C-terminal mechano growth factor protects dopamine neurons: A novel peptide that induces heme oxygenase-1

Abstract

To assess potential efficacy of mechano growth factor (MGF) for chronic neurodegenerative disorders, we studied whether MGF protects dopamine (DA) neurons subjected to neurotoxic stress. We show that a short 24-amino acid C-terminal peptide of MGF (MGF24) upregulates heme oxygenase-1 (HO-1) expression and protects SH-SY5Y cells against apoptosis and cell loss induced by three DA cell-specific neurotoxins: 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP⁺), and rotenone. MGF24 maintains the mitochondrial membrane potential and blocks the release of mitochondrial apoptotic-inducing factor into the cytoplasm induced by 6-OHDA, MPP⁺, and rotenone. Chemical inhibition of HO-1 with zinc protoporphyrin-IX prevents neuroprotection by MGF24 against the three neurotoxins. MGF24 does not activate Akt signaling nor does Akt inhibition block MGF24 protection of SH-SY5Y cells. In 6-OHDA-lesioned rats, central or peripheral MGF24 administration protects against the development of contralateral forelimb under-utilization, reduces ipsilateral nigral DA cell body loss, and attenuates tyrosine hydroxylase fiber loss in the ipsilateral striatum, independent of IGF-1 receptor activation. Peripheral MGF24 administration upregulates HO-1 expression in striatal and midbrain tissue. This report is the first to demonstrate that a small peptide, MGF24, upregulates HO-1, an important cell defense mediator, and protects DA cells, suggesting new strategies for neuroprotection in Parkinson's disease.

Introduction

Parkinson's disease (PD) is a common neurodegenerative disorder for which only symptomatic therapies are available. A major medical need is the identification of therapies that arrest or reverse neuronal loss in this disorder. Although much has been learned about intracellular mechanisms implicated in dopamine (DA) neuron loss in PD, and factors identified that influence those processes, no therapies that are potent, selective, practical, and safe have been developed. Potent neuroprotective small peptides that enter the brain on peripheral administration would fulfill this medical need if achievable. Neurotrophins have long intrigued PD researchers as potent neuroprotectants and potential promoters of neural regeneration. Mechano growth factor (MGF) is a splice variant of IGF-1, also termed IGF-1Ec in humans and IGF-1Eb in rodents, whose expression in muscle is upregulated by stretch (McKoy et al., 1999, Yang et al., 1996). Although IGF-1 is only a 70-amino acid peptide, its gene at 90 kb is quite large (Winn et al., 2002). Elaborate alternative splicing of IGF-1 exons results in multiple distinct circulating and tissue-specific isoforms with different functions, including involvement in neuronal maintenance (Cheng et al., 2003, D'Ercole et al., 1996, Fukudome et al., 2003, Vicario-Abejon et al., 2003). All isoforms of IGF-1 encode the 70-amino acid peptide but differ in the E-domain region. In rodents, the E-domain of MGF is created by a 52-base pair insert from exon 5, which causes a reading frame shift encoding a unique E-domain region that distinguishes it from the predominant IGF-1Ea isoform. Cleavage of this prohormone form by cellular proprotein convertases yields the mature IGF-1 peptide and the E-domain (Shavlakadze et al., 2005, Winn et al., 2002).

MGF initially was appreciated as exerting post-mitotic reparative effects in skeletal muscle, but more recently neuroprotective actions have been demonstrated in acute injury models: The 24-amino acid C-terminal peptide of MGF protects cultured hippocampal cells from oxidative stress and glutamate-dependent excitotoxicity. Intra-carotid injection of modified MGF C-terminal peptide reduces ischemic CA1 hippocampal cell loss in gerbils. In this model, ischemia increases endogenous MGF expression in the ischemia-resistant CA2 region, raising the possibility that MGF upregulation in some neurons is a defense response (Dluzniewska et al., 2005). We previously showed that nigral DA neurons, which abundantly express IGF-1 receptors (Quesada et al., 2007), are protected by IGF-1 from neurotoxicity in rat (Quesada and Micevych, 2004) via the phosphoinositol-3-kinase (PI3K)/Akt pathway (Quesada et al., 2008). These previous findings suggest that the IGF-1 system is important in protecting nigral DA neurons. We thus investigated whether the splice variant of IGF-1, MGF 24-amino acid C-terminal fragment (MGF24), could protect DA neurons in models of a chronic neurological disease, Parkinson's.

When challenged by adverse conditions such as oxidative stress, specific cell defense proteins may be upregulated (Calabrese et al., 2005). Heme oxygenase-1 (HO-1), also known as heat shock protein 32 (HSP32), is a small stress protein involved in heme catabolism, leading to the generation of biliverdin, free iron, and carbon monoxide (Choi and Alam, 1996, Foresti and Motterlini, 1999, Motterlini et al., 2002). Three isoforms of HO, products of individual genes, have been identified: the inducible HO-1 and the constitutive HO-2 and HO-3 (Schipper et al., 1995). Chemical induction of HO-1 or over-expression confers protection against oxidative and nitrosative stress (Alves da Costa et al., 2006, Busiguina et al., 2000, Fauconneau et al., 2002, Yang et al., 2003). In the brain, HO-1 levels are normally low, but are readily induced by various stressors (Scapagnini et al., 2002). Evidence suggests that the HO-1 pathway acts as an important defense mechanism for neurons and modulation of HO-1 expression may represent a novel target for therapeutic intervention. This is the first report showing that MGF24 powerfully protects DA neurons both in vivo and in vitro, and that this peptide upregulates the important neuroprotection mediator HO-1, effects that are independent of IGF-1 receptor/Akt activation.