To study miRNAs, first author Nan Liu and colleagues mutated the loquacious gene, rendering flies unable to properly process the small oligonucleotides. These animals died earlier than wild-type, and had striking brain deterioration, leading the authors to surmise that perhaps one or more miRNAs were responsible. The team pored over the miRNAs expressed in the aging fly brain and noticed that while most either showed steady expression or decreased with age, miR-34 expression increased as flies got older.

Next, Liu and colleagues generated miR-34 knockouts. Compared to controls, these flies had dramatically accelerated brain deterioration marked by large brain vacuoles, and died early. At just 20 days of age, the flies had trouble climbing and were much more sensitive to stress than were age-matched controls. The researchers partially rescued these deficits by restoring limited miR-34 expression; bumping up miR-34 expression in wild-type flies also increased survival by about 10 percent. The 20-day-old knockout flies expressed a range of genes typically activated in older animals.

"This is the first case, outside of worms, where somebody has knocked out a microRNA and shown that the animal has a lifespan defect," said Frank Slack, Yale University, New Haven, Connecticut, who did not participate in the research. Slack's lab previously reported that miR-34 ramps up with age in C. elegans, and that knocking it out shortens lifespan (see de Lencastre et al., 2010), while upregulation of lin-4, another C. elegans miRNA, extends the worm's lifespan (see Boehm and Slack, 2005).

However, for Liu and colleagues the question remained, What harmful mRNAs does miR-34 target to slow aging and neurodegeneration? Using computer algorithms, the researchers predicted that the mRNA for the gene Eip74EF, a component of steroid hormone signaling pathways, would bind miR-34. The gene encodes two protein forms, but Northern blot tests showed that only one isoform of E74A was present in adults, so the scientists focused on that. Despite strong E74A mRNA expression in the adult fly heads, miR-34 kept protein translation quiet. In flies without miR-34, however, E74A protein ran amok and flies aged faster. Adding miR-34 back to the genome rescued the premature demise. Flies engineered to express additional Eip74EF at high temperatures also showed neurodegeneration and a shorter lifespan when raised at 29 degrees Celsius. In contrast, fruit flies with a partial loss-of-function E74A mutation aged more normally in the absence of miR-34. The authors concluded that Eip74EF, which is critical during fruit fly development, has negative effects on the adult fly, and that miR-34 mitigates those. However, Eip74EF is just one of a potential pool of targets for miR-34, note the authors.

"Further understanding of mir-34 and its targets may provide key mechanistic insight into how age-related events are linked to integrity of the brain," said Bonini. She said her team plans to look for more of miR-34's targets and seek other microRNAs that are differentially expressed with age. There are three isoforms (a to c) of 21, 22, and 24 nucleotides in length, and it is not clear if they have independent or redundant effect.

In addition to accelerated aging and neurodegeneration, fruit flies lacking miR-34 also showed an increase in protein misfolding, as evident by inclusions that immunostained for stress chaperones. The team used transgenes to boost the amount of miR-34 in flies that expressed ataxin-3 with a polyglutamine (polyQ) expansion. This mutation is responsible for one form of spinocerebellar ataxia (see ARF related news story and ARF news story). These flies had fewer inclusions, more soluble polyQ, and less neural degeneration, although the E74A gene seemed uninvolved in this process.

MiR-34 is conserved across species. Roundworms, fruit flies, mice, and humans all have it. In fact, it is one of only 14 microRNAs known to be so highly conserved, said Martin Bushell, University of Leicester, U.K. Does miR-34 influence human aging and neurodegeneration? It abounds in the hippocampi of mouse models of Alzheimer's disease (AD) and people with AD, and inhibiting one of its isoforms—miR-34c—rescues memory deficits in APPPS1-21 and aged wild-type mice (see ARF related news story on Zovoilis et al., 2011). MiR-34 appears to be pleiotropic; for example, it prevents cell division. For that reason, tissue-specific delivery will be key if it is ever to be used therapeutically, noted Bushell. "That's the major hurdle to overcome," he said. "You might get off-target effects in tissue where upregulation of miR-34 is bad." A number of labs and pharmaceutical companies are working on this problem now, he added.—Gwyneth Dickey Zakaib.

Reference:
Liu N, Landreh M, Cao K, Abe M, Hendriks GJ, Kennerdell JR, Zhu Y, Wang LS, Bonini NM. The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila. Nature. 2012 Feb 15. Abstract