Keystone: More Than Mere Nucleotides—miRNAs as Master Regulators, Part 2

Series - Keystone Symposium: Neurodegenerative Diseases: New Molecular Mechanisms:

MicroRNAs were a common emerging theme at the recent Keystone Symposium, “Neurodegenerative Diseases: New Molecular Mechanisms,” held 17-22 February at Keystone, Colorado. These small non-coding RNAs generally dial down gene expression by silencing messenger RNAs, and there is some hope they may eventually offer new ways of treating a wide variety of disorders, including neurodegenerative diseases such as Alzheimer’s. For example, at Keystone, Bart De Strooper, KU Leuven, Belgium, reviewed evidence from his lab linking miRNAs to regulation of BACE activity, and Valina Dawson, Johns Hopkins University, Baltimore, Maryland, showed a role for miRNAs in regulating pathways that protect cells from stress (see Part 1 of this news story).

One neurodegenerative disease that might benefit from activating pro-survival pathways is Parkinson’s. It has already been linked to non-coding RNAs. These regulate dopamine receptor levels (see ARF related news story) and developmental pathways that lead to a PD-like phenotype when perturbed (see ARF related news story). MicroRNA miR133b is reduced in PD (Kim et al., 2007), while an FGD20 gene mutation found in some PD patients is in a binding site for miR433 (see Wang et al., 2008). In his short talk, Wim Mandemakers, KU Leuven, Belgium, offered a link between microRNAs and α-synuclein, the major component of Lewy body pathology. Since elevated expression of α-synuclein by itself is sufficient to cause Parkinson’s, and microRNAs generally suppress expression, the notion that microRNAs might be involved in α-synuclein regulation is worth exploring, suggested Mandemakers.

Mandemakers used a bioinformatics screen to search for microRNAs that might bind the α-synuclein (SNCA) sequence. This identified miR7 and miR153 as possible matches. Using a luciferase gene construct containing the SNCA 3’ untranslated region, Mandemakers showed that both microRNAs suppressed luciferase expression in HeLa cells and miR153 also suppressed expression of the reporter in SH-SY5Y neuroblastoma cells, suggesting the microRNA/SNCA interaction may be functionally significant. Then he showed that overexpressing miR153 in either SH-SY5Y or PC12 cells suppressed α-synuclein.

Whether miR153 has any influence on Parkinson pathology is as yet unclear. Mandemakers did show that the microRNA is expressed in the substantia nigra, a small region of the brain where Parkinson pathology is rampant, but the microRNA is more robustly expressed in the total brain. Analysis of miR153 knockdown mice is underway to determine how the microRNA affects α-synuclein expression in vivo, he said.

Fiona Doetsch, Columbia University, New York, reported how different microRNAs, in different places in the brain, are crucial for adult neurogenesis, a phenomenon that holds promise for treating neurodegenerative conditions in the future. Doetsch showed that at least three microRNAs are involved—181a, 181b, and 124—each apparently playing a different role in the neurogenesis process. This process starts in the subventricular zone (SVZ) with astrocytic stem cells, which generate transit amplifying cells that wind their way via the rostral migratory stream (RMS), ending up as neuroblasts in the olfactory bulb. The three miRNAs may have specific roles in each of the three cells, suggested Doetsch: In the subventricular zone the astrocytic neural precursors predominantly express miR181b; the rapidly dividing transit-amplifying cells express miR181a; and the neuroblasts express miR124. It was the role of this miRNA that Doetsch focused on. She showed that it was not simply the site of miR124 expression that was crucial, but also the timing. Some of this work recently appeared in the March 15 Nature Neuroscience online (see Cheng et al., 2009).

Doetsch reported that injecting an miR124-expressing retrovirus into the SVZ led to an induction of neurogenesis in mice, while adding antisense miR124 to purified stem cells in culture caused a significant increase in the number of dividing cells but a decrease in the number of neurons—cell survival was unchanged. The results suggested that miR124 promotes differentiation into neurons. To test if miR124 regulates the process or its timing, Doetsch and colleagues again turned to in-vivo experiments. They completely ablated neurogenesis by using miniature osmotic pumps to flood the brain for six days with the anti-mitotic agent Ara-C, a cytosine analog that blocks DNA replication. After this treatment, astrocytic stem cells in the SVZ that were not dividing during the treatment survive and begin to repopulate the RMS and the olfactory bulb with neuroblasts, which begin to appear after four and a half to five days. However, if antisense miR124 is pumped into the brain immediately after Ara-C treatment, then the number of neuroblasts is almost zero five days later, but the number of transit amplifying cells is roughly doubled. This is in keeping with miR124 acting as a pro-differentiation switch. Interestingly, another two days later, there is a huge increase in the number of neuroblasts formed. These results indicate that miR124 controls not the differentiation into neurons, but the timing of that differentiation, said Doetsch.

How does miR124 exert its control? To address this next question, Doetsch studied targets of the microRNA, including the genes Dix-2, Jag-1, Sox-9, and Pea-15. All of these were knocked down in the presence of the microRNA. Sox-9 is of particular interest to Doetsch because it is expressed in astrocytes, and its mRNA, but not the protein, is found in neuroblasts. Using a Sox-9 gene construct lacking the region that binds miR124, Doetsch showed that overexpression of the gene abolished neurogenesis, and adding miR124 did not rescue this effect. The results suggest that the major effect of miR124 on neurogenesis is probably through suppression of Sox-9, Doetsch said.

Whether these findings will lead to future therapies that boost neurogenesis in the adult brain remains to be seen. Other miRNAs, by contrast, have already been studied in this regard. Beverly Davidson, University of Iowa, Iowa City, reported that artificial miRNA strategies may be one way to go in the treatment of Huntington disease (HD). Davidson noted some of her previous work showing that the short hairpin RNA sh2.4, which targets the second exon of the huntingtin mRNA, reduces expression of the protein when placed in an miRNA expression system. This artificial miRNA protected striatal neurons against huntingtin toxicity in a mouse model of Huntington disease (see McBride et al., 2008).

One advantage of miRNAs is that they can target different alleles of the same gene, be they mutants or transgenes. Davidson showed more recent evidence that the sh2.4 artificial miRNA can counteract both human and mouse huntingtin in a transgenic mouse model of the disease established by David Borchelt (see Schilling et al., 1999). Injecting the RNA at seven weeks of age silenced both mRNAs by 20 weeks, and the mice showed a benefit on the rotarod. While control mice progressively deteriorated between 14 and 18 weeks, the treated mice maintained their performance during this time. They also lived longer, said Davidson. Some of this work recently appeared in the February 24 Molecular Therapy online (see Boudreau et al., 2009).

It is too early to say whether the study of miRNAs will identify additional novel pathways or drug targets that can actually be exploited as treatments for AD or other neurodegenerative diseases. In the meantime, the talks at the Keystone symposium illustrated just how manifold the roles of these small actors are in the brain. Given that this field is still young, that microRNAs are strictly regulatory, and that they have pleiotropic effects, it may well turn out that these micro players have a macro impact on the field.—Tom Fagan

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References

News Citations

  1. Keystone: More Than Mere Nucleotides—miRNAs as Master Regulators, Part 1
  2. Local Control: Dendritic BC1 RNA Regulates Dopamine Receptor Levels
  3. Research Brief: Do MicroRNAs Cause Parkinson Disease?

Paper Citations

  1. Kim J, Inoue K, Ishii J, Vanti WB, Voronov SV, Murchison E, Hannon G, Abeliovich A. A MicroRNA feedback circuit in midbrain dopamine neurons. Science. 2007 Aug 31;317(5842):1220-4. PubMed.
  2. Wang G, van der Walt JM, Mayhew G, Li YJ, Züchner S, Scott WK, Martin ER, Vance JM. Variation in the miRNA-433 binding site of FGF20 confers risk for Parkinson disease by overexpression of alpha-synuclein. Am J Hum Genet. 2008 Feb;82(2):283-9. PubMed.
  3. Cheng LC, Pastrana E, Tavazoie M, Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci. 2009 Apr;12(4):399-408. PubMed.
  5. Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG, Price DL, Ross CA, Borchelt DR. Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet. 1999 Mar;8(3):397-407. PubMed.
  6. Boudreau RL, McBride JL, Martins I, Shen S, Xing Y, Carter BJ, Davidson BL. Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington's disease mice. Mol Ther. 2009 Jun;17(6):1053-63. PubMed.

Further Reading

Primary Papers

  1. Cheng LC, Pastrana E, Tavazoie M, Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci. 2009 Apr;12(4):399-408. PubMed.
  2. Boudreau RL, McBride JL, Martins I, Shen S, Xing Y, Carter BJ, Davidson BL. Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington's disease mice. Mol Ther. 2009 Jun;17(6):1053-63. PubMed.

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