HIF prolyl hydroxylase inhibition increases cell viability and potentiates dopamine release in dopaminergic cells.

Hypoxia-inducible factor (HIF) controls the expression of genes that adapts the cellular condition to accommodate oxidative stress. The potential beneficial effect of HIF up-regulation in ischemia has recently gained interest substantiated by the known HIF-regulation of erythropoietin and other hypoxia accommodating genes. So far the perspectives for HIF up-regulation has been focused on anemia and ischemia related diseases but little information is available about the relevance of HIF biology for neurodegenerative disease like Parkinson's disease. We therefore sought out to characterize the effect of HIF-up-regulation on survival and dopamine homeostasis in dopaminergic cells. We used a low molecular weight HIF prolyl hydroxylase (HPH) inhibitor and lentiviral based shRNA knockdown of HPH subtypes as molecular tools to increase HIF protein level and downstream HIF-regulated genes. We show that HIF induction results in protection against oxidative stress in cellular models based on PC12 cells and LUHMES cells. In addition, HPH inhibition elevates tyrosine hydroxylase expression and activity, which causes increased dopamine synthesis and release in both PC12 cells and a primary rat ventral mesencephalic cell culture. All together these findings suggest that prolyl hydroxylases may represent novel targets for therapeutic intervention in disorders characterized by dopamine homeostasis dysregulation like Parkinson's disease.

HIF prolyl hydroxylase inhibition augments dopamine release in the rat brain in vivo.

The transcription factor hypoxia-inducible factor (HIF) is essential for the activation of several genes that promote the survival of cells exposed to oxidative stress. Expression of tyrosine hydroxylase (TH), which is the rate-limiting enzyme in the dopamine (DA) synthesis, is one of the genes that are positively regulated by HIF. Accordingly, HIF induction results in elevated DA release in various cell lines in vitro. HIF prolyl hydroxylase (HPH) is critically involved in the negative regulation of HIF levels. We investigated the in vivo effects of the HPH inhibitor FG0041 on brain DA function in rats by microdialysis in freely moving rats, locomotor activity, and Western blot analysis. Administration of FG0041 (10 mg/kg i.p.), as an acute (single injection), or as subchronic (once daily for 6 days) treatment and cobalt chloride (CoCl2) (60 mg/kg s.c.) potentiated potassium (K+) induced increases in extracellular levels of DA levels in the rat striatum. The increase in extracellular DA of freely moving rats was sought in relationship to locomotor activity in rats. A significant increase in locomotor activity was observed in FG0041-treated rats compared with vehicle on a cocaine challenge. In support of these findings, protein levels of TH in the rat brain stem were increased after treatment with FG0041. These data indicate that FG0041 augments DA function in the rat brain. Inhibition of HPH enhances DA function by increasing DA release, which has implications for the use of HIF induction in the treatment of neurodegenerative diseases.

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

An 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.

Efficient in vivo protection of nigral dopaminergic neurons by lentiviral gene transfer of a modified Neurturin construct.

Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member Neurturin (NTN) have demonstrated neuroprotective effects on dopaminergic (DA) neurons, which are selectively lost during Parkinson's disease (PD). However, unlike GDNF, NTN has not previously been applied in PD models using an in vivo gene therapy approach. Difficulties with lentiviral gene delivery of wild type (wt) NTN led us to examine the role of the pre-pro-sequence, and to evaluate different NTN constructs in order to optimize gene therapy with NTN. Results from transfected cultured cells showed that wt NTN was poorly processed, and secreted as a pro-form. A similarly poor processing was found with a chimeric protein consisting of the pre-pro-part from GDNF and mature NTN. Moreover, we found that the biological activity of pro-NTN differs from mature NTN, as pro-NTN did not form a signaling complex with the tyrosine kinase receptor Ret and GFRalpha2 or GFRalpha1. Deletion of the pro-region resulted in significantly higher secretion of active NTN, which was further increased when substituting the wt NTN signal peptide with the immunoglobulin heavy-chain signal peptide (IgSP). The enhanced secretion of active mature NTN using the IgSP-NTN construct was reproduced in vivo in lentiviral-transduced rat striatal cells and, unlike wt NTN, enabled efficient neuroprotection of lesioned nigral DA neurons, similar to GDNF. An in vivo gene therapy approach with a modified NTN construct is therefore a possible treatment option for Parkinson's disease that should be further explored.