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.

Reference:
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

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Comments on News and Primary Papers

  1. This article by Lehmann et al. presents interesting results in mice regarding induction of neuronal death by let-7 miRNA through TLR-7 on neurons. They confirmed the results in Tlr7-/- mice, including the Tlr7-/- mice from fetuses transfected by TLR7. This mode of neuronal death could be implicated in patients with Alzheimer's disease (AD) who suffered brain trauma, which is one of the risk factors of AD. The relevance to patients with sporadic AD is difficult to evaluate at present. Their study included 13 patients with MMSE 18-28 and 11 control subjects, but the incidence of brain trauma and other associated conditions, such as stroke and diabetes, was not listed. The significance of let-7 differences in the CSF was stated as 0.0139, but it does not seem that the data were evaluated by the Levene Test for Equality of Variances. Thus, the results remain an interesting link of let -7 to neurodegeneration in an animal model, but with only weak connection to AD neuropathology and amyloid-β. It would be interesting to evaluate let-7 in the CSF of patients with brain trauma, which could provide much better comparison (acute brain trauma vs. no trauma vs. meningitis, etc.).

    View all comments by Milan Fiala
  2. There is growing evidence showing that Toll-like receptors (TLRs) play a key role in the pathogenesis of Alzheimer¹s disease (AD): 1) TLRs are upregulated in AD patients and AD animal models; 2) CD14, the co-receptor of TLR4, and TLR2, has been observed to directly interact with aggregated amyloid-β peptide (Aβ) and trigger microglial neurotoxic inflammatory activation; 3) experimental manipulations to activate TLR4 or TLR9, and to inhibit CD14, TLR2, TLR4, or MyD88, a signaling molecule downstream of TLRs, have been shown to change AD-like pathology, for example, neuroinflammation, Aβ load, and neuronal degeneration in amyloid precursor protein (APP) transgenic mice. In these studies, scientists mainly focus on: 1) effects of microglial TLRs, (although the effects of neuronal TLRs cannot be excluded in experiments cross-breeding TLR2- or TLR4-knockout mice and APP transgenic mice); 2) Aβ as the ligand of TLRs, although other ligands, for example, high-mobility group box-1, could also activate TLRs.

    This publication in Nature Neuroscience by Lehnardt and her colleagues indicates that TLR7 could be a new innate immune receptor promoting AD pathogenesis. The endogenous ligand for this receptor is a sequence motif (GUUGUGU) contained in microRNAs. Here, microRNAs induce neurodegeneration instead of their conventional role of regulating gene transcription.

    However, the most interesting finding in this publication is that activation of neuronal TLR7 directly causes neuronal death, whereas activation of microglial TLR7 triggers an inflammatory response with secondary neurotoxic effects. These findings extend the concept of innate immunity, as neurons also exert innate immune functions, although the immune signaling cascades in neurons could be different in microglia or macrophages. Whether TLR7 really plays an important role in AD pathogenesis still needs further animal experiments, for example, conditionally ablating neuronal TLR7 in AD mouse models. Whether the concentration of extracellular microRNAs is sufficient to activate TLR7 should also be further investigated.

    View all comments by Klaus Fassbender
  3. The study by Lehmann and colleagues provides a fascinating and unprecedented look at microRNA function in the brain. Typically, microRNAs function to regulate gene expression at the post-transcriptional level by targeting messenger RNAs, with known roles in neuronal maintenance, function, and survival. Here, however, the authors convincingly demonstrate that extracellular let-7 family members and, in particular, let-7b, function as ligands for the TLR7 receptor, known to play a fundamental role in pathogen recognition and activation of innate immunity. The TLR7 signaling cascade is activated through the conserved GUUGUGU motif located in let-7. Incubation of neurons with let-7b induced a dose- and time-dependent cell loss both in vitro and in vivo, an effect blocked in TLR7 knockout models. The authors identified caspase-3 as a mediator of neurodegeneration. Interestingly, neuronal loss could be induced using medium isolated from let-7 overexpressing HEK293 cells. Moreover, neuronal degeneration stimulated endogenous let-7 secretion, possibly contributing to a deleterious feedback loop. Finally, let-7b was significantly increased in CSF samples taken from Alzheimer’s disease (AD) patients. Obviously, this last observation raises several questions with regard to the potential role of let-7 family members, in addition to caspase activation and inflammation, in AD development. Previous studies have shown that let-7i was downregulated in AD brain and in biological models, likely a downstream effect of amyloid overproduction (1,2). On the other hand, and consistent with the current study, let-7f was previously shown to be upregulated in AD CSF (3). The possibility that neuronal microRNAs are secreted into the CSF under either physiological and/or pathological conditions remains to be fully explored. Clearly, this study adds to the growing list of publications suggesting that soluble microRNAs could provide important biomarkers for neurological diseases (3-5). The fact that microRNAs could function outside the cell adds to the complexity of this already multifaceted field.
     

    View all comments by Sebastien S. Hebert

References

News Citations

  1. A Toll on Memory: New Role for Immune-Related Receptor in Brain

Paper Citations

  1. . Toll-like receptor 3 inhibits memory retention and constrains adult hippocampal neurogenesis. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15625-30. PubMed.
  2. . Toll-like receptor 3 is a potent negative regulator of axonal growth in mammals. J Neurosci. 2007 Nov 21;27(47):13033-41. PubMed.
  3. . Toll-like receptor 7 mediates pruritus. Nat Neurosci. 2010 Dec;13(12):1460-2. PubMed.
  4. . Analysis of cellular and behavioral responses to imiquimod reveals a unique itch pathway in transient receptor potential vanilloid 1 (TRPV1)-expressing neurons. Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3371-6. PubMed.
  5. . Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. J Cell Biol. 2006 Oct 23;175(2):209-15. PubMed.
  6. . TLR8: an innate immune receptor in brain, neurons and axons. Cell Cycle. 2007 Dec 1;6(23):2859-68. PubMed.
  7. . MicroRNA identification in plasma and serum: a new tool to diagnose and monitor diseases. Expert Opin Biol Ther. 2009 Jun;9(6):703-711. PubMed.
  8. . Cerebrospinal fluid biomarkers for Alzheimer's disease: the present and the future. Neurodegener Dis. 2011;8(6):413-20. PubMed.
  9. . Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat Commun. 2011;2:282. PubMed.
  10. . An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat Neurosci. 2012 May 20; PubMed.

Further Reading

Papers

  1. . Resveratrol mitigates lipopolysaccharide- and Aβ-mediated microglial inflammation by inhibiting the TLR4/NF-κB/STAT signaling cascade. J Neurochem. 2012 Feb;120(3):461-72. PubMed.
  2. . Toll-like receptor 4 promotes α-synuclein clearance and survival of nigral dopaminergic neurons. Am J Pathol. 2011 Aug;179(2):954-63. PubMed.
  3. . Toll-like receptor signaling in neural plasticity and disease. Trends Neurosci. 2011 May;34(5):269-81. PubMed.
  4. . Soybean isoflavone alleviates β-amyloid 1-42 induced inflammatory response to improve learning and memory ability by down regulation of Toll-like receptor 4 expression and nuclear factor-κB activity in rats. Int J Dev Neurosci. 2011 Aug;29(5):537-42. PubMed.
  5. . No association of toll-like receptor 2 polymorphisms with Alzheimer's disease in Han Chinese. Neurobiol Aging. 2011 Oct;32(10):1924.e1-3. PubMed.
  6. . An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat Neurosci. 2012 May 20; PubMed.
  7. . Common variants in toll-like receptor 4 confer susceptibility to Alzheimer's disease in a Han Chinese population. Curr Alzheimer Res. 2012 Jan 23; PubMed.

Primary Papers

  1. . An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat Neurosci. 2012 May 20; PubMed.