Animals resistant to drunken behavior offer clues to alcoholism's roots

Animals with a remarkable ability to hold their liquor may point the way toward the genetic underpinnings of alcohol addiction, two separate research teams reported in the October 6, 2006 issue of the journal Cell. Earlier studies have shown that people with a greater tolerance for alcohol have a greater risk of becoming alcoholics, according to the researchers.

In one study, researchers at the Fondazione Istituto FIRC di Oncologia Molecolare in Italy found that mice lacking a gene that influences the cellular "skeleton" become less susceptible to the intoxicating effects of alcohol. The animals consequently drink more ethanol than the average mouse, they reported. They further showed that the mutant animals' neurons became less sensitive to "remodeling" of the cytoskeleton that otherwise follows ethanol exposure.

In the second paper, a team of researchers led by Adrian Rothenfluh and Ulrike Heberlein at the University of California, San Francisco characterized flies that carried a mutation rendering them invulnerable to drunken behavior. They discovered that the ethanol-resistant flies failed to produce a regulatory protein underlying the sedative effects of alcohol. The lacking protein is one of three encoded by a single gene and is also suspected to influence the cytoskeleton of neurons, though indirectly.

"Most researchers in the alcohol field have focused on cell surface receptors--with little attention to the role of the cytoskeleton," said Ulrike Heberlein at UCSF, who is an author on both papers. "Now, these two papers have, through very different means, come to highlight a possible role of the actin cytoskeleton in behavioral responses to ethanol."

Cytoskeletal defects may have the ability to simultaneously change the dynamics of multiple receptors, she said. "These animals are tremendously resistant to alcohol. There seems to be something very central about the observed changes to their behavior."

Cytoskeletal modification is generally important for the development and later flexibility of brain cells, the researchers noted. Moreover, such flexibility, or "plasticity," of nerve connections in the brain represents the cellular basis for learning and memory.

"A growing body of evidence suggests that addiction is, in essence, a maladaptive form of learning," Heberlein added.

Alcoholism affects approximately 300 million people worldwide, Di Fiore's group said in their article. Resistance to the acute intoxicating effects of ethanol is a risk factor for the development of alcoholism and is genetically determined, at least in part. Thus, the researchers said, an understanding of the molecular mechanisms underlying ethanol resistance might provide important clues about alcohol addiction.

The Italian researchers, who normally study cancer, simply stumbled onto the findings now reported, Di Fiore said. They developed mice lacking Eps8 out of an interest in its cytoskeletal role only to find that the animals had no immediately apparent abnormalities.

Knowing that other genes can substitute for Eps8, the team decided to look for parts of the body that lacked activity in any of the other "backup" genes. That search led them to the brain, and specifically to the cerebellum. The cerebellum is responsible for coordinating fine muscle movements and maintaining balance and is known to play a role in the loss of coordination characteristic of drunkenness.

Indeed, the researchers found, their mutant mice were resistant to the "hypnotic and motor-incoordinating effects of ethanol." The "knockout" mice also drank more alcohol.

"The phenotype is quite striking," said Di Fiore. " The mice can consume a considerable amount of ethanol without becoming intoxicated. The differences are remarkable and they hold across the sexes."

Further investigation showed that ethanol exposure normally affects the distribution of Eps8, leading to a pronounced loss of actin support structures in the cytoskeleton of neurons. Those rapid and reversible responses to ethanol seen in normal mice were lacking in the Eps8-deficient animals.

By contrast, Heberlein's group set out to find genes involved in drug responses, said Adrian Rothenfluh, also of UCSF. Their genetic screen of many fly mutants uncovered several with a genetic disruption that left them particularly resistant to ethanol, he said. The team named the mutations white rabbit (whir) for their role in regulating responses to abused drugs as described in the song "White Rabbit" by Jefferson Airplane.

"Normally when you expose flies to ethanol vapor, they become hyperactive at first," Rothenfluh said. "Within five to ten minutes, they start to keel over." By contrast, whir flies just kept running around, he said.

Teasing out such differences in behavior usually requires careful analysis of videotaped behavior, he added. However, the ethanol resistance they observed in this case was immediately obvious.

Further examination of their resistant flies led them to a gene called RhoGAP18B. RhoGAPs negatively regulate enzymes called Rho GTPases, which are involved in dynamics of the cytoskeleton.

Scientists knew that the gene represented a RhoGAP based on its sequence, but it had otherwise not been previously characterized, Rothenfluh said. Further analysis revealed that the single gene actually encodes three different RhoGAP protein transcripts.

They determined that the resistance to alcohol stemmed from the loss of just one of those transcripts, RC, implicating it in the sedative effects of ethanol. They further reported evidence that the effects of RC loss stemmed from shifts in the levels of cytoskeleton-influencing RhoGTPases.

Moreover, they showed, a second transcript of the gene, called RA, is responsible for the early hyperactivity induced by alcohol. The distinct behavioral effects of the two transcripts are mediated by RhoGAP18B function in the same subset of adult neurons, they reported.

"Curiously, different RhoGAP18B transcripts, RA and RC, regulate the stimulant and sedating effects of ethanol, respectively," the researchers said. While the exact mechanisms underlying these distinct effects remain uncertain, "our data clearly show that Rho-type GTPases are intimately involved in the regulation of behavioral responses to ethanol exposure, thus implicating actin dynamics in the process."

Di Fiore said his group will next sequence human DNA samples in search of evidence that Eps8 variants might play a role in alcoholism. If that hypothesis is borne out, drugs that moderate the cytoskeleton might offer a new method for treating addiction, he suggested.

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Rothenfluh et al.

The researchers include Adrian Rothenfluh, Robert J. Threlkeld, Roland J. Bainton, and Linus T.-Y. Tsai of University of California, San Francisco in San Francisco, California; Ulrike Heberlein of University of California, San Francisco and Ernest Gallo Clinic in Emeryville, California; Amy W. Lasek of Ernest Gallo Clinic and Research Center in Emeryville, California.

This research was supported by the Swiss Society for Biomedical Stipends (A.R.) and NIH grants AA10035, AA13105, and DA14809 (U.H.).

Offenhäuser et al.

The researchers include Nina Offenhäuser, Daniela Castelletti, Blanche Ekalle Soppo, Arianna Tocchetti, and Benedetta Pozzi of Fondazione Istituto FIRC di Oncologia Molecolare in Milan, Italy; Lisa Mapelli, Paola Rossi, and Egidio D'Angelo of Università di Pavia in Pavia, Italy; Maria Cristina Regondi and Carolina Frassoni of Istituto Nazionale Neurologico C. Besta in Milan, Italy; Alida Amadeo of Università degli Studi di Milano in Milan, Italy; Andrea Disanza and Giorgio Scita of Fondazione Istituto FIRC di Oncologia Molecolare and Istituto Europeo di Oncologia in Milan, Italy; Douglas Guarnieri and Ulrike Heberlein of University of California, San Francisco in San Francisco, California; Christer Betsholtz Karolinska Institutet in Stockholm, Sweden; Pier Paolo Di Fiore of Fondazione Istituto FIRC di Oncologia Molecolare, Istituto Europeo di Oncologia, and Università degli Studi di Milano in Milan, Italy.

This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, Ministero della Salute, MIUR, and Fondazione Monzino to P.P.D.F.; NIH grants AA13105 and AA10035 to U.H.; and grants from the European Community (VI Framework) to P.P.D.F. and E.D.

Rothenfluh et al.: "Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B Isoforms." Publishing in Cell 127, 199–211, October 6, 2006 DOI 10.1016/j.cell.2006.09.010.

Offenhäuser et al.: "Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics." Publishing in Cell 127, 213–226, October 6, 2006 DOI 10.1016/j.cell.2006.09.011.


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