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,1
* Ernest Gallo Research Center,
Department of Neurology, University of California, San Francisco, Emeryville, California, USA
1Correspondence: 5858 Horton St. Ste. 200, Emeryville, CA 94608, USA. E-mail: dorit.ron{at}ucsf.edu
SPECIFIC AIMS
Ibogaine is a psychoactive indole alkaloid extracted from the root bark of the African shrub Tabernanthe Iboga. Human and rodent studies suggest that Ibogaine administration produces a long-lasting reduction in consumption, craving, and withdrawal symptoms for multiple drugs of abuse, including alcohol. Despite its potential use as an anti-addiction agent, Ibogaine is not used in the U.S. to treat addiction because of its undesirable side effects. We recently found that up-regulation of the glial cell line-derived neurotrophic factor (GDNF) pathway in the dopaminergic ventral tegmental area mediates the Ibogaine-induced reduction in voluntary ethanol consumption. Human anecdotal reports and studies in rodents have suggested that a single treatment produced long-lasting attenuation of addictive phenotypes. We hypothesized that the long-lasting actions of Ibogaine are mediated at least in part via initiation of prolonged activation of the GDNF pathway. We therefore used the neuroblastoma dopaminergic-like SHSY5Y cells as a model system and determined whether, and how, short-term exposure of cells with Ibogaine produced a long-lasting increase in GDNF expression and signaling.
PRINCIPAL FINDINGS
Ibogaine treatment induces a persistent increase in GDNF expression and Ret phosphorylation
Treatment of cells with Ibogaine resulted in a sustained increase in GDNF mRNA (239±26%, n=3, P<0.01) and Ret phosphorylation that lasted for 48 h after Ibogaine incubation (338±78%, n=5, P<0.05). Next, we determined whether acute treatment with Ibogaine results in a prolonged increase in GDNF mRNA levels. We found that short-term incubation with Ibogaine resulted in a persistent increase in GDNF mRNA levels even 12 h after Ibogaine removal (Fig. 1
A). To test whether the long-term increase in GDNF message after short-term exposure of cells to Ibogaine is due to an increase in the stability of GDNF mRNA, cells were treated with Ibogaine for 3 h, washed, and fresh media was added with or without actinomycin D (A/D), an inhibitor of transcription. As shown in Fig. 1B
, A/D inhibited the long-lasting increase in GDNF mRNA levels, suggesting that Ibogaine does not affect the stability of the GDNF message. Next, we determined whether the initial increase in GDNF expression upon Ibogaine exposure is required for the long-lasting increase in GDNF mRNA levels. To test this, cells were treated with Ibogaine for 3 h, then incubated after Ibogaine washout with fresh media containing anti-GDNF-neutralizing antibodies. Incubation of cells with anti-GDNF antibodies, which sequester GDNF and thus inhibit the GDNF signaling pathway, abolished the increase in the expression of GDNF (Fig. 1C
). These results suggest that Ibogaine triggers the long-lasting increase in GDNF expression in a GDNF-dependent manner.
Autoregulation of GDNF expression
Neurotrophins have been reported previously to self-regulate their expression and secretion. We therefore tested the possibility that Ibogaine may mediate its long-lasting activities via autoregulation of GDNF expression and function. We found that incubation of cells with recombinant GDNF induced a sustained increase in GDNF expression (Fig. 2
A), suggesting a positive feedback mechanism in which activation of the GDNF pathway results in an increase in the message of the growth factor. To confirm this, we incubated cells stably expressing the empty pUSE vector (CM-C) with conditioned media from cells that stably overexpress GDNF and secrete high levels of the growth factor (CM-GDNF) or with media form CM-C. Incubation of cells with CM-GDNF but not CM-C induced a continuous increase in GDNF expression (Fig. 2B
). Next we determined whether the induction of GDNF expression requires ligation of the secreted polypeptide to its receptors. Cells were treated with PI-PLC, which hydrolyzes the GPI link of the GDNF coreceptor GFR
1 and thus blocks GDNF signaling, or with anti-GDNF neutralizing antibodies, followed by addition of CM-GDNF. As shown in Fig. 2C, D
(lanes 2 vs. 4), both inhibitory reagents prevented the increase in GDNF expression. In addition, similar to Ibogaine’s effect, a short treatment with CM-GDNF induced a long-lasting increase in the message of the growth factor even after CM-GDNF was removed (Fig. 2E
), and this was inhibited in the presence of A/D (Fig. 2F
). Finally, we found that blockade of ERK activity by U0126 inhibited the induction of GDNF expression in cells given 3 h exposure to either Ibogaine (99±1% inhibition, n=3, P<0.01) or CM-GDNF (67±14% inhibition, n=3, P<0.01); this effect was also observed when the MEK inhibitor was added after Ibogaine washout (88±6% inhibition, n=3, P<0.01).
Long-term, but not short-term, up-regulation of GDNF expression depends on protein synthesis
Our data imply that acute exposure of cells to Ibogaine results in an increase in GDNF mRNA levels, followed by secretion of the polypeptide to up-regulate its own message, initiating a GDNF autoregulatory cycle (Fig. 3
). If so, then increases in GDNF mRNA on short-term Ibogaine exposure should not be dependent on protein synthesis. As predicted, a brief (0.5 h) incubation of cells with Ibogaine was insensitive to the protein synthesis inhibitor, cycloheximide (no significant inhibition was observed, n=3, P<0.33), whereas longer (3 h) Ibogaine-mediated induction of GDNF expression was sensitive to cycloheximide treatment (94±9% inhibition, n=3, P<0.01).
CONCLUSIONS AND SIGNIFICANCE
Our results suggest that Ibogaine exposure leads to an increase in GDNF message, followed by the translation and subsequent secretion of the polypeptide, resulting in the activation of the GDNF receptor Ret, which activates the MAP kinase pathway to further up-regulate the GDNF message (Fig. 3)
. To our knowledge this is the first report of the up-regulation of GDNF expression via GDNF itself, and an intriguing possibility is that the positive feedback cycle of GDNF expression and sustained activation of the GDNF pathway contribute to long-term processes such as synaptic plasticity. As GDNF has been shown to be a critical mediator of the development and survival of midbrain dopaminergic neurons, this autoregulatory pathway could have implications for the use of GDNF as a therapeutic target for neurodegenerative diseases such as Parkinson’s disease (PD). Finally, this GDNF-mediated autoregulatory positive feedback mechanism may explain the long-lasting actions of Ibogaine to reduce drug and alcohol self-administration and may have implications for the treatment of addiction. GDNF acts as a negative regulator of biochemical and behavioral adaptations to drugs of abuse and alcohol. Therefore, agents that activate the GDNF pathway and/or increase GDNF message may be useful drugs to treat addiction, and our current work implies that short-term treatment with such agents may result in long-lasting changes in addictive phenotypes.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6394fje
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 R Maciulaitis, V Kontrimaviciute, F. Bressolle, and V Briedis Ibogaine, an anti-addictive drug: pharmacology and time to go further in development. A narrative review Human and Experimental Toxicology, March 1, 2008; 27(3): 181 - 194. [Abstract] [PDF]
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