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NIAD Researcher Expects to Advance In Vitro RNAi Study of Parkinson s Gene Into Animals

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Following the successful development and application of siRNAs targeting three genes associated with the dopaminergic system of the ventral mesencephalon, a researcher from the National Institute on Drug Abuse is preparing to advance her work into live animals.

If successful, the effort, led by Cristina Backman, is expected to shed light on the genetics of Parkinson’s disease and other neurological disorders.

About a year ago, Backman and colleague Andreas Tomac published a paper in the Journal of Neuroscience Methods describing the use of short-interfering RNAs to suppress the expression of tyrosine hydroxylase, Nr4a2 (Nurr1), and cRet in cultured mammalian cells.

TH is an enzyme associated with the synthesis of catecholamine neurotransmitters including dopamine, a deficiency of which has been linked to the symptom’s of Parkinson’s disease. Nurr1 is a transcription factor expressed in the central nervous system and essential for the development of dopaminergic neurons. CRet is a high-affinity receptor for glial cell line-derived neurotrophic factor (GDNF), which plays a role in the survival of dopaminergic nerves.

In their experiments, Backman and Tomac, who has since left the NIDA to pursue a medial degree at the University of Rochester, used a variety of siRNA constructs that had been fused in tandem to a modified firefly luciferase reporter gene. Gene-expression knockdown was observed for all three targets, and now Backman is planning to begin using the most efficient siRNAs from this earlier work in mice.

While cRet and TH are both important research targets, “we’re [primarily] interested in Nurr1, because it is essential for the development of dopaminergic neurons, and dopaminergic neurons die [as a result of] Parkinson’s disease,” Backman told RNAi News this week.

But because Nurr1 is key to the neuron-development process, researchers have not been able to create Nurr1-knockout animals, hindering research into the role the gene plays, Backman noted.

“When you have a knockout of the Nurr1 gene, when you take away the Nurr1 gene, the animals die when they are born and the dopaminergic neurons are not there,” she said. “Is this gene related only to development, or does it have a function in adult animals, too? Because the animals die when they are born, nobody knows yet the function of [the gene] during adulthood.”

To sidestep this problem, Backman is considering using a single plasmid tetracycline-inducible expression system, developed with Tomac, to create transgenic animals. The design of this system was detailed in another Journal of Neuroscience Methods paper published in October. Alternatively, she is considering directly introducing the siRNAs into adult mice.

“We had to develop the tools first,” she said. “Now we’re putting them together. But we may just [knock down] Nurr1 directly — it takes so much time to make transgenics and we have the [siRNA] constructs ready now.”

As for cRet and TH, Backman said that they are on the list, but Nurr1 is at the top.

“People working in Parkinson’s [disease] are very interested in Nurr1,” she said. “It is not only expressed in dopamine neurons of the ventral mesencephalon … but it’s expressed in many other brain areas like the olfactory bulb and the hippocampus. However, when you knock out this gene … it appears to only affect the development of the dopamine neurons in the ventral mesencephalon.”

As a result, “many people are trying to answer [the questions]: What is really the function of Nurr1, especially in an adult system? Does it have protective effects? Is it essential for survival?” Backman said.

“Also, it has been shown that there are some mutations in the Nurr1 gene … in some familial cases of Parkinson’s disease,” she noted. “So it seems like there might be an involvement of this molecule in this disease. And even if it is not directly involved, it may be a protector — if you enhance its function it may help cells survive or degenerate more slowly.”

But before any of this can be determined, researchers need to understand the basics of Nurr1’s role in the adult animal. “It will be very interesting. … If we can [knock out Nurr1] in adulthood, we will be able to answer many of these questions,” Backman said. Later on, experiments geared towards Parkinson’s disease, such as ones involving stressing the mice with brain lesions, can be conducted, she added.

“As I see it today, [RNAi] for [creating] conditional animals and doing knockdowns later on is very powerful,” she added. “The question is whether we can really knock down the entire production of this protein by using silencing RNA. We’ll try and see what we get.”

In in vitro experiments, Backman said that she and her colleagues have at best achieved 43-percent knockdown. “You have to keep in mind that our Nurr1 production [was induced by the] CMV promoter, which is very strong — much stronger than the promoters that you have endogenously,” she said. “So we think that in a mouse system … there won’t be some much Nurr1 … and hopefully we’ll be able to silence everything.”

— DM