100‐day‐old G93A Tg rats for four weeks. At 130 days old, the average numbers of motor neurons in the ventral horn were: non‐Tg rats, 19.2±3.3; vehicle only, 2.9±1.3; 40 µg hrHGF, 6.3±2.1; 200 µg hrHGF, 11.2±4.2. Significantly more motor neurons survived in hrHGF‐treated (40 µg, p<0.01; 200 µg, p<0.001) than in vehicle‐treated G93A Tg rats. hrHGF prevented motor neuron death in G93A Tg rats in a dose‐dependent manner.
Administration of hrHGF to 115‐day‐old G93A Tg rats for four weeks. There were no statistically significant differences in the onset between the groups. In contrast, 200 µg hrHGF extended mean survival by 11 days compared to vehicle‐treated G93A Tg rats (p = 0.0135), although G93A Tg rats show very rapid disease progression and die within 20 days of disease onset. The average periods from the onset to death were 16.9±8.17 and 27.5±11.1 days in vehicle (n = 8) and hrHGF (n = 8) groups, respectively. The latter represented an increase of 63% relative to vehicle‐treated controls even started at the ALS onset.
Discussion and conclusions: The intrathecal administration of hrHGF attenuated motor neuron degeneration, and prolonged the duration of the disease by 63% even when administered from the onset of paralysis. We claim therapeutic effects of continuous intrathecal administration of hrHGF in Tg rats. The results should prompt further clinical trials in ALS using continuous intrathecal administration of hrHGF.
C54 INTRACEREBELLAR INJECTION OF AAV‐IGF‐1 IMPROVES MOTOR FUNCTION AND EXTENDS SURVIVAL IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Dodge JC1, Passini MA1, Clarke J1, Yang W1, Grissett L2, Kim SH2, Wen R2, Cheng SH1, Kaspar BK2, Shihabuddin LS1
1Genzyme Corporation, Framingham, MA, USA, 2Columbus Children's Research Institute, Columbus, Ohio, USA
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by loss of motor neurons in the cortex, brainstem and spinal cord. While there are numerous hypotheses for disease onset and progression, neurotrophic factors such as insulin‐like growth factor 1 (IGF‐1) have shown promise in delaying motor neuron death in ALS. Several studies have shown the benefit of using muscle‐specific IGF‐1 that have led to significant life extension and improved motor function in ALS transgenic mice. An important question raised by these studies is whether IGF‐1's mode of action was on the muscle, the neuromuscular junction or the motor neuron cell body. It is also becoming increasingly clear that multiple cell types other than motor neurons contribute to the pathogenesis of ALS. This raises the question as to whether potential therapies should be directed not only to motor neurons, but also to the surrounding cellular environment for maximal therapeutic efficiency.
Objectives: We sought to determine the effects of CNS delivered IGF‐1 without targeting the muscle and neuromuscular junction. We accomplished this by delivering IGF‐1 expressing AAV vectors (that are capable of retrograde transport) to a region of the brain that has extensive connections with the brainstem and spinal cord – the deep cerebellar nuclei (DCN) of the cerebellum. By targeting the DCN we also tested whether IGF‐1 expression by cells that comprise a motor neuron's surrounding cellular environment is sufficient to modify disease progression in SOD1 mice.
Methods: In this experiment we evaluated the efficacy of bilateral delivery of AAV1–IGF‐1 and AAV2‐IGF‐1 to the DCN in symptomatic SOD1G93A mice. Starting at 80 days of age, SOD1G93A mice underwent rotarod and grip strength testing to assess motor function. At 90 days of age mice received stereotaxic injections of AAV1‐IGF‐1 (n = 26), AAV2‐IGF‐1 (n = 27), AAV1‐GFP (n = 26) or AAV2‐GFP (n = 25) aimed at the DCN.
Results: We found that AAV‐IGF‐1 treatment (regardless of serotype) significantly reduced gliosis throughout the brainstem and spinal cord, promoted motor neuron survival, improved motor performance in both rotarod and grip strength tests and significantly extended lifespan. IGF‐1 expression was detected throughout the brainstem and spinal cord using PCR and ELISA. Treatment with AAV‐GFP had no effect on any of the parameters mentioned above. Positive GFP fibers and/or cell bodies were seen in the cortex, brainstem (i.e. motor trigeminal nucleus, hypoglossal nucleus and facial nucleus) and in each division of the spinal cord.
Conclusion: Our results indicate that direct administration of AAV‐IGF‐1 into the DCN modifies disease progression in a mouse model of ALS.
C55 CHARACTERIZATION OF COLIVELIN‐MEDIATED NEUROPROTECTION AGAINST ALS‐RELEVANT INSULTS
Chiba T1, Yamada M1, Sasabe J1, Hashimoto Y2, Terashita K2, Aiso S1, Matsuoka M2, Nishimoto I2
1KEIO University, School of Medicine, Department of Anatomy, 2KEIO University, School of Medicine, Department of Pharmacology, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. Currently, there is no cure for ALS. We have previously reported that intracerebroventricular (i.c.v.) injection of Colivelin, a hybrid peptide consisting of activity‐dependent neurotrophic factor (ADNF) and a potent humanin (HN) derivative termed AGA‐(C8R)HNG17, improves motor performance and prolonged survival of G93A‐SOD1 transgenic (Tg) mice (1–3).
Objectives: To determine the neuroprotective mechanism of Colivelin, we compared the effect of ADNF, AGA‐(C8R)HNG17, or Colivelin on motor performance and the lifespan of G93A‐SOD1 Tg mice in vivo and on death of NSC34 motor neuronal cells overexpressing G93A‐SOD1‐ in vitro.
Methods: G93A‐SOD1 Tg mice, implanted with a cannula, were i.c.v. injected with 10 pmol/mouse of ADNF, AGA‐(C8R)HNG17 or Colivelin every other day from the age of 80 days. Motor performance was evaluated in the rotarod test performed every three days and mean ages of death were compared among the groups. NSC34 cells were transfected with G93A‐SOD1 cDNA, together with or without dominant negative forms (dn) of Ca2+/calmodulin‐dependent protein kinase IV (CaMKIV) or STAT3, by lipofection and cultured in the presence or absence of the peptides at 100 nM. Seventy‐two hours after transfection, cell mortality was measured by WST‐8 assay.
Results: Mean survivals of control (vehicle)‐, ADNF‐, AGA‐(C8R)HNG17‐, and Colivelin‐treated mice were 142.0±2.2, 143.6±1.7, 147.1±1.8, and 151.5±1.8 days, respectively. Colivelin significantly prolonged the life span vs. control (p = 0.0009), while neither ADNF nor AGA‐(C8R)HNG17 treatment did so. Death induced by G93A‐SOD1 was completely suppressed by 100 pM ADNF, AGA‐(C8R)HNG17, or Colivelin. Neuroprotective effect of ADNF was antagonized by dominant‐negative (dn) CaMKIV, while that of AGA‐(C8R)HNG17 was antagonized by dnSTAT3. Colivelin‐mediated neuroprotection was antagonized only when both dnCaMKIV and dnSTAT3 were simultaneously expressed.
Discussion and conclusions: Colivelin significantly prolonged survival of ALS mice, while administration of the same amount of ADNF or AGA‐(C8R)HNG17 did not, indicating that fusion of the two peptides gives rise to an additional advantage in neuroprotection in vivo. Colivelin protected NSC34 cells from death induced by overexpression of G93A‐SOD1 via activating CaMKIV and STAT3 in vitro. Thus, we conclude that Colivelin is a promising neurotrophic factor for treatment of ALS.
References
- Chiba T, Hashimoto Y, Tajima H, et al. J Neurosci Res. 2004;78:542–52.
- Chiba T, Yamada M, Hashimoto Y, et al. J Neurosci. 2005;25:10252–61.
- Chiba T, Yamada M, Sasabe J, et al. Biochem Biophys Res Commun. 2006;343:793–8.
C56 NGR ANTAGONIZES P75NTR‐DEPENDENT MOTOR NEURON DEATH: IMPLICATIONS FOR ALS
Rene F1, Dupuis L1, Pehar M2, Cassina P3, Castellanos R2, Rouaux C1, Dimou L4, Schwab M4, Loeffler JP1, Barbeito L2, Gonzalez de Aguilar JL1
1Laboratoire de Signalisations Moléculaires et Neurodégénérescence, INSERM U‐692, Université Louis Pasteur, Strasbourg, France, 2Departamento de Neurobiología Celular y Molecular, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay, 3Departamento de Histología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 4Brain Research Institute, University of Zurich and Department of Biology, ETH, Zurich, Switzerland
E‐mail address for correspondence: [email protected]
Background: Spinal motor neurons express the neurotrophin receptor p75NTR during the embryonic period of naturally occurring cell death but this expression gradually ends after birth. Adult motor neurons, however, can re‐express p75NTR following axotomy or in ALS, which has been involved in triggering the motor neuron death characteristic of these conditions. Indeed, our recent in vitro findings revealed that NGF secreted by reactive astrocytes as well as spinal cord lysates from symptomatic ALS SOD1(G93A) mice stimulate motor neuron death. On the other hand, growing evidence shows that p75NTR also interacts with the glycosyl‐phosphatidyl‐inositol‐anchored central nervous system Nogo‐66 receptor NgR to inhibit axon repair after spinal cord lesion. Therefore, p75NTR is able to trigger either neuronal death, upon binding to neurotrophins such as NGF and their precursor forms, or neurite outgrowth inhibition when bound to NgR; albeit, the crosstalk between these two pathways has not been explored to date.
Objectives: In the present study, we asked whether NgR activation, via binding to specific ligands, could regulate motor neuron survival by modulating p75NTR‐induced cell death under conditions in which both receptor partners appear coexpressed.
Methods: To assess whether previously characterized NgR ligands, Pep4 and NEP1‐40, can affect motor neuron survival, we administered these ligands to embryonic rat motor neuron cultures challenged with NGF in the presence of a low steady state concentration of nitric oxide or cocultured on reactive astrocytes secreting NGF. We also evaluated the ability of both NgR ligands to modulate motor neuron death induced by spinal cord lysates from symptomatic SOD1(G93A) mice. Finally, we tested whether Pep4 and NEP1‐40 were able to promote motor neuron survival in vivo following neonatal sciatic nerve axotomy.
Results: Pep4 and NEP1‐40 counteracted the death action of p75NTR in the various in vitro experimental paradigms used in this study. Surprisingly, they exhibited similar protection against p75NTR‐mediated cell death, which clearly contrasts with their antagonistic effects on neurite outgrowth. Most importantly, both NgR ligands abolished post‐axotomy ipsilateral motor neuron loss compared to vehicle‐treated animals.
Conclusion: The results of this study show for the first time that Pep4 and NEP1‐40, two NgR ligands typically reported as regulators of neurite outgrowth, are able to promote motor neuron survival in vitro and in vivo. We therefore describe an as yet unknown function of NgR in maintaining neuronal survival against the death‐promoting effect of p75NTR. These findings may have relevance when addressing future strategies to protect motor neurons from ALS.
C57 VEGF RESCUES THE LOSS OF ACTIVATED AKT PRECEDING MOTOR NEURON DEGENERATION IN ALS
Dewil M1, Kiraly D1, Sciot R2, Shaw PJ5, Ince PG4, Robberecht W3, van den Bosch L1
1Laboratory for Neurobiology, Experimental Neurology, 2Laboratory for Morphology and Molecular Pathology, University of Leuven, Leuven, Belgium, 3Department of Neurology, Leuven, Belgium, 4Neuropathology, 5Academic Neurology Unit, University of Sheffield, School of Medicine and Biomedical Sciences, Sheffield, UK
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis is one of the most devastating neurodegenerative disorders. The pathogenesis of this disease that results in a relentless loss of motor function remains unknown. We recently reported that treatment of mutant SOD1G93A overexpressing rats with VEGF resulted in a significant increase in survival compared to the control group (1). The precise mechanism of the neuroprotective effect of VEGF in ALS remains to be elucidated, but modulation of the phosphoinositol‐3‐kinase/Akt cascade is one of the most likely targets.
Objectives: In the present study, we aimed to investigate the pathophysiological role of Akt in ALS and the influence of VEGF on this important anti‐apoptotic protein.
Methods and results: Immunohistochemical studies showed that motor neurons of both sporadic, familial SOD1 and familial non‐SOD1 ALS patients lack phospho‐Akt, compared to control patients. In mutant SOD1G93A overexpressing mice, Western blot analysis showed that activated protein kinase B or phospho‐Akt is indeed lost very early in the disease. Immunohistological experiments showed that this loss is confined to motor neurons, supporting the pathogenetic relevance of the potent anti‐apoptotic kinase Akt. In vitro transfection experiments with constitutively active Akt revealed the neuroprotective effect of Akt in mutant dependent cyclosporin A‐induced cell death in SOD1G37R overexpressing N2A cells. To evaluate the relevance of the Akt pathway in the neuroprotective effect of VEGF, Western blot analysis was performed in spinal cord homogenates of VEGF‐treated SOD1G93A rats. These results showed that treatment with VEGF resulted in a significant increase in the levels of phospho‐Akt.
Conclusions: Our data support the relevance of Akt to the selective loss of motor neurons in ALS and provide evidence for its role as a target of VEGF. Therefore, further development of therapeutic strategies targeting the phosphoinositol‐3‐kinase/Akt pathway may be useful.
References
- Storkebaum E, Lambrechts D, Dewerchin M, et al. Nat Neurosci. 2005;8:85–92