Elsevier

Brain Research

Volume 1111, Issue 1, 21 September 2006, Pages 187-195
Brain Research

Research Report
Adenoviral gene transfer of hepatocyte growth factor prevents death of injured adult motoneurons after peripheral nerve avulsion

https://doi.org/10.1016/j.brainres.2006.06.104Get rights and content

Abstract

Hepatocyte growth factor (HGF) exhibits strong neurotrophic activities on motoneurons both in vitro and in vivo. We examined survival-promoting effects of an adenoviral vector encoding human HGF (AxCAhHGF) on injured adult rat motoneurons after peripheral nerve avulsion. The production of HGF in COS1 cells infected with AxCAhHGF and its bioactivity were confirmed by ELISA, Western blot and Madin-Darby canine kidney (MDCK) cell scatter assay. The facial nerve or the seventh cervical segment (C7) ventral and dorsal roots of 3-month-old Fischer 344 male rats were then avulsed and removed from the stylomastoid or vertebral foramen, respectively, and AxCAhHGF, AxCALacZ (adenovirus encoding β-galactosidase gene) or phosphate-buffered saline (PBS) was inoculated in the lesioned foramen. Treatment with AxCAhHGF after avulsion significantly prevented the loss of injured facial and C7 ventral motoneurons as compared to AxCALacZ or PBS treatment and ameliorated choline acetyltransferase immunoreactivity in these neurons. These results indicate that HGF may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Introduction

Hepatocyte growth factor (HGF) was initially identified and purified as a potent mitogen of primary cultured hepatocytes (Nakamura et al., 1984, Nakamura et al., 1989). HGF is a heterodimeric protein composed of α and β chains and induces proliferation, migration, differentiation of target cells as well as organogenesis and neovascularization (Funakoshi and Nakamura, 2003). In the nervous system, HGF exhibits strong neurotrophic activities for motoneurons both in vitro and in vivo (Caton et al., 2000, Ebens et al., 1996, Funakoshi and Nakamura, 2003, Honda et al., 1995, Koyama et al., 2003, Maina and Klein, 1999, Naeem et al., 2002, Novak et al., 2000, Okura et al., 1999, Sun et al., 2002, Wong et al., 1997, Yamamoto et al., 1997). There have been no reports, however, concerning the neurotrophic effects of HGF on adult motoneuron death after proximal nerve injury. In animal models of adult motoneuron injury, avulsion of cranial and spinal nerves causes marked motoneuron degeneration in adult rats (Koliatsos et al., 1994, Moran and Graeber, 2004, Ruan et al., 1995, Sakamoto et al., 2000, Sakamoto et al., 2003a, Sakamoto et al., 2003b, Søreide, 1981, Watabe et al., 2000, Watabe et al., 2005, Wu, 1993), so that these animal models can be useful for therapeutic evaluation of neurotrophic factors or neuroprotective molecules against adult motoneuron death (Ikeda et al., 2003, Sakamoto et al., 2000, Sakamoto et al., 2003a, Sakamoto et al., 2003b, Watabe et al., 2000, Watabe et al., 2005). We have recently shown that adenoviral gene transfer of glial-cell-line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), transforming growth factor-β2 (TGFβ2) and growth inhibitory factor (GIF)/metallothionein-III (MT-III) prevented the death of adult rat facial and spinal motoneurons after facial nerve and cervical spinal root avulsion (Sakamoto et al., 2000, Sakamoto et al., 2003a, Sakamoto et al., 2003b, Watabe et al., 2000). In the present study, we investigated whether HGF protects injured motoneurons after facial nerve or spinal root avulsion by using a recombinant adenoviral vector encoding human HGF.

Section snippets

Bioassay of recombinant human HGF

In this study, we constructed a recombinant adenoviral vector encoding human HGF (AxCAhHGF). To test the ability of AxCAhHGF to induce human HGF expression in vitro, COS1 cells were infected with AxCAhHGF and the conditioned media (CMs) were harvested at 3 days postinfection. The levels of human HGF in uninfected and infected CMs analyzed by enzyme-linked immunosorbent assay (ELISA) were 1.9 ±  0.4 ng/ml and 2004.8 ± 160 ng/ml, respectively (n = 3). Western blot analysis of the CM harvested at 3 days

Discussion

HGF binds to tyrosine kinase receptor c-Met and triggers diverse biological responses that include cell motility, proliferation, morphogenesis, neurite extension and anti-apoptotic activities in a variety of cells (Funakoshi and Nakamura, 2003, Maina and Klein, 1999). Although the function of HGF in the nervous system has not been fully elucidated, it has recently been shown that HGF plays a strong neuroprotective role for motoneurons both in vitro and in vivo (Caton et al., 2000, Ebens et al.,

Adenovirus preparation

The human HGF cDNA was excised from pBS-hHGF with deletion of 15 base pairs (Seki et al., 1990) and subsequently cloned into SwaI cloning site of a cassette cosmid pAxCAwt (TaKaRa, Osaka, Japan) carrying an adenovirus type-5 genome lacking the E3, E1A and E1B regions to prevent the virus replication. The cosmid pAxCAwt contains the CAG (cytomegalovirus-enhancer-chicken β-actin hybrid) promoter on the 5′ end and a rabbit globin poly (A) sequence on the 3′ end. The cosmid was then cotransfected

Acknowledgments

We are grateful to Dr. Jing-Song Shen (Jikei University School of Medicine) for adenovirus preparation. This work was supported by Grants-in-Aid for Ministry of Education, Culture, Sports, Science and Technology, Japan, and Research on Psychiatric and Neurological Diseases and Mental Health, H16-kokoro-017, Ministry of Health, Labor and Welfare, Japan.

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