Inhibiting BDNF expression by antisense oligonucleotide infusion causes loss of nigral dopaminergic neurons

https://doi.org/10.1016/j.expneurol.2004.11.030Get rights and content

Abstract

Brain derived neurotrophic factor (BDNF) expression is significantly reduced in the Parkinson's disease substantia nigra. This neurotrophin has potent affects on dopaminergic neuron survival protecting them from the neurotoxins MPTP and 6-hydroxydopamine (6-OHDA) commonly used to create animal models of Parkinson's disease and also promoting dopaminergic axonal sprouting. In this study, we demonstrate that an antisense oligonucleotide infusion (200 nM for 28 days) to prevent BDNF production in the substantia nigra of rats mimics many features of the classical animal models of Parkinson's disease. 62% of antisense treated rats rotate (P ≤ 0.05) in response to dopaminergic receptor stimulation by apomorphine. 40% of substantia nigra pars compacta tyrosine hydroxylase immunoreactive neurons are lost (P ≤ 0.00001) and dopamine uptake site density measured by 3H-mazindol autoradiography is reduced by 34% (P ≤ 0.005). Loss of haematoxylin and eosin stained nigral neurons is significant (P ≤ 0.0001) but less extensive (34%). These observations indicate that loss of BDNF expression leads both to down regulation of the dopaminergic phenotype and to dopaminergic neuronal death. Therefore, reduced BDNF mRNA expression in Parkinson's disease substantia nigra may contribute directly to the death of nigral dopaminergic neurons and the development of Parkinson's disease.

Introduction

Brain derived neurotrophic factor (BDNF) is a neurotrophin which is both synthesised by dopaminergic neurons in the adult nigrostriatal pathway (Schmidt-Kastner et al., 1996, Seroogy et al., 1994) and used by the same cells as a potent survival and growth factor (Hyman et al., 1991).

Application of BDNF (by intrathecal infusion, striatal injection or implantation of BDNF excreting fibroblasts) protects monkey and rat substantia nigra pars compacta (SNpc) dopaminergic neurons from the neurotoxic effects of MPTP (Tsukahara et al., 1995) and 6-OHDA (Levivier et al., 1995, Shults et al., 1995) respectively, improving the rotational behaviours induced by asymmetric stimulation of dopamine receptors with apomorphine in these models of Parkinson's disease. BDNF also promotes sprouting of dopaminergic axons. This occurs both at the site of BDNF injection into the striatum (Shults et al., 1995) and when striatal injury leads to increased striatal BDNF production (Wong et al., 1997) by activated microglia (Batchelor et al., 1999). The role of BDNF in these in vivo sprouting responses has been confirmed by demonstrating that intrastriatal BDNF antisense infusions both reduce wound related BDNF mRNA and protein expression and inhibit periwound dopaminergic sprouting (Batchelor et al., 2000). These animal data suggest that BDNF acts as an autocrine or paracrine survival and growth factor for dopaminergic neurons. A reciprocal accumulation of BDNF protein in the substantia nigra and depletion in the striatum after colchicine treatment to inhibit anterograde axonal transport (Altar and DiStefano, 1998) suggests either that BDNF is needed locally to maintain dopaminergic terminals or the possibility of an additional role in support of striatal neurons. However, lack of extensive striatal neuronal loss in Parkinson's disease raises doubt about the significance of the later suggestion (Agid et al., 1989).

The dopaminergic neurons which survive in the SNpc of Parkinson's disease patients express variable levels of BDNF mRNA (Howells et al., 2000) with a significant population of neurons expressing extremely low levels of BDNF protein (Mogi et al., 1999, Parain et al., 1999). These observations led to the hypothesis that reduced expression of BDNF by nigral dopaminergic neurons might contribute to their own death (Howells et al., 2000). Unfortunately, the analysis of post-mortem human tissue does not lend itself to determining whether BDNF loss causes neuronal injury or whether neural injury coincidentally causes the loss of BDNF. Furthermore, systemic deficits of BDNF expression in genetically modified animals which die soon after birth (Dluzen et al., 1999) also shed little light on this matter.

The aim of this study therefore was to determine whether reducing SNpc BDNF expression by antisense oligonucleotide infusion into the lateral substantia nigra pars compacta causes the death of SNpc dopaminergic neurons and behavioural changes characteristic of Parkinsonism in rats.

Section snippets

Animal surgery and oligonucleotide infusions

Male Sprague–Dawley rats (210–280 g, aged 6-7 weeks) were housed in a 12 h light/dark cycle with free access to food and water. The animals were anaesthetised (Ketamine hydrochloride 1 mg/kg and Acepromazine 5 mg/kg, i.p.) and placed in a stereotaxic frame. A burr hole was created over the SNpc at coordinates AP 5.3 mm and L 2.5 mm relative to bregma. A 6.8 mm long cannula was inserted through the burr hole until its opening was just above the SNpc and was fixed to the skull with cyanacrylate

Results

Infusing antisense, sense or reverse oligonucleotides into the SNpc of the rat for 28 days did not affect the daily life of the animals. All were able to feed, drink and move freely around their enclosure. BDNF antisense infusions did result in significant weight loss during the first few days of infusion. However, this loss was regained quickly and by 28 days all animals in the study had gained approximately 150 g in weight (Fig. 1).

Discussion

The inhibition of BDNF gene expression by antisense oligonucleotides (Batchelor et al., 2000, Lau et al., 1998) was used in an attempt to determine if the loss of BDNF expression in the substantia nigra resulted in neuronal death and thus resulted in a syndrome akin to Parkinson's disease as predicted from our previous finding that the surviving neurons in the SNpc of Parkinson's disease patients have a decreased BDNF mRNA expression (Howells et al., 2000). The results presented here

Conclusions

The infusion of an antisense oligonucleotide specific to the neurotrophic factor BDNF into the lateral tip of the substantia nigra pars compacta resulted in anatomical, neurochemical and behavioural deficits characteristic of neurotoxic models of Parkinson's disease in rodents. These results support the hypothesis that reduced BDNF expression is likely to contribute to the death of dopaminergic neurons in Parkinson's disease.

Acknowledgments

This study was supported by the National Health and Medical Research Council of Australia, the Austin Hospital Medical Research Foundation, Parkinson's Victoria, Bethlehem Griffiths Research Foundation and the Australian Brain Foundation.

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