23 May 2012. In the May 20 Nature Neuroscience online, researchers report that the let-7 family of miRNAs seeks out Toll-like receptor 7, quickly obliterating neurons. The paper provides the first evidence that miRNAs can act as extracellular death signals, said senior author Seija Lenhardt of Charité-Universitaetsmedizin Berlin, in Germany. The researchers also detected excess let-7b miRNA in the spinal fluid of people with Alzheimer’s disease. Lenhardt is now investigating a possible role for the miRNA in AD and other diseases using more human samples as well as mouse models.
To experts in the field, the paper comes a bit out of left field, because receptor ligand is a new role for miRNAs, as is neural function for Toll-like receptors (TLRs). These receptors normally function in innate immunity—for example, TLR7 is activated by an HIV RNA sequence—though researchers recently did see them modulating central nervous system (CNS) tasks such as learning and memory (see ARF related news story on Okun et al., 2010), and axon growth (Cameron et al., 2007). Could TLR7, known to exist on peripheral neurons (Liu et al., 2010; Kim et al., 2011), function in the brain as well?
First author Sabrina Lehmann and colleagues tested whether TLR7 was expressed in the CNS. In the brains of mice, they observed TLR7 mRNA by in-situ hybridization, and protein by immunohistochemistry, in cortical and hippocampal neurons. The mRNA localized to endosomes in cultured neurons, astrocytes, and microglia. Curiously, this contrasts with Western blot and immunostaining experiments by former colleagues of Lenhardt’s, which indicated that cortical neurons contain no TLR7 (Ma et al., 2006; Ma et al., 2007). Lenhardt was unsure of the reason for the discrepancy.
The study team wondered what TLR7 might be doing in the cortex and hippocampus. Since the short, single-stranded HIV ssRNA40 binds TLR7 via a GUUGUGU motif, they looked for endogenous RNAs that might also serve as a ligand for this receptor. Let-7b is common in neurons and possesses the same GU motif. When applied to cortical neurons in culture, let-7b killed them. This toxicity was similar to that of a known TLR7 ligand called loxoribine, a guanosine derivative that potently activates the innate immune system. Altering let-7b’s GU sequence rendered the miRNA non-toxic, whereas other miRNAs with GU-rich sequences, including let-7 family members, destroyed neurons.
Let-7b miRNA was neurotoxic in vivo. The researchers injected the miRNA into the spinal canal in mice. Within three days, axons started to degenerate in the cortex and striatum, and neurons became apoptotic. Mice lacking TLR7 were resistant to the miRNA’s effects, and let-7b without the GU motif was innocuous. Pre-injecting a let-7b inhibitor also protected neurons. The combined data suggesting the toxicity signaling role for let-7b are “convincing,” commented Mark Mattson of the National Institute on Aging in Bethesda, Maryland, who was not involved in the research. “The idea that a microRNA released from cells can trigger death in other cells is very novel,” he said.
Lenhardt’s team does not know how let-7b reaches and activates TLR7. “We think that in a normal healthy neuron, let-7 is in the cytosol and TLR7 in endosomes, so there is no contact,” Lenhardt said. Injured neurons, she hypothesizes, somehow release let-7b into the extracellular space. If it is then endocytosed, it will meet TLR7 in the endosome. It is not yet confirmed if the miRNA directly binds TLR7 or activates it as part of a protein complex.
The scientists were surprised to see let-7b had lasting effects because RNA is notoriously instable. That seems to be not always true, however. Researchers recently discovered that miRNAs persist in spinal fluid and blood (Cortez and Calin, 2009; Ghidoni et al., 2011). “Nobody knows why microRNAs seem to be stable in extracellular fluids,” Lenhardt said. Others have described miRNAs traveling from cell to cell in small membrane-bound exosomes (Mittelbrunn et al., 2011), although naked mRNA appeared to work in Lehmann’s experiments.
How do the neurons die? While both glia and neurons expressed TLR7, let-7b only killed the latter. The downstream cell-death cascade that commenced with TLR7 activation was unusual. NF-B, traditionally part of the TLR7 pathway, was not activated. Caspase-3 was, but the signals that turned on this enzyme remain to be elucidated.
The scientists wondered if let-7b might participate in human neurodegeneration and neuroinflammation. They are examining several conditions, and included the first dataset, on Alzheimer’s, in the paper. Thirteen people with AD had higher let-7b in their CSF when compared to 11 cognitively healthy controls. However, there was plenty of overlap in the data, and the difference could not serve as a biomarker, Mattson said. Looking at the statistical analysis, one can see that the data offer “only a weak connection to AD neuropathology and amyloid-,” agreed Milan Fiala of the University of California, Los Angeles, who was not involved in the research, in an e-mail to ARF (see full comment below).
“Whether TLR7 really plays an important role in AD pathogenesis still needs further animal experiments,” wrote Yang Liu and Klaus Fassbender, of Saarland University in Saarbrücken, Germany, in an e-mail to ARF (see full comment below). “Whether the concentration of extracellular microRNAs is sufficient to activate TLR7 should also be further investigated,” they added. Neither was involved in the research. For her part, Lenhardt is analyzing animal models of Alzheimer’s and Parkinson’s, as well as looking for let-7b in samples from people with Parkinson’s, Huntington’s, and multiple sclerosis, she told ARF.
“Particularly interesting and intriguing is the hypothesis that miRNAs may function not only as key regulators of gene expression at the post-transcriptional level, but also can act as signals for membrane receptor activation,” wrote Eleonora Aronica of the Academisch Medisch Centrum in Amsterdam, The Netherlands, who was not part of the research team, in an e-mail to ARF. This role of miRNAs might also extend to ALS, she suggested.—Amber Dance.
Lehmann SM, Krüger C, Park B, Derkow K, Rosenberger K, Baumgart J, Trimbuch T, Eom G, Hinz M, Kaul D, Habbel P, Kälin R, Franzoni E, Rybak A, Nguyen D, Veh R, Ninnemann O, Peters O, Nitsch R, Heppner FL, Golenbock D, Schott E, Ploegh HL, Wulczyn FG, Lehnardt S. An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat Neurosci. 2012 May 20. Abstract