Vesicular monoamine transporter 2 regulates the sensitivity of rat dopaminergic neurons to disturbed cytosolic dopamine levels

Abstract

A B S T R A C TAn abnormal accumulation of cytosolic dopamine resulting in reactive oxygen species and dopamine-quinone products may play an important role in the rather selective degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons in Parkinson's disease. The neuronal-specific vesicular monoamine transporter (VMAT2), responsible for uptake of dopamine into vesicles, has been shown to play a central role both in intracellular dopamine homeostasis and sequestration of dopaminergic neurotoxins. Direct or indirect enhancement of VMAT2 activity could therefore have neuroprotective effects by decreasing cytosolic dopamine levels. Here, we demonstrate that transfection of VMAT2 in the dopaminergic cell line, PC12, increases intracellular dopamine content, augments potassium-induced dopamine release and attenuates cell death induced by the cytosolic dopamine enhancer, methamphetamine, suggesting an enhancement in vesicular dopamine storage. In rat ventral mesencephalic cultures highly enriched for dopaminergic neurons, lentiviral delivery of recombinant VMAT2 using a neuronal-specific promoter also resulted in elevated intracellular dopamine content and neurotransmitter release after depolarization. The opposite was seen after downregulation of VMAT2 using virally delivered shRNAs. Furthermore, using this VMAT2 knockdown model, we are the first to report a direct link between enhanced cytoplasmic dopamine levels, measured following mild permeabilization of the plasma membrane using digitonin, and neurite degeneration in primary dopaminergic neurons. In conclusion, our data support the hypothesis that an increase in vesicular sequestration of dopamine by modulation of VMAT2 activity could restore neuronal function and enhance dopaminergic cell survival in conditions of dysregulated dopamine homeostasis such as Parkinson's disease.

PACAP38 increases vesicular monoamine transporter 2 (VMAT2) expression and attenuates methamphetamine toxicity

“…Also, it has been shown that women who possess VMAT2 promoter variants which increase vesicular dopamine uptake are relatively protected from late-onset Parkinson's disease (Glatt, et al 2006). Furthermore, a recent study reported two intriguing findings: that reducing VMAT2 in midbrain cultures by shRNA leads to terminal loss and that increasing VMAT2 expression by the use of viral vectors in PC-12 cells attenuates METH cytotoxicity (Vergo, et al 2007). However, no studies investigating the consequence of elevated VMAT2 on METH neurotoxicity in vivo have yet been reported.…”

“…METH neurotoxicity depends on the transporter proteins that package and recycle dopamine and PACAP38 has been suggested to alter this transport (Fumagalli, et al 1998, Takei, et al 1998, Fumagalli, et al 1999, Vergo, et al 2007). Dysfunction of the dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) caused by METH leads to a loss of dopamine from the neuron and oxidative stress Hastings 1999, Miyazaki, et al 2006).…”

Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases

“…The cells were transformed using the calcium-phosphate precipitation method. For this, a mixture of 27 µg lentiviral vector (Leander Johansen et al, 2005), 12 µg pMDG (envelope vector, http://www.addgene.com), 19 µg pBRΔ8.91 (packaging vector) (Vergo et al, 2007;Scholz et al, 2011), and 215 µl 2 M CaCl2 in a total volume of 1.75 ml was added dropwise to a 2x HBS solution (50 mM HEPES, 280 mM NaCl, and 1.5 mM Na2HPO4; pH 6.95) while vortexing. Precipitation was allowed by incubating the mixture at RT for 35 min.…”

The Effects of Locus Coeruleus and Norepinephrine in Methamphetamine Toxicity

“…The causal link between cytosolic DA levels and METH toxicity is supported by several studies. In fact, overexpression of VMAT2, which stores DA in the vesicles, significantly protects PC12 against METH toxicity [165], while a mutant strain of mice, which possesses only 5-10% of the VMAT2 expressed by wild-type animals, undergo enhanced METH-induced striatal neurotoxicity [166]. For the same reasons, DAT inhibitors protect against METH-induced striatal DA damage [74, 83, 167].…”