Current animal models with restricted genetic modifications, such as knockout or transgenic mice, play an important role in understanding the mechanics of disease, but are limited by developmental effects, genetic compensation, and lack of regional specificity. But according to a paper in this week’s Nature Medicine, RNA interference may offer a better option, given the success a group of researchers from the University of Texas Southwestern Medical Center at Dallas had with using the technology to specifically knock down a gene related to Parkinson’s disease in the brains of adult mice.
In the context of neuroanatomy, “the development of efficient techniques and strategies for the generation of conditional mutants is essential for gaining a better understanding of gene function,” Ralph DiLeone and colleagues at UT Southwestern wrote in the journal report. “Although modern microarray and proteomic strategies are resulting in the identification of many regulated genes and proteins, efficient in vivo tests of function will be needed to convert this data into a mechanistic understanding of systems biology.”
The problem with transgenic mice, they wrote, is that not only are they difficult to produce with precision, but when it comes to knocking out brain function, the transgene insertion sites themselves often influence gene expression patterns, interfering with the “generation of transgenic lines expressing recombinase proteins in specified brain regions.”
Additionally, while the transgenic approach is powerful, DeLeone told RNAi News that it requires “a heavy resource commitment — it’s very time consuming [and] expensive. You can only do a few genes at a time, because of how extensive an effort it is,” he said. “For mutant mice … if you’re starting from scratch, you’ve got to create a knockout floxed line and all that — the whole process can take years. … With the RNAi, we can basically design the vector and have it into animals within weeks and start getting data.”
DiLeone et al. set their sights on generating a specific knockdown of Th, the gene encoding the dopamine synthesis enzyme tyrosine hydroxylase, in the midbrain neurons of adult mice. Dopamine is a neurotransmitter involved in a wide range of behaviors including food intake, addiction, and control of movement, they noted, adding that the degeneration of dopaminergic neurons in the midbrain substantia nigra compacta is the primary cause of Parkinson’s disease.
Further, they pointed out, mouse knockouts of Th are lethal and genetic removal of tyrosine hydroxylase from the dopamine system results in hypogenic mice that die in three weeks.
The researchers generated two adeno-associated viral vectors(AAV-shTH1 and AAV-shTH2) that expressed either one of two U6-promoter-driven short hairpin RNAs targeting distinct sequences of Th, as well as a control virus with a scrambled sequence. The vectors were also designed to express enhanced green fluorescent protein (EGFP) so that infected neurons could be detected.
Using sterotaxic injections, AAV- shTH1 was delivered to one side of the substantia nigra compacta of nine-week-old C57BL/6J mice. The control vector was delivered to the other side. After 12 days, a substantial reduction in tyrosine hydroxylase staining in AAV-shTH1-infected cells was observed, while cells infected with the control vector showed normal expression of the enzyme. Similar results were observed in experiments with AAV-shTH2.
Additional experiments, which all used the AAV-shTH1 virus, showed persistent and increasing knockdown of tyrosine hydroxylase through day 50, as well as a substantial decrease in tyrosine hydroxylase mRNA in cells from the ventral tegmental area (VTA) infected with the shRNA versus the control sequence.
“Because tyrosine hydroxylase regulates dopamine production in synaptic terminals, we [also] assessed the consequences of midbrain [AAV-shTH1] infection on tyrosine hydroxylase levels in the forebrain,” DiLeone and his colleagues wrote. Stereotaxic injections were used to deliver AAVshTH1 to one side of the VTA, and the control vector to the other, in nine-week-old mice. Two weeks later, an analysis of the nucleus accumbens — a forebrain target of VTA dopamine neurons — showed a significant reduction in tyrosine hydroxylase levels in the AAV-shTH1-infected side versus the control-infected side.
“Regions with high concentrations of EGFP-labeled axons showed a reduction in tyrosine hydroxylase, whereas regions innervated with [axons infected by the control vector] did not,” they wrote. “This demonstrates the utility of the coexpressed EGFP marker for analysis of gene knockdown effects at relevant neuronal projection sites.”
RNAi-induced reduction of tyrosine hydroxylase also led to behavioral defects, DiLeone and colleagues wrote.
To evaluate the effects of Th knockdown in the VTA on the locomotor response to amphetamine, a well-established dopamine-dependent behavior, mice were given bilateral injections of either AAV-shTH1 or the control vector, and then tested after 16 days. In response to amphetamine injections, the knockdown mice showed a significant attenuation in locomotor response, they stated.
Using rotarods, the researchers also measured motor activity in the mice that had received bilateral injections of the two vectors in the substantia nigra compacta. After 17 days, the mice were evaluated twice a day for three days. As they continued training, the mice with a bilateral reduction in tyrosine hydroxylase showed a significant deficit in rotarod performance, a phenotype similar to neurotoxin-induced rodent models of Parkinson’s disease, they wrote. This suggests “that attenuation of dopamine synthesis is sufficient to mimic motor deficiencies caused by neurodegeneration of the substantia nigra compacta.”
“Notably, the average weights of the Th knockdown mice were not significantly different from those of control mice after targeting both the substantia nigra compacta … and the VTA,” the researchers added, “suggesting that overall health was not affected by gene knockdown.”
According to DiLeone et al., the experiments demonstrate the efficacy of RNAi for generating conditional mutations and gene knockdowns in multiple brain regions.
“Even if you had all the resources you could dream of having, nowadays, if you want to knock down gene X from four different regions of the brain, those resources don’t exist,” DiLeone said. “They don’t have Cre-driver lines for all different parts of the brain.” But with RNAi, he said, “we’re delivering the virus directly through an injection and by doing that, we can … direct the gene knockdown to very specific regions of the brain — to different sets of midbrain dopamine neurons, even.”
DiLeone said that the work detailed in the Nature Medicine paper was a proof-of-principle experiment, and that he and his colleagues are now planning to use the RNAi approach to study the role of brain reward pathways in feeding.
— DM