- Email: [email protected]
Post-transcriptional gene silencing in neurons
Post-transcriptional gene silencing in neurons Henry C Zeringue and Martha Constantine-Paton The techniques evolving from the rapidly developing field of small RNAs promise accessible approaches to dissecting cellular and molecular mechanisms of higher brain function. Here, a current overview of the technology is presented, along with an outline of how these approaches might help neuroscientists to more rapidly uncover the cellular and molecular bases of behavior.
subjective view of the promise of these techniques specifically as tools for those investigators who seek to bridge the gap between the in vivo system level, function, development and pathogenesis of the nervous system and the explosion of information generated by the field of cellular and molecular neurobiology.
Background Addresses Department of Biology, McGovern Institute for Brain Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:654–659 This review comes from a themed issue on New technologies Edited by Winfried Denk and Liqun Luo Available online 11th September 2004 0959-4388/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.08.009
Abbreviations DCX dsRNA GFP miRNA PTGS RNAi shRNA siRNA
doublecortin double-stranded RNA green fluorescent protein microRNA post-transcriptional gene silencing RNA interference small hairpin RNA short-interfering RNA
Introduction The story of post-transcriptional gene silencing (PTGS) is rapidly unfolding in multiple model systems. PTGS is an evolutionarily conserved phenomenon that is frequently referred to as cosuppression in plants [1], RNA interference (RNAi) in animals [2,3] and quelling in fungi [4]. An in-depth examination of PTGS mechanisms has been presented by many other authors [5,6–8]. Figure 1 presents, in cartoon overview, the generally agreed upon processes of PTGS. An in-depth treatment of the biochemistry and molecular biology involved is beyond the scope of this article. Here, we try to clarify the phenomena for neurobiologists. We describe the techniques used, the controls necessary and the advantages over earlier procedures of capitalizing on these processes. These advantages explain the rapidly growing applications of PTGS to problems in neuroscience. We close with a brief Current Opinion in Neurobiology 2004, 14:654–659
PTGS encompasses two related mechanisms. One, RNAi, involves double-stranded short interfering RNAs (siRNAs), of approximately 21–26 basepairs, which degrade transcripts with the complementary sequence and can have very high specificity [8]. The second utilizes endogenously encoded, single-stranded micro-RNAs (miRNAs) of similar sizes, which bind homologous but not identical sequences of the 30 -untranslated region in target mRNAs and disrupt translation [7,9]. Many miRNAs have been found in genome screens [10–13] and it is clear that a single miRNA can modulate the expression of multiple gene products. However, the identity of these targets is largely unknown. The two PTGS mechanisms share pathways, associated proteins, regulating systems [6,7,14] and nomenclatures that can frequently but not always describe the same process. Here, we use the following terms: RNAi incorporates siRNAs for the highly specific downregulation of a target message achieved usually, although not exclusively [15], through experimental induction of double-stranded RNA (dsRNA)-mediated, sequence-specific messenger RNA (mRNA) degradation. miRNA silencing refers to the regulation achieved through the endogenous singlestranded miRNA-mediated, homology-dependent reduction in protein expression achieved by interfering with productive translation. However, because the molecular details of these processes are not fully understood, it is possible that an siRNA can decrease protein without affecting transcripts, by acting in the same manner as a miRNA [16,17], and it is similarly possible that miRNAs can specifically complement transcripts and thus act in the same manner as siRNA [18].
Experimental use of siRNA Introducing siRNA into cells
The techniques used to achieve specific siRNA knockdown in neurons depend crucially on the organism that houses the neurons and when, in the organisms life cycle, the interference is required. In Caenorhabditis elegans [2] and Drosophila [19], the siRNA precursor can be a long dsRNA precursor that is directly incorporated into cells and then broken down into shorter sequences. In mammalian cells, however [20], immunological reaction to long dsRNA severely compromises this approach [21–23]. www.sciencedirect.com
Post-transcriptional gene silencing in neurons Zeringue and Constantine-Paton 655
Figure 1
Long dsRNA
shRNA
Dicer
miRNA precursor
Dicer
miRNA
siRNA
Dicer
Dicer Activated RISC
Degrade mRNA
Activated RISC
Dicer
Dicer
Block mRNA
Current Opinion in Neurobiology
A schematic of the two major categories of PTGS. (a) siRNA: this method involves the dsRNA-dependent endonuclease Dicer cleaving dsRNA (either long dsRNA or shRNA into short 21–25 basepair siRNAs). The siRNA combines with and activates an RNA-induced silencing complex (RISC). Once joined with RISC, the siRNA denatures such that only a single strand is held in place by RISC. This single stranded RNA determines the sequence specificity of the RISC activity. The activated RISC binds with an mRNA complementary to the bound RNA and facilitates degradation of the transcript. (b) The same sequence occurs when a genetically encoded miRNA precursor is expressed. However, complementarity is not complete in these RNA sequences. In these cases, Dicer and the RISC complex continue to process the short RNAs but without precise complementarity, allowing for the function of more than one mRNA sequence to be disrupted. With miRNA, interference is not by degradation of the transcript but rather at some stage during translation of the transcript into a mature protein.
Thus, in mammalian cells, introduction into cells for transient expression is accomplished by transfection or electroporation of either the specific siRNA itself or small hairpin RNA (shRNA) in which the hair loop is rapidly cleaved to produce siRNA and the specific siRNA response. For stable, longer-term suppression, a DNA plasmid containing a construct coding for the shRNA [24,25] can be applied. Additional advantages of the DNA approach include the ability to apply viral vectormediated delivery of the construct to a spatially defined region and to use conditional promoters to regulate the expression of the construct [26]. Use of lenti-viral vectors is a relatively new and potentially more powerful technical development in the viral vector field. Using incapacitated lenti-viruses, both dividing and mature cells can be infected. The construct is incorporated in low copy number, generally in a single site on chromosomes, and cells appear to survive for long periods, if not indefinitely, following infection. Lenti-virus vectors are generally capable of carrying DNA coding for the shRNA, the U6 promoter region for transcription of the shRNA, and a second strong promoter to drive expression of a www.sciencedirect.com
reporter gene. In addition to direct injection into brain, these vectors can be used to generate shRNA expressing transgenic lines with extremely high efficiency [26]. siRNA advantages
The utility of the siRNA approach is manifold. First, there is rapid, relatively inexpensive and efficient knockdown, generally 90% of protein product, in transgenic animals when compared with animals with specific gene knockouts generated using homologous recombination [27,28]. Second, the function of proteins whose knockouts are lethal when expressed throughout the brain can still be studied by introducing siRNA [28,29] into only a subset of cells. The third advantage is the possibility that knockdown, as compared with complete knockout, of a protein might not induce rescue pathways seen with some knockouts [30]. Experimental controls
Despite the successful and robust knockdown achievable with RNAi, there are several possibilities for nonspecific effects. Therefore, good controls are necessary to Current Opinion in Neurobiology 2004, 14:654–659
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determine if the observed loss of function is actually restricted to effects on the gene product of interest or on off-target genes. Unfortunately, many reports involving this technology applied either no control or an overexpressed housekeeping gene control. The editors of Nature Cell Biology outlined a series of ‘appropriate’ controls for the siRNA researcher and generated a first attempt at codifying ‘proof’ of specificity [31]. The importance of such controls with siRNA is becoming increasingly apparent because even short RNAs are being shown to act nonspecifically and alter several proteins [32]. For example, Persengiev et al. [32] recently reported nonspecific siRNA effects in mammalian cells as a result of expression of a common ‘control’ siRNA sequence. The siRNA, directed against the luciferase reporter gene, was transfected into HeLa cells. HeLa cell transcript expression was subsequently determined with an Affymetrix gene chip. Of 33 000 genes tested, over 1100 increased and almost 700 decreased by 2.5-fold. These transcripts covered a broad range of cellular functions. Such findings demonstrate the importance of multiple controls when using RNAi because even an exogenous sequence does not necessarily offer ‘control’ expression. This generalized disruption resulting from exposure of cells to ‘naked’ siRNA might be caused by endosomal uptake, which has recently been shown in cultured primary neurons to produce broad metabolic impairment [33]. Nevertheless, one specific cautionary note from the results of Persengiev et al. is that the expression of glyceraldehydes 3-phosphate dehydrogenase (GAPDH), a ‘housekeeping’ gene frequently used to demonstrate the specificity of an siRNA ‘knockdown’ of an unrelated transcript or protein in mammalian cells, was not substantially affected. This demonstrates the very low sensitivity obtained by no change in GAPDH. Unfortunately, a common ‘RNAi-sensitive’ gene has not been identified. In fact, it is likely that no single transcript can be identified for the enormous range of siRNA sequences and cell types that can be employed with this technology. Nonspecific effects can also be obtained simply as a result of increased RNA load or disruption of unknown proteins downstream in a pathway. However, using a null-expressing vector or trying to detect differences in overexpressed products does nothing to control for nonspecific effects of siRNA activity. One suggested approach is to use several different siRNAs against the same target or to inspect products of several genes known to be associated with the actual target in both sequence homology and functional cellular pathways [34]. By looking at only a change in the product of interest using only one siRNA, researchers might miss the well-documented, and poorly understood, nonspecific effects of RNAi, particularly as it applies to vertebrate neurons.
RNAi in vitro Much of the work on RNAi in neurons has been performed in vitro. Using a variety of systems, investigators Current Opinion in Neurobiology 2004, 14:654–659
have examined neurite growth [35–38], voltage-dependent channels [39], apoptosis [27,28], cellular differentiation and growth [34,40] and a variety of signaling pathways [41–43]. One of the more common controls used for in vitro siRNA in mammalian cells is a scrambled sequence with no known homology in the genome being explored [28,37]. For example, Higuchi et al. [37] successfully used siRNA specifically to knockdown the p75 neurotrophin receptor in primary neuron culture. They studied neurite growth inhibition resulting from myelinassociated glycoprotein (MAG). The RNAi-induced suppression of p75 caused MAG-treated neurites to grow almost as long as controls, eliminating a significant decrease in neurite length when wild type neurons were treated with MAG. Suppression of p75 also decreased nerve growth factor-induced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelling (TUNEL) analysis. In one of the first studies to examine synaptic function after siRNA application, scrambled control siRNA and siRNA against CPl-17, a specific inhibitor of myosin/moesin phosphatase (MMP), have been introduced into primary cultured cerebellar neurons. Along with immunological analysis showing specific knockdown, the CPl-17 siRNA was demonstrated using electrophysiological analysis of synaptic currents to completely eliminate granule cell to Purkinje cell long-term depression, whereas long-term depression remained present in Purkinje cells transfected with scrambled CPl-17 siRNA [44].
RNAi in vivo Many more examples of elegant culture work could be cited here. However, the ultimate goal of many neurobiologists is to determine how mechanisms defined in vitro are orchestrated to function in vivo in the dynamic environment of the intact nervous system. Difficulties of in vivo neurobiology research ranging from the blood– brain barrier to redundancy at system [45] and molecular [46] levels necessitate ever-more-sophisticated assays. The majority of in vivo siRNA work reported thus far has been in C. elegans. Because first, RNAi was first discovered in this organism [2], second, there is no immunological response to dsRNA, and third, RNAi can be easily implemented in the animal by feeding or injection. We cite only a small portion of the rich RNAi literature performed in C. elegans. Genetic screening methods employing siRNA in C. elegans have recently yielded insight into a ubiquitin pathway-related protein that is localized at the neuromuscular junction [47], a K+ channel-associated ortholog of the mammalian MinK-related peptide [48], a pathway known to affect axon guidance [49], an aminopeptidase localized to neurons [50] and a protein thought to act within the RNAi pathway itself [51]. A useful tool in integrating this vast amount of information is a web-based functional genomics repository based on much of the work in C. elegans [52]. As more experimental www.sciencedirect.com
Post-transcriptional gene silencing in neurons Zeringue and Constantine-Paton 657
data becomes available, an enabling tool for continued research will be a central repository to house RNAi information about mammalian neuronal models. Drosophila, like C. elegans, does not exhibit an interferon response. However, to obtain a system-wide infection, a shRNA-coding DNA has to be introduced into the genome. A good example of the work being carried out in the fly is illustrated by Yang et al. [53]. These investigators explored the role of the ubiquitin ligase Parkin in neurotoxicity. They showed that pan-neuronal expression of Pael-R, a Parkin substrate, causes an age-associated degeneration of Drosophila dopamine neurons. Coexpression of Pael-R and human Parkin rescues these cells. Moreover, expressing shRNA against Drosophila Parkin (dParkin) in the dopamine neurons, along with the panneuronal Pael-R, promoted Pael-R accumulation and augmented neurotoxicity. Thus, using siRNA together with rescue by an exogenous protein, Yang et al. [53] built a strong case for the cellular interaction between Parkin and Pael-R during dopaminergic neuron cell death. A crucial goal of biomedical neuroscience research is to understand the human nervous system. However, the power of RNAi has only recently been applied to mammalian systems, and robust implementation has proven complicated. Only a few studies have reported in vivo siRNA in mammalian neurons. Several utilized electroporation of a shRNA-expressing DNA plasmid [30,54,55]. For example, Matsuda et al. [55] explored the phenotypic changes due to knockdown of the retinal transcription factors Crx and Nrl. Plasmids containing DNA constructs for shRNA against Crx or Nrl transcripts were coelectroporated into the retina of newborn mice along with green fluorescent protein (GFP)-expressing plasmids. After differentiation, GFP-positive cells in the outer nuclear layer of the retina had structural changes consistent with the knockout phenotypes, as demonstrated by immunohistochemistry of retinal sections. Bai et al. [30] used a similar system to explore the role of doublecortin (DCX) during development of the neocortex in rat. They performed cotransfection of plasmids producing GFP and an anti-DCX shRNA by in utero electroporation of embryonic mice. Control mice were transfected with GFP–plasmid alone. Using fluorescence and immunohistochemistry, they showed arrested migration of cerebral neocortical neurons when shRNA against DCX was expressed. Antibody staining against DCX was used to detect knockdown. DCX knockdown cells remained scattered among the layers of the cortex, whereas control cells were highly concentrated in cortical layers two and three. Overall, these reports suggest that siRNA applied in vivo might adequately substitute for time- and resource- intensive conditional knockout techniques currently applied to the mammalian brain. www.sciencedirect.com
Prospectus and conclusion It is hoped that this mini-review has explained why the discovery of PTGS has so galvanized large segments of biology that nearly every biology journal carries papers studying the phenomenon of using, or purporting to use, siRNA, for loss-of-function assays. In the field of neuroscience, this technology has opened the door to entirely new approaches to studying the intact brain. For example, viral vectors carrying DNA constructs for shRNA against specific proteins expected to be active during particular processes could be injected into the specific brain regions suspected to be involved before triggering the process. These processes could range from learning, to attention, to decision making, to fear or to a developmental event such as the activity-dependent refinement of synapses. The structural, physiological and behavioral differences between animals carrying a control shRNA, perhaps scrambled shRNA, and the targeted shRNA can then be compared using any of the numerous assay techniques available to neuroscientists today. Ideally, such constructs should be designed to express, in specific neuron types, a requirement that might be difficult but not beyond the toolbox available to molecular neurobiologists. Indeed, many efforts are underway to target siRNAs against disease genes to particular cells or tissues. Collaborations will be important to success in all of these endeavors. Additionally, many such ‘system’ level experiments will have to await the intermediate steps of siRNA expression in intact model systems, such as rodents, songbirds or ferrets, in which single-cell anatomy and single-cell physiology can be applied to examine individual transfected cells among populations of otherwise normal cells or circuits to determine whether the siRNA ‘knockdown’ induces the expected effect and whether any effect remains cell autonomous or alters the entire circuit or cell population in which the affected cell resides. In fact, for many neuroscientists working at the interface of molecular, cellular and systems neurobiology, the latter effort — to define what a small subset of neurons with a specific molecular defect, located among normal or control-infected neurons, can accomplish or fail to accomplish — is a significant goal in and of itself. The excitement over siRNA is that such studies are now within both our technical and financial capabilities.
Acknowledgements We would like to thank HR Horvitz and EA Miska (Massachusetts Institute of Technology) for their helpful suggestions on this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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25. Yu JY, DeRuiter SL, Turner DL: RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002, 99:6047-6052. 26. Rubinson DA, Dillon CP, Kwaitkowski AV, Sievers C, Yang L, Kopinja J, McManus MT, Gertler FB, Scott ML, Van Parijs L: A lentivirus-based system to silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003, 33:401-406. 27. Sarkar SN, Das HK: Regulatory role of presenilin-1 and nicastrin in neuronal differentiation during in vitro neurogenesis. J Neurochem 2003, 87:333-343. 28. Trulzsch B, Davies K, Wood M: Survival of motor neuron gene downregulation by RNAi: towards a cell culture model of spinal muscular atropy. Brain Res Mol Brain Res 2004, 120:145-150. 29. Barnes JW, Tischkau SA, Barnes JA, Mitchell JW, Burgoon PW, Hickok JR, Gillette MU: Requirement of mammalian Timeless for circadian rhythmicity. Science 2003, 302:439-442. 30. Bai J, Ramos RL, Ackman JB, Thomas AM, Lee RV, LoTurco JJ: RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci 2003, 6:1277-1283. 31. Whither RNAi? (editorial). Nature Cell Biology 2003, 5:489-490. The editors describe some of the pitfalls associated with RNAi in mammals and outline some methods to control for nonspecific effects. It should be noted that their suggestion of center mismatch controls is debatable, given more recent data concerning miRNA mechanisms. 32. Persengiev SP, Zhu X, Green MR: Nonspecific, concentrationdependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). RNA 2004, 10:12-18. 33. Lingor P, Michel U, Scholl U, Bahr M, Kugler S: Transfection of ‘‘naked’’ siRNA results in endosomal uptake and metabolic impairment in cultured neurons. Biochem Biophys Res Commun 2004, 315:1126-1133. 34. Zhao C, Ma H, Bossy-Wetzl E, Lipton SA, Zhang Z, Feng G-S: GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2. J Biol Chem 2003, 278:34641-34653. 35. Hering H, Sheng M: Activity-dependent redistribution and essential role of cortactin in dendritic spine morphogenesis. J Neurosci 2003, 23:11759-11769. 36. Passafaro M, Nakagawa T, Sala C, Sheng M: Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2. Nature 2003, 424:677-681. 37. Higuchi H, Yamashita T, Yoshikawa H, Tohyama M: Functional inhibition of p75 receptor using a small interfering RNA. Biochem Biophys Res Commun 2003, 301:804-809. 38. Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemoto-Kimura S, Kimura K, Nozaki K, Hashimoto N, Narumiya S: Control of axon elongation via an SDF-1a/Rho/mDia pathway in cultured cerebellar granule neurons. J Cell Biol 2003, 161:381-391. 39. McCrossan ZA, Lewis A, Panaghie G, Jordan PN, Christini DJ, Lerner DJ, Abbott GW: MinK-related peptide 2 modulates Kv2.1 and Kv3.1 potassium channels in mammalian brain. J Neurosci 2003, 23:8077-8091. 40. Vojtek AB, Taylor J, DeRuiter SL, Yu JY, Figueroa C, Kwok RPS, Turner DL: Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation. Mol Cell Biol 2003, 23:4417-4427. 41. Leucht C, Simoneau S, Rey C, Vana C, Rieger R, Lasmezas CI, Weiss S: The 37 kDa/67 kDa laminin receptor is required fro PrPsc propagation in scrapie-infected neuronal cells. EMBO Rep 2003, 4:290-295. 42. Zhang W-B, Zhang A, Ni Y-X, Wu Y-L, Pei G: A novel function of Goa: mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells. J Neurochem 2003, 86:1213-1222. www.sciencedirect.com
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43. Lin S-L, Chang D, Yu D-Y, Ying S-Y: A novel RNA splicingmediated gene silencing mechanism potential for genome evolution. Biochem Biophys Res Commun 2003, 310:754-760.
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53. Yang Y, Nishimura I, Imai Y, Takahashi R, Lu B: Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila. Neuron 2003, 37:911-924. The authors performed in vivo RNAi against dParkin in Drosophila. They demonstrated neurotoxic death in neurons only when dParkin suppression was combined with Pael-R expression. To further strengthen their argument, the authors rescued neuron numbers by coexpression of the exogenous human Parkin.
46. Varga EV, Yamamura HI, Rubenzik MK, Stropova D, Navratilova E, Roeske WR: Molecular mechanisms of excitatory signaling upon chronic opioid agonist treatment. Life Sci 2003, 74:299-311. 47. Schulze E, Altmann ME, Adham IM, Schulze B, Frode S, Engel W: The maintenance of neuromuscular function requires UBC-25 in Caenorhabditis elegans. Biochem Biophys Res Commun 2003, 305:691-699. 48. Bianchi L, Kwok S-M, Driscoll M, Sesti F: A potassium channel-MiRP complex controls neurosensory function in Caenorhabditis elegans. J Biol Chem 2003, 278:12415-12424. 49. Yau DM, Yokoyama N, Goshima Y, Siddiqui ZK, Siddiqui SS, Kozasa T: Identification and molecular characterization of the Galpha12-Rho guanine nucleotide exchange factor pathway in Caenorhabditis elegans. Proc Natl Acad Sci USA 2003, 100:14748-14753. 50. Brooks DR, Hooper NM, Isaac RE: The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction. J Biol Chem 2003, 278:42795-42801.
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54. Konishi Y, Stegmuller J, Matsuda T, Bonni S, Bonni A: Cdh1-APC controls axonal growth and patterning in the mammalian brain. Science 2004, 303:1026-1030. The authors performed a series of RNAi studies using a broad range of systems, from primary cultures to intact rat brain. A plasmid expressing shRNA against Cdh1 was compared with a null-expressing plasmid as the control. 55. Matsuda T, Cepko CL: Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA 2004, 101:16-22. The authors used a siRNA-expressing plasmid construct electroporated into retinal cells of newborn rodent pups to explore loss-of-function neuronal phenotypes. RNAi plasmids were coelectroporated with GFP plasmids. Immunocytochemistry of dissociated retinal cells showed efficient knockdown and coelectroporation. Immunohistochemistry of retinal slices demonstrated expected structural phenotypes in knockdown cells.
Current Opinion in Neurobiology 2004, 14:654–659
subjective view of the promise of these techniques specifically as tools for those investigators who seek to bridge the gap between the in vivo system level, function, development and pathogenesis of the nervous system and the explosion of information generated by the field of cellular and molecular neurobiology.
Background Addresses Department of Biology, McGovern Institute for Brain Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:654–659 This review comes from a themed issue on New technologies Edited by Winfried Denk and Liqun Luo Available online 11th September 2004 0959-4388/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.08.009
Abbreviations DCX dsRNA GFP miRNA PTGS RNAi shRNA siRNA
doublecortin double-stranded RNA green fluorescent protein microRNA post-transcriptional gene silencing RNA interference small hairpin RNA short-interfering RNA
Introduction The story of post-transcriptional gene silencing (PTGS) is rapidly unfolding in multiple model systems. PTGS is an evolutionarily conserved phenomenon that is frequently referred to as cosuppression in plants [1], RNA interference (RNAi) in animals [2,3] and quelling in fungi [4]. An in-depth examination of PTGS mechanisms has been presented by many other authors [5,6–8]. Figure 1 presents, in cartoon overview, the generally agreed upon processes of PTGS. An in-depth treatment of the biochemistry and molecular biology involved is beyond the scope of this article. Here, we try to clarify the phenomena for neurobiologists. We describe the techniques used, the controls necessary and the advantages over earlier procedures of capitalizing on these processes. These advantages explain the rapidly growing applications of PTGS to problems in neuroscience. We close with a brief Current Opinion in Neurobiology 2004, 14:654–659
PTGS encompasses two related mechanisms. One, RNAi, involves double-stranded short interfering RNAs (siRNAs), of approximately 21–26 basepairs, which degrade transcripts with the complementary sequence and can have very high specificity [8]. The second utilizes endogenously encoded, single-stranded micro-RNAs (miRNAs) of similar sizes, which bind homologous but not identical sequences of the 30 -untranslated region in target mRNAs and disrupt translation [7,9]. Many miRNAs have been found in genome screens [10–13] and it is clear that a single miRNA can modulate the expression of multiple gene products. However, the identity of these targets is largely unknown. The two PTGS mechanisms share pathways, associated proteins, regulating systems [6,7,14] and nomenclatures that can frequently but not always describe the same process. Here, we use the following terms: RNAi incorporates siRNAs for the highly specific downregulation of a target message achieved usually, although not exclusively [15], through experimental induction of double-stranded RNA (dsRNA)-mediated, sequence-specific messenger RNA (mRNA) degradation. miRNA silencing refers to the regulation achieved through the endogenous singlestranded miRNA-mediated, homology-dependent reduction in protein expression achieved by interfering with productive translation. However, because the molecular details of these processes are not fully understood, it is possible that an siRNA can decrease protein without affecting transcripts, by acting in the same manner as a miRNA [16,17], and it is similarly possible that miRNAs can specifically complement transcripts and thus act in the same manner as siRNA [18].
Experimental use of siRNA Introducing siRNA into cells
The techniques used to achieve specific siRNA knockdown in neurons depend crucially on the organism that houses the neurons and when, in the organisms life cycle, the interference is required. In Caenorhabditis elegans [2] and Drosophila [19], the siRNA precursor can be a long dsRNA precursor that is directly incorporated into cells and then broken down into shorter sequences. In mammalian cells, however [20], immunological reaction to long dsRNA severely compromises this approach [21–23]. www.sciencedirect.com
Post-transcriptional gene silencing in neurons Zeringue and Constantine-Paton 655
Figure 1
Long dsRNA
shRNA
Dicer
miRNA precursor
Dicer
miRNA
siRNA
Dicer
Dicer Activated RISC
Degrade mRNA
Activated RISC
Dicer
Dicer
Block mRNA
Current Opinion in Neurobiology
A schematic of the two major categories of PTGS. (a) siRNA: this method involves the dsRNA-dependent endonuclease Dicer cleaving dsRNA (either long dsRNA or shRNA into short 21–25 basepair siRNAs). The siRNA combines with and activates an RNA-induced silencing complex (RISC). Once joined with RISC, the siRNA denatures such that only a single strand is held in place by RISC. This single stranded RNA determines the sequence specificity of the RISC activity. The activated RISC binds with an mRNA complementary to the bound RNA and facilitates degradation of the transcript. (b) The same sequence occurs when a genetically encoded miRNA precursor is expressed. However, complementarity is not complete in these RNA sequences. In these cases, Dicer and the RISC complex continue to process the short RNAs but without precise complementarity, allowing for the function of more than one mRNA sequence to be disrupted. With miRNA, interference is not by degradation of the transcript but rather at some stage during translation of the transcript into a mature protein.
Thus, in mammalian cells, introduction into cells for transient expression is accomplished by transfection or electroporation of either the specific siRNA itself or small hairpin RNA (shRNA) in which the hair loop is rapidly cleaved to produce siRNA and the specific siRNA response. For stable, longer-term suppression, a DNA plasmid containing a construct coding for the shRNA [24,25] can be applied. Additional advantages of the DNA approach include the ability to apply viral vectormediated delivery of the construct to a spatially defined region and to use conditional promoters to regulate the expression of the construct [26]. Use of lenti-viral vectors is a relatively new and potentially more powerful technical development in the viral vector field. Using incapacitated lenti-viruses, both dividing and mature cells can be infected. The construct is incorporated in low copy number, generally in a single site on chromosomes, and cells appear to survive for long periods, if not indefinitely, following infection. Lenti-virus vectors are generally capable of carrying DNA coding for the shRNA, the U6 promoter region for transcription of the shRNA, and a second strong promoter to drive expression of a www.sciencedirect.com
reporter gene. In addition to direct injection into brain, these vectors can be used to generate shRNA expressing transgenic lines with extremely high efficiency [26]. siRNA advantages
The utility of the siRNA approach is manifold. First, there is rapid, relatively inexpensive and efficient knockdown, generally 90% of protein product, in transgenic animals when compared with animals with specific gene knockouts generated using homologous recombination [27,28]. Second, the function of proteins whose knockouts are lethal when expressed throughout the brain can still be studied by introducing siRNA [28,29] into only a subset of cells. The third advantage is the possibility that knockdown, as compared with complete knockout, of a protein might not induce rescue pathways seen with some knockouts [30]. Experimental controls
Despite the successful and robust knockdown achievable with RNAi, there are several possibilities for nonspecific effects. Therefore, good controls are necessary to Current Opinion in Neurobiology 2004, 14:654–659
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determine if the observed loss of function is actually restricted to effects on the gene product of interest or on off-target genes. Unfortunately, many reports involving this technology applied either no control or an overexpressed housekeeping gene control. The editors of Nature Cell Biology outlined a series of ‘appropriate’ controls for the siRNA researcher and generated a first attempt at codifying ‘proof’ of specificity [31]. The importance of such controls with siRNA is becoming increasingly apparent because even short RNAs are being shown to act nonspecifically and alter several proteins [32]. For example, Persengiev et al. [32] recently reported nonspecific siRNA effects in mammalian cells as a result of expression of a common ‘control’ siRNA sequence. The siRNA, directed against the luciferase reporter gene, was transfected into HeLa cells. HeLa cell transcript expression was subsequently determined with an Affymetrix gene chip. Of 33 000 genes tested, over 1100 increased and almost 700 decreased by 2.5-fold. These transcripts covered a broad range of cellular functions. Such findings demonstrate the importance of multiple controls when using RNAi because even an exogenous sequence does not necessarily offer ‘control’ expression. This generalized disruption resulting from exposure of cells to ‘naked’ siRNA might be caused by endosomal uptake, which has recently been shown in cultured primary neurons to produce broad metabolic impairment [33]. Nevertheless, one specific cautionary note from the results of Persengiev et al. is that the expression of glyceraldehydes 3-phosphate dehydrogenase (GAPDH), a ‘housekeeping’ gene frequently used to demonstrate the specificity of an siRNA ‘knockdown’ of an unrelated transcript or protein in mammalian cells, was not substantially affected. This demonstrates the very low sensitivity obtained by no change in GAPDH. Unfortunately, a common ‘RNAi-sensitive’ gene has not been identified. In fact, it is likely that no single transcript can be identified for the enormous range of siRNA sequences and cell types that can be employed with this technology. Nonspecific effects can also be obtained simply as a result of increased RNA load or disruption of unknown proteins downstream in a pathway. However, using a null-expressing vector or trying to detect differences in overexpressed products does nothing to control for nonspecific effects of siRNA activity. One suggested approach is to use several different siRNAs against the same target or to inspect products of several genes known to be associated with the actual target in both sequence homology and functional cellular pathways [34]. By looking at only a change in the product of interest using only one siRNA, researchers might miss the well-documented, and poorly understood, nonspecific effects of RNAi, particularly as it applies to vertebrate neurons.
RNAi in vitro Much of the work on RNAi in neurons has been performed in vitro. Using a variety of systems, investigators Current Opinion in Neurobiology 2004, 14:654–659
have examined neurite growth [35–38], voltage-dependent channels [39], apoptosis [27,28], cellular differentiation and growth [34,40] and a variety of signaling pathways [41–43]. One of the more common controls used for in vitro siRNA in mammalian cells is a scrambled sequence with no known homology in the genome being explored [28,37]. For example, Higuchi et al. [37] successfully used siRNA specifically to knockdown the p75 neurotrophin receptor in primary neuron culture. They studied neurite growth inhibition resulting from myelinassociated glycoprotein (MAG). The RNAi-induced suppression of p75 caused MAG-treated neurites to grow almost as long as controls, eliminating a significant decrease in neurite length when wild type neurons were treated with MAG. Suppression of p75 also decreased nerve growth factor-induced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelling (TUNEL) analysis. In one of the first studies to examine synaptic function after siRNA application, scrambled control siRNA and siRNA against CPl-17, a specific inhibitor of myosin/moesin phosphatase (MMP), have been introduced into primary cultured cerebellar neurons. Along with immunological analysis showing specific knockdown, the CPl-17 siRNA was demonstrated using electrophysiological analysis of synaptic currents to completely eliminate granule cell to Purkinje cell long-term depression, whereas long-term depression remained present in Purkinje cells transfected with scrambled CPl-17 siRNA [44].
RNAi in vivo Many more examples of elegant culture work could be cited here. However, the ultimate goal of many neurobiologists is to determine how mechanisms defined in vitro are orchestrated to function in vivo in the dynamic environment of the intact nervous system. Difficulties of in vivo neurobiology research ranging from the blood– brain barrier to redundancy at system [45] and molecular [46] levels necessitate ever-more-sophisticated assays. The majority of in vivo siRNA work reported thus far has been in C. elegans. Because first, RNAi was first discovered in this organism [2], second, there is no immunological response to dsRNA, and third, RNAi can be easily implemented in the animal by feeding or injection. We cite only a small portion of the rich RNAi literature performed in C. elegans. Genetic screening methods employing siRNA in C. elegans have recently yielded insight into a ubiquitin pathway-related protein that is localized at the neuromuscular junction [47], a K+ channel-associated ortholog of the mammalian MinK-related peptide [48], a pathway known to affect axon guidance [49], an aminopeptidase localized to neurons [50] and a protein thought to act within the RNAi pathway itself [51]. A useful tool in integrating this vast amount of information is a web-based functional genomics repository based on much of the work in C. elegans [52]. As more experimental www.sciencedirect.com
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data becomes available, an enabling tool for continued research will be a central repository to house RNAi information about mammalian neuronal models. Drosophila, like C. elegans, does not exhibit an interferon response. However, to obtain a system-wide infection, a shRNA-coding DNA has to be introduced into the genome. A good example of the work being carried out in the fly is illustrated by Yang et al. [53]. These investigators explored the role of the ubiquitin ligase Parkin in neurotoxicity. They showed that pan-neuronal expression of Pael-R, a Parkin substrate, causes an age-associated degeneration of Drosophila dopamine neurons. Coexpression of Pael-R and human Parkin rescues these cells. Moreover, expressing shRNA against Drosophila Parkin (dParkin) in the dopamine neurons, along with the panneuronal Pael-R, promoted Pael-R accumulation and augmented neurotoxicity. Thus, using siRNA together with rescue by an exogenous protein, Yang et al. [53] built a strong case for the cellular interaction between Parkin and Pael-R during dopaminergic neuron cell death. A crucial goal of biomedical neuroscience research is to understand the human nervous system. However, the power of RNAi has only recently been applied to mammalian systems, and robust implementation has proven complicated. Only a few studies have reported in vivo siRNA in mammalian neurons. Several utilized electroporation of a shRNA-expressing DNA plasmid [30,54,55]. For example, Matsuda et al. [55] explored the phenotypic changes due to knockdown of the retinal transcription factors Crx and Nrl. Plasmids containing DNA constructs for shRNA against Crx or Nrl transcripts were coelectroporated into the retina of newborn mice along with green fluorescent protein (GFP)-expressing plasmids. After differentiation, GFP-positive cells in the outer nuclear layer of the retina had structural changes consistent with the knockout phenotypes, as demonstrated by immunohistochemistry of retinal sections. Bai et al. [30] used a similar system to explore the role of doublecortin (DCX) during development of the neocortex in rat. They performed cotransfection of plasmids producing GFP and an anti-DCX shRNA by in utero electroporation of embryonic mice. Control mice were transfected with GFP–plasmid alone. Using fluorescence and immunohistochemistry, they showed arrested migration of cerebral neocortical neurons when shRNA against DCX was expressed. Antibody staining against DCX was used to detect knockdown. DCX knockdown cells remained scattered among the layers of the cortex, whereas control cells were highly concentrated in cortical layers two and three. Overall, these reports suggest that siRNA applied in vivo might adequately substitute for time- and resource- intensive conditional knockout techniques currently applied to the mammalian brain. www.sciencedirect.com
Prospectus and conclusion It is hoped that this mini-review has explained why the discovery of PTGS has so galvanized large segments of biology that nearly every biology journal carries papers studying the phenomenon of using, or purporting to use, siRNA, for loss-of-function assays. In the field of neuroscience, this technology has opened the door to entirely new approaches to studying the intact brain. For example, viral vectors carrying DNA constructs for shRNA against specific proteins expected to be active during particular processes could be injected into the specific brain regions suspected to be involved before triggering the process. These processes could range from learning, to attention, to decision making, to fear or to a developmental event such as the activity-dependent refinement of synapses. The structural, physiological and behavioral differences between animals carrying a control shRNA, perhaps scrambled shRNA, and the targeted shRNA can then be compared using any of the numerous assay techniques available to neuroscientists today. Ideally, such constructs should be designed to express, in specific neuron types, a requirement that might be difficult but not beyond the toolbox available to molecular neurobiologists. Indeed, many efforts are underway to target siRNAs against disease genes to particular cells or tissues. Collaborations will be important to success in all of these endeavors. Additionally, many such ‘system’ level experiments will have to await the intermediate steps of siRNA expression in intact model systems, such as rodents, songbirds or ferrets, in which single-cell anatomy and single-cell physiology can be applied to examine individual transfected cells among populations of otherwise normal cells or circuits to determine whether the siRNA ‘knockdown’ induces the expected effect and whether any effect remains cell autonomous or alters the entire circuit or cell population in which the affected cell resides. In fact, for many neuroscientists working at the interface of molecular, cellular and systems neurobiology, the latter effort — to define what a small subset of neurons with a specific molecular defect, located among normal or control-infected neurons, can accomplish or fail to accomplish — is a significant goal in and of itself. The excitement over siRNA is that such studies are now within both our technical and financial capabilities.
Acknowledgements We would like to thank HR Horvitz and EA Miska (Massachusetts Institute of Technology) for their helpful suggestions on this manuscript.
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Current Opinion in Neurobiology 2004, 14:654–659