Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Abstract

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1 μM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24–48 h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI₃K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.

Introduction

Dysfunction of dopamine (DA) neurons has been implicated in several neurodegenerative and neuropsychiatric disorders, including Parkinson's disease, addiction, bipolar disorder, and depression. DA neurons in the substantia nigra (SN) degenerate in Parkinson's disease, whilst ventral tegmental area (VTA) DA neurons are thought to be involved in addiction and mood disorders. The magnitude and duration of dopaminergic signaling are defined by the amount of DA released, the sensitivity of the DA receptors, and the efficiency of DA clearance.

Vesicular release of DA is dependent on vesicular monoamine transporter-2 (VMAT2) activities of DA neurons in the CNS. VMAT2 is expressed on the monoaminergic secretory vesicles in the CNS (Erickson et al., 1992, Erickson and Eiden, 1993) and is responsible for packaging DA, noradrenalin, serotonin, and histamine in the neuronal cytoplasm into synaptic vesicles (Johnson, 1988, Henry et al., 1994, Liu et al., 1992, Erickson et al., 1992). VMAT2-knockout (KO) mice completely lacking VMAT2 survive less than a few days after birth (Fon et al., 1997, Takahashi et al., 1997, Wang et al., 1997), whereas mice that express approx. 5% of the normal VMAT2 level are viable into adulthood (Mooslehner et al., 2001). The brains of VMAT2-KO mice show extremely low contents of catecholamines including serotonin, noradrenalin, and DA (Fon et al., 1997). Lack of VMAT2 causes depletion of DA in synaptic vesicles, and cytoplasmic DA is immediately metabolized by monoamine oxidase.

In heterozygous VMAT2-KO mice, the administration of the MPP⁺ precursor N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine produces more than twice the DA cell loss found in wild-type mice treated with the precursor (Takahashi et al., 1997). Also, VMAT2 has been implicated in effecting methamphetamine-induced DA deficits. Methamphetamine-induced dopaminergic neurotoxicity is increased in the striatum of VMAT2-KO mice compared with that in wild-type mice, as revealed by a more consistent DA and metabolite depletion and a greater decrease in DA transporter (DAT) expression (Fumagalli et al., 1999). The DAT is largely responsible for regulating DA clearance (Giros et al., 1996) and is a main target of amphetamines.

Of interest, morphometric analysis of cultured midbrain DA neurons from wild-type and VMAT2-KO mice has revealed that the ultrastructure of DA terminals and synaptic vesicles in the neurons from VMAT2-KO mice is indistinguishable from that of those in wild-type cultures (Croft et al., 2005). Also, VMAT2-KO does not influence the total number of synaptic vesicles or their ability to cycle (Croft et al., 2005). These studies using VMAT2-KO mice have yielded an abundance of valuable information; however, aside from the neurotoxicity caused by genetic deletion of VMAT2, the effects of this deletion on dopaminergic cell functions are not clear.

Here, we investigated dopaminergic neurons more precisely by using midbrain cultures derived from fetuses of wild-type and VMAT2-KO (heterozygous and homozygous) mice. We found that DAT activity was reduced by genetic deletion or pharmacological blockade of VMAT2. In addition, the results of cDNA array experiments showed a difference in the gene expression profile between wild-type and VMAT2-KO mice.