MicroRNAs can degrade or repress translation of their targets, and also upregulate mRNA expression in some cases. The short sequences, 21 to 23 nucleotides, have been linked to neurodegenerative diseases such as Alzheimer’s (see ARF related news story and ARF news story); for review, see Bushati and Cohen, 2008), and Rademakers predicts that miRNA dysfunction will be implicated in many more conditions.

Rademakers’ research is the first evidence for miRNA involvement in frontotemporal dementia. She and colleagues found that the human miRNA miR-659 binds to the 3’-untranslated region of the progranulin mRNA, blocking translation. People with a progranulin mutation that enhances this binding can have significantly lowered progranulin levels, leading to frontotemporal dementia, Rademakers said, making the mutation a major risk factor for disease.

Scientists had previously linked frontotemporal lobe degeneration with ubiquitin- and TDP-43-positive inclusions (FTLD-U) to mutations in the progranulin gene (PGRN) (Baker et al., 2006, Cruts et al., 2006, and see ARF related news story). (Another form of frontotemporal dementia exhibits tau-positive inclusions.) All of the more than 60 PGRN mutations previously associated with FTLD-U are dominant, loss-of-function mutations that cause premature termination of the PGRN mRNA (Gass et al., 2006). These mutations cut progranulin protein levels by approximately 50 percent in heterozygotes, which is sufficient to cause disease (Van Damme et al., 2008).

Progranulin’s role in the nervous system is unclear. It acts as an anti-inflammatory in its full-length form but, when proteolytically cleaved to form granulins, is pro-inflammatory. Progranulin is present in neurons and microglia. In the periphery, progranulin is involved in wound healing (for review, see He and Bateman, 2003).

Rademakers and colleagues discovered the link between miRNA-659 and progranulin when they sequenced PGRN genes from 378 FTLD patients. Among 339 patients with no PGRN coding mutation, they found a polymorphism, rs5848, in the 3’UTR that did not exhibit Hardy-Weinberg equilibrium: more of the patients (55) were TT homozygous at this position than would be predicted by chance. TT homozygotes made up 16.2 percent of the FTLD patients, compared to 9.3 percent of control subjects. Among 59 FTLD-U patients without other FTLD-linked mutations, the rs5848 TT genotype frequency was 25.4 percent. All TT subjects showed the same neuropathology, with neuronal cytoplasmic inclusions and short, thin neurites in the cortex. The similar pathology suggests a common cause of disease among these patients. While the coding-sequence PGRN mutations are dominant, heterozygotes for rs5848 TT did not have increased susceptibility to FTLD-U, suggesting the mutation is recessive.

The location of the mutation, outside the coding sequence, suggests that it affects PGRN expression rather than function. On Western blots, extracts from rs4858 TT patients had progranulin levels reduced by approximately 30 percent compared to CC homozygous control extracts. However, quantitative RT-PCR found no difference in the PGRN mRNA levels between CC and TT individuals. “That was the first time we thought maybe microRNAs are involved,” Rademakers said. Using computer analysis, Rademakers predicted that a single miRNA, miR-659, could bind to the site of the mutation. In silico, changing the cytosine to a thymine altered the PGRN mRNA-miRNA binding, shifting miR-659’s position and allowing it to bind the transcript more tightly, with three additional nucleotide pairings. After confirming that miR-659 is expressed in human brain tissue, including the frontal and temporal neocortex, Rademakers hypothesized that miR-659 binds to PGRN mRNA, blocking translation, and that the mutant mRNA binds miR-659 more tightly. This knockdown of PGRN, in turn, could cause FTLD.

To test this hypothesis, the scientists turned to cell experiments. When they transfected human M17 neuroblastoma cells with miR-659, they expressed less progranulin, while cells transfected with a nonspecific control miRNA had normal PGRN levels. To further quantify miR-659’s activity, Rademakers and colleagues engineered a luciferase reporter with the PGRN 3’-UTR. In mouse N2A neuroblastoma cells (used to avoid any effects from endogenous human miR-659), miR-659 decreased luciferase expression in a dose-dependent manner, further suggesting miR-659 acts on the GRN 3’-UTR to knock down expression.

Rademakers posits that the lower one’s progranulin levels, the higher the risk of developing FTLD-U. A single T allele appears not to lower PGRN translation below the acceptable threshold, but two mutations can repress enough GRN translation to cause disease.

“The data do look convincing,” said Alison Goate, a geneticist at Washington University in St. Louis. “They provide pretty good evidence that there is an increased risk of FTLD-U associated with people who are homozygous for this variant.” However, she noted that the patient numbers were small—only 59 subjects in the second genetic analysis, and only 14 for the GRN expression studies. “In some ways, they were pretty lucky they found it,” Goate said.

MicroRNAs appear poised to be major players in neurodegenerative disorders, and Rademakers said her research is more evidence for that hypothesis. However, she recognizes that other scientists may take some convincing. “People are very skeptical of these novel mechanisms that become popular,” she said. Goate also noted that TDP-43 is implicated in several neurodegenerative diseases, and that perhaps GRN has a role in promoting TDP-43-based diseases other than FTLD-U. In the future, Rademakers intends to analyze sequence variation and expression levels of miRNAs to further characterize their role in FTLD.—Amber Dance.

Reference:
Rademakers R, Eriksen JL, Baker M, Robinson T, Ahmed Z, Lincoln SJ, Finch N, Rutherford NJ, Crook RJ, Josephs KA, Boeve BF, Knopman DS, Petersent RC, Parisi JE, Caselli RJ, Wszolek ZK, Uitti RJ, Feldman H, Hutton ML, Mackenzie IR, Graff-Radford NR, Dickson DW. Common variation in the miR-659 binding-site of GRN is a major risk factor for TDP43-positive frontotemporal dementia. Hum. Mol. Genet. 2008 December 17(23):3631-3642. Abstract