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Complete RNAi rescue of neuronal degeneration in a constitutively active Drosophila TRP channel mutant
Biochimica et Biophysica Acta 1674 (2004) 91 – 97 www.bba-direct.com
Complete RNAi rescue of neuronal degeneration in a constitutively active Drosophila TRP channel mutant Chaoxian Geng 1, Ann Pellegrino, John Bowman, Liqin Zhu, William L. Pak * Lily Hall of Life Science, Department of Biological Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907-2054, USA Received 13 April 2004; received in revised form 28 May 2004; accepted 4 June 2004 Available online 26 June 2004
Abstract RNA interference has been widely used to reduce the quantity of the proteins encoded by the targeted genes. A constitutively active, dominant allele of trp, TrpP365, causes massive degeneration of photoreceptors through a persistent and excessive Ca2 + influx. Here we show that a substantial reduction of the TRP channel protein by RNAi in TrpP365 heterozygotes completely rescues the neuronal degeneration and significantly improves the light-elicited responses of the eye. The reduction need not be complete, suggesting that rescue of degeneration may be possible with minimal side effects arising from overdepletion of the target protein. D 2004 Elsevier B.V. All rights reserved. Keywords: Neuronal degeneration; RNAi rescue; TRP channel; Unregulated Ca2+ influx; Ion channel hypothesis; Drosophila
1. Introduction Calcium homeostasis is vital for cell function and survival. Both genetic and environmental factors may disrupt cellular Ca2 + regulation. For example, pathological peptides arising from mutations or oxidative stress may be incorporated into the plasma membrane causing unregulated ion flow. The hypothesis that cell death can be caused by such unregulated ion flow through channels formed from the pathological peptides is referred to as ‘‘ion channel hypothesis’’ (reviewed in Ref. [1]). The nervous system appears to be particularly susceptible to damage by cellular ion imbalance [2]. Indeed, a large body of experimental evidence supports the ‘‘ion channel hypothesis’’ in certain forms of human neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases [3,4]. Furthermore, it has been recently shown that sustained increase in the cytosolic Ca2 + concentration causes production of intraneuronal amyloidh1– 42 (Abeta1 – 42) and induces neuronal death [5]. In spite of intense search for cures, effective prevention * Corresponding author. Tel.: +1-765-494-8202; fax: +1-765-4940876. E-mail address: [email protected] (W.L. Pak). 1 Present address: Cambria Biosciences, 8A Henshaw St., Woburn, MA 01801, USA. 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.002
and management strategies are lacking for age-related human neurodegenerative diseases [6]. In Drosophila melanogaster, it has been demonstrated that excessive Ca2 + influx can cause massive neuronal degeneration [7]. The Drosophila trp gene [8,9], predominantly expressed in photoreceptors in the eye, encodes a subunit of a highly Ca2 +-permeable TRP channel, which is primarily localized in the rhabdomeres, a specialized structure for phototransduction consisting of densely packed microvilli. Photostimulation of rhodopsin, a G-protein-coupled receptor, triggers a cascade of reactions that lead to the opening of the TRP channels on the photoreceptor plasma membrane. An autosomal dominant mutation TrpP365 (Phe550Ile) in the trp gene causes the TRP channels to be constitutively active resulting in excessive Ca2 + influx into the photoreceptor cells, and this excessive Ca2 + influx causes photoreceptor degeneration [7,10]. Degeneration caused by TrpP365 is greatly ameliorated by another mutation, inaF [11]. The inaF gene encodes a highly eye-enriched protein, INAF, of unknown function. Several lines of evidence [11] suggest that INAF protein might have a regulatory role on the TRP channel function. However, it was also found [11] that the inaF mutation causes a significant reduction in quantity of the TRP protein. Thus, it was unclear as to how much of the amelioration of the TrpP365-caused degeneration was attributable to the
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reduction of the TRP channel protein and how much to shutting down the channel by affecting channel regulation. In particular, we wanted to determine whether reduction in the quantity of the TRP channel alone was sufficient to suppress degeneration. It has been recently reported that anoxia-induced cell death of cultured murine cortical neurons is mediated by the activation of TRPM7 [12], a Ca2 +-permeable cation channel of the TRP superfamily, demonstrating that cell death by overactivation of TRP channels can also occur in mammalian neurons. These investigators also showed that suppression of TRPM7 by RNAi inhibits Ca2 + uptake and partially rescues the neurons from anoxia-induced cell death [12]. Here we show, using whole organisms, complete rescue of neuronal degeneration caused by persistent and excessive Ca2 + influx in Drosophila by RNAi that directly targets the native transcript for the Ca2 + channel protein TRP.
2. Experimental procedures 2.1. RNAi construct To make the RNAi construct, the 5V portion of the trp gene that contains the first intron (834 bp) was ligated tailto-tail with the corresponding 5Vportion of trp cDNA (826 bp) (Fig. 1A). The native upstream regulatory region of the trp gene was placed at the two ends of the RNAi construct in opposite orientations so that transcription can start from both ends. Such a construct is advantageous because double-stranded RNA molecules can form either from complementary RNA strands transcribed by the two oppositely oriented promoters or by snapping back of a single strand transcribed by either of the two promoters. The intron was included to break the perfect palindrome because long perfect palindromes are usually eliminated from host bacteria [13], rendering low yields of plasmid DNA. The RNAi construct was subcloned into a P element vector (pCaSpeR4) for germline transformation. It should be noted that the RNAi construct does not specifically target the Trp365 mutant allele. Instead, it is intended to target mutant and wild-type alleles equally because the construct utilizes the first exon and a part of the second exon sequences (Fig. 1A) while the mutation is in the eighth exon of the trp gene. 2.2. Electroretinogram (ERG) recording ERGs were recorded as previously described [14], using glass electrodes filled with Hoyle’s saline. The light stimuli originating from a tungsten halogen lamp (Bausch & Lomb) were delivered to the flies with a fiber optic light guide. The unattenuated intensity at the level of the fly eye was approximately 47 mW/cm2. The light was attenuated with neutral density filters as needed and filtered with a sharp cut
Fig. 1. (A) Schematic diagram of the RNAi construct. The arrows indicate the directions of transcription. (B, C) Western analyses showing reduction in quantity of the TRP protein by RNA interference (RNAi). Twenty-fourto thirty-six-hour-old flies (day 1 post-eclosion) were used in these experiments. (B) Lanes 1 – 5 were loaded with protein extracts from wildtype heads and diluted to 100%, 40%, 20%, 10%, and 5% wild-type equivalents, respectively. Lane 6, Ri/Ri; lane 7, Ri/+; lane 8, wild type at 100% equivalent. Note the reduction in the quantity of TRP protein by RNAi construct is dose dependent. (C) Lanes 1 – 7 were loaded with protein extracts from wild-type heads at 10%, 15%, 20%, 25%, 30%, 35%, and 40% equivalents, respectively; lane 8, Ri/Ri. One hundred percent wildtype and RNAi lanes were loaded with extracts from two heads per lane.
orange filter (Corning CS 2 –73) for orange stimuli. The flies were dark adapted for two min before each recording. 2.3. Western blot analysis Ten fly heads were homogenized in 40 Al of SDS-PAGE sample buffer containing 50 mM DTT and proteinase inhibitors of recommended concentrations (CompleteR, Roche Molecular Biochemicals). The homogenate was boiled for 5 min and centrifuged (12,000 g for 3 min), and 8 Al of the supernatant were loaded onto each lane of 8% SDS-polyacrylamide gels. For quantity estimation, protein extracts from wild-type fly heads were diluted in the sample buffer by known amounts to achieve various fractional concentrations of the wild-type amount. Western blots were generated using a standard protocol and the primary anti-TRP antibody (monoclonal, the Benzer Laboratory, California Institute of Technology) was used at 1:3000 dilution.
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2.4. Deep pseudopupil (dpp) measurement Flies were reared and aged in a 12-h light/12-h dark cycle. Newly emerging adult flies collected within 24 h of eclosion were considered 0 day post-eclosion. FlyNap (Carolina Biological) was used to immobilize the flies, and the dpp [15] was observed under a dissecting microscope. At least 30 flies were examined for each data point, and the flies were discarded after each observation. 2.5. Confocal microscopy Fly eyes were dissected in a fixative (4% paraformaldehyde in phosphate-buffered saline with 0.3% Triton X-100), and the dissected retinas (photoreceptor layers) were kept in the same fixative for 1 h. After incubation in phosphatebuffered saline that contained 4% normal goat serum, the fixed retinas were stained with phalloidin-tetramethylrhodamine B isothiocyanate (Sigma) to label filamentous actin in the rhabdomeres. Transverse optical sections of f 1-Am thickness were taken f 6 Am from the distal tips of the rhabdomeres for observation.
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stimulus of 4-s duration, the ERG waveform of the flies harboring RNAi was indistinguishable from that of wild type (Fig. 2). In chemically induced trp mutants with no detectable amount of TRP, on the other hand, the same stimulus would have generated a strong mutant phenotype, described above. With a bright stimulus of 30-s duration, a small but noticeable decay of the photoreceptor potential was observed during light stimulus in flies homozygous for RNAi (RNAi/RNAi) (Fig. 2, second trace). Increasing the number of RNAi copies to four, by generating flies that carry the RNAi harboring P element on both the second and third chromosomes (RNAi/RNAi;RNAi/RNAi), resulted in a further reduction in the TRP protein and a further decay of the ERG. The quantity of the TRP protein was approximately 18% of the wild-type amount (not shown) in these flies, and the ERG decayed, during 30-s stimuli, to a level much closer to baseline than with two RNAi copies (Fig. 2, cf. second and third traces). Under the same conditions, wild type maintained a sustained response (Fig. 2, first trace). Thus, both the reduction in the TRP amount and the level of ERG decay were RNAi copy-number-dependent, suggesting that RNAi is indeed responsible for both these effects. The ERG phenotype induced by RNAi, on the other hand, was relatively mild.
3. Results 3.1. RNAi causes a reduction in the quantity of the TRP protein By P-element-mediated germline transformation, five Drosophila transformant lines carrying the RNAi transgene were obtained. Of the five, two lines had P element insertion on the second chromosome, and the remaining three, on the third chromosome. No obvious adverse effects of transgenes on fitness of the flies were observed in any of the lines. To determine whether the RNAi transgene causes degradation of the trp transcripts, and thereby reduces the amount of the TRP protein, we analyzed the relative quantities of the TRP protein in the RNAi flies by Western blot analysis. The quantity of the TRP protein in flies heterozygous for the RNAi transgene (RNAi/+) was reduced to approximately 60% of that in wild type (Fig. 1B, lane 7), and in flies homozygous for the RNAi transgene (RNAi/RNAi), it was further reduced to approximately 30% of the wild-type amount (Fig. 1B,C). We next tested whether the reduction in quantity of the TRP protein by RNAi would cause an alteration in lightinduced responses of the eye. In most of the chemically induced trp mutants previously reported, no detectable amount of TRP is present [8,9]. Light-induced responses of these trp mutants are characterized by smaller than normal amplitudes and a rapid decay of the photoreceptor potential to baseline even during a brief but strong stimulus, whereas, in wild type, a sustained component is maintained throughout the stimulus. We used the ERG to measure lightinduced mass responses of the eye extracellularly. With a
Fig. 2. ERG showing a mild but distinctly noticeable decay (arrow) of the photoreceptor potential caused by the RNAi transgene during strong, prolonged illumination. Ri/Ri designates flies carrying two copies of the RNAi transgene on the second chromosome, whereas Ri/Ri;Ri/Ri designates flies carrying four copies of the RNAi transgene on the second and third chromosomes. Note the rate of decay of the photoreceptor potentials during stimulus is correlated with the copy number of the RNAi transgene. The decrease in ERG amplitudes during 30-s illumination was quantified by taking the ratio of the amplitude at light offset to that at light onset. These ratios are: wild type, 79 F 8% (n = 4); Ri/Ri, 41 F 6% (n = 7); and Ri/Ri;Ri/Ri, 28 F 6% (n = 4). The numbers below the stimulus protocol trace at the bottom refer to stimulus intensities. 0: unattenuated intensity; 2 and 4: attenuated by 2 and 4 log units, respectively. Or: unattenuated orange stimulus, obtained by interposing a Corning orange filter in the light path; 4/Or: orange stimulus attenuated by 4 log units, obtained by interposing both an orange filter and a neutral density filter. See Experimental procedures for filters used.
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3.2. RNAi completely rescues photoreceptor cell degeneration in TrpP365/+ To test whether a reduction of TRP by RNAi can rescue photoreceptor degeneration caused by TrpP365, the RNAi construct was introduced into a heterozygous TrpP365 background by crossing RNAi to TrpP365 flies. The integrity of the rhabdomeres of photoreceptor cells was monitored both by using confocal microscopy and by examining the dpp. Because actin filaments thread the microvilli comprising the rhabdomeres, the rhabdomeres can be visualized in confocal microscopy by staining the filamentous actin molecules with rhodamine-phalloidin. The dpp is a virtual image of superposed neighboring rhabdomeres. When the superposition fails either because of disappearance or disarrangement of the rhabdomeres, the dpp disappears. On day 0 post-eclosion, all flies heterozygous for TrpP365 have apparently normal dpp. However, by day 6 post-eclosion, all of these flies become dpp-negative (Fig. 3). In contrast, nearly 100% of flies carrying the RNAi construct in a TrpP365/+ background are dpp-positive at least up to 28 days post-eclosion. Some of the very young RNAi flies (0 to 2 days posteclosion), however, appear to be dpp-negative. This phenomenon is probably related to delayed development of the rhabdomeres or misalignment of some of the rhabdomeres at an early age because the flies become dpp-positive as they become older (Fig. 3). Consistent with the dpp analysis, confocal microscopy also demonstrated that photoreceptor degeneration in TrpP365/+ was completely rescued by the RNAi transgene (Fig. 4). In TrpP365/+, all seven rhabdomeres were present at day 0 post-eclosion, but no intact rhabdomeres were detectable by 8 days post-eclosion. By contrast, in TrpP365/+ flies carrying the RNAi transgene, all rhabdomeres were still intact even at 28 days post-eclosion, just as in control flies that do not carry the mutation TrpP365 (Ri/Ri;Ri/+).
Fig. 4. Confocal microscopic analyses showing rapid degeneration of photoreceptor cells in P365/+ (bottom row) and preservation of rhabdomere structures with the introduction of the RNAi construct (top row). Ri/Ri;Ri/+ (middle row) was included as a control. These flies carry three copies of RNAi transgene as in the test flies (top row). Unlike the test flies, they have no P365, but carry instead two copies of trp+.
3.3. RNAi substantially rescues the ERG phenotype In addition to maintaining the integrity of the rhabdomere structure, the RNAi transgene substantially rescued the photoreceptor physiology (Fig. 5) of TrpP365/+ flies, as monitored by ERG recording. Compared with those of wild type, the ERGs of TrpP365/+ (Fig. 5) are characterized by (1) a significantly reduced amplitude, (2) markedly slower termination kinetics at stimulus off, and (3) nearly or completely absent on-transients. Very small transients are sometimes present in young TrpP365/+ flies, but they disappear by day 7 post-eclosion. The ERG transients arise in L1/L2 cells in the lamina as a result of synaptic inputs from the majority class of photoreceptors, R1 – 6. Photoreceptors of the minority class, R7/8, bypass the lamina and
Fig. 3. Deep pseudopupil (dpp) analysis to assess the integrity of rhabdomeres of photoreceptor cells. Note the rapid disappearance of dpp in flies heterozygous for TrpP365 (P365/+) whereas nearly 100% of the flies with RNAi transgene in a P365/+ background were dpp-positive even at day 28 post-eclosion. Ri/Ri;Ri/P365 flies carry one copy of wild-type trp and one copy of TrpP365, whereas Ri/Ri;Ri/+ flies carry two copies of trp+ on the third chromosome.
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Fig. 5. ERG analyses showing functional rescue of the photoreceptor cells in P365/+ by an RNAi transgene. The wild-type and P365/+ flies were 7 days old, while the RNAi/RNAi;RNAi/P365 flies were 28 days old. Note the presence of the on- and off-transients (arrow heads) and the improved amplitude of the receptor potential component of the ERG in flies carrying the RNAi transgene. The labels for stimulus protocol at the bottom of the figure are as explained in Fig. 2.
synapse in the medulla, and thus do not contribute to the transients. Therefore, the absence of transients is an indication of disrupted synaptic communication between R1 –6 photoreceptors and their postsynaptic target neurons in the lamina [16]. The small ERG amplitudes and the absence of the transients in TrpP365/+ together indicate that R1 – 6 photoreceptors are almost completely nonfunctional in these mutants (Fig. 5, third trace). The residual responses are almost entirely from R7/8 photoreceptors because they do not contribute to the transients. Introducing the RNAi construct into TrpP365/+ restored the ERG amplitudes to nearly the wild-type levels and, in addition, brought back both the on- and off-transients even at 28 days post-eclosion (Fig. 5). The results indicate that R1 – 6 photoreceptors, which were previously nonfunctional, are now functional and contribute to the near-normal ERG amplitudes and to the generation of the transients. However, the termination time course of the responses at stimulus offset was still slower than in wild type, indicating that rescue is not complete.
4. Discussion We have shown in this work that RNAi targeting the native trp transcript completely rescues the photoreceptor degeneration in TrpP365 heterozygotes at least up to 28-day post-eclosion. Because the TrpP365 mutation causes the TRP channels to be constitutively active, leading to persistent and excessive Ca2 + influx, the above results suggest that the Ca2 + influx is brought back to a tolerable range [17] by RNAi, thereby protecting the photoreceptors from degeneration.
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The expression of RNAi transgene in this study was under the control of the native trp promoter and cisregulatory sequences. Therefore, expression of RNAi and that of mutant TrpP365 were synchronized and colocalized. Whenever and wherever TrpP365 was transcribed, so also was the RNAi transgene to permit effective degradation of TrpP365 transcripts. Significantly, total elimination of the TRP protein is not necessary to achieve complete rescue of degeneration. Even with four copies of RNAi, a significant amount (18%) of TRP remains, and the ERG phenotype caused by the protein reduction is relatively mild, becoming apparent only with very prolonged, bright illuminations. It thus appears that RNAi does not completely prevent expression of TRP although the native trp promoter was used in the RNAi construct (see Experimental procedures). The reason might be that some of the endogenous transcripts reach the translation complex without being degraded because of the concentration-dependent interaction between double-stranded interfering RNA and native target RNA. Complete or near-complete elimination of the TRP protein would cause a strong mutant phenotype [8,9,11,17], which would have undesirable side effects on the fly. Thus, the results demonstrate that complete rescue of degeneration can be achieved by RNAi without significant adverse side effects on the organism. Rescue of the ERG phenotype by the RNAi transgene is also substantial, although not complete (Fig. 5). It may be that the amount of the mutant TRPP365 protein in these flies is still sufficiently large to cause a larger-than-normal Ca2 + influx. Furthermore, because the TRPP365 channel is constitutively active, the Ca2 + influx would persist even in the absence of light stimulus. Nevertheless, the level of Ca2 + influx in these flies appears to be in a near-normal range so that the ERG is substantially rescued and the integrity of the neurons is preserved. Conceivably, one can lower the quantity of the TRPP365 protein further to an optimal range so that both the photoreceptor degeneration and ERG phenotypes are completely rescued. One might achieve this goal, for example, by genetically increasing the copy number of the RNAi transgene and/or by using an inducible promoter to control the RNAi transgene expression in time, space, and quantity. Photoreceptor degeneration in TrpP365 proceeds in total darkness because the mutant channels are constitutively active [7]. In TrpP365 homozygotes, any additional effects of light cannot be determined because degeneration is already nearly complete at eclosion with or without light present. However, using TrpP365 heterozygotes, in which degeneration proceeds more slowly, Yoon et al. [7] showed that light exacerbates the degeneration probably by activating the TRP channels formed from wild-type subunits, causing an additional increase in Ca2 + influx into photoreceptor cells. Light-accelerated degeneration in TrpP365/+ flies is an example of synergistic interactions of genetic and environmental factor(s) that cause alterations in Ca2 +
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homeostasis to contribute to the altered onset and progression of neurodegeneration. Imbalance of cellular ions has been considered an etiological basis of many diseases [2,18,19]. This imbalance may arise from genetically altered endogenous ion channels, channels formed from pathological peptides [1,20 –22], disrupted plasma membrane [2], altered ion pump activity, or altered internal stores. Cellular ion imbalance triggers disturbances in cell physiology, including altered enzymatic reactions and gene expression, leading to neurodegeneration. Thus, degeneration in TrpP365 mutants may be but one manifestation of more commonly and widely occurring forms of neurodegeneration. As has been previously discussed by Yoon et al. [7], even that subclass of such degenerations caused by overactive TRP-related channels may not be limited to Drosophila but occurs much more widely. For example, a large number of mammalian TRPrelated channels has been identified. Many of them are abundantly expressed in the brain, and some are highly Ca2 +-permeable (reviewed in Ref. [23]). A certain class of mutations in the genes encoding these channels [7] or certain perturbations in environmental conditions could overactivate the channels to cause neurodegeneration, as in TrpP365. The recent demonstration by Aarts et al. [12] that anoxic cell death of cultured murine cortical neurons is mediated by the activation of TRPM7, a member of the TRP cation channel superfamily, suggests that neuronal cell death by overactivation of TRP channels does, indeed, occur in mammalian neurons. By showing that photoreceptor degeneration in TrpP365 can be rescued through RNAi targeting of a class of highly Ca2 +-permeable channels, TRP, we have provided a feasible model system for controlling neuronal Ca2 + influx and degeneration in cases where the disease-causing protein is either overexpressed or overactive. Once fine tuned, the system described here may be useful for studying the consequences of perturbed Ca2 + homeostasis in neurons and its implications on the progression of certain forms of age-related human neurodegenerative diseases. Furthermore, it may be useful for examining the interplay of genetic determinants and environmental factors in the development of such diseases. For such studies, mammalian mutations implicated in neurodegeneration could be expressed in the Drosophila eye to study their consequences, thus taking advantage of genetic tractability of Drosophila [24 – 26].
Acknowledgements The authors wish to thank the reviewers for their constructive comments and suggestions. This work was supported by a grant from the National Eye Institute, NIH (EY00033) to W.L.P.
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Complete RNAi rescue of neuronal degeneration in a constitutively active Drosophila TRP channel mutant Chaoxian Geng 1, Ann Pellegrino, John Bowman, Liqin Zhu, William L. Pak * Lily Hall of Life Science, Department of Biological Sciences, Purdue University, 915 W. State St., West Lafayette, IN 47907-2054, USA Received 13 April 2004; received in revised form 28 May 2004; accepted 4 June 2004 Available online 26 June 2004
Abstract RNA interference has been widely used to reduce the quantity of the proteins encoded by the targeted genes. A constitutively active, dominant allele of trp, TrpP365, causes massive degeneration of photoreceptors through a persistent and excessive Ca2 + influx. Here we show that a substantial reduction of the TRP channel protein by RNAi in TrpP365 heterozygotes completely rescues the neuronal degeneration and significantly improves the light-elicited responses of the eye. The reduction need not be complete, suggesting that rescue of degeneration may be possible with minimal side effects arising from overdepletion of the target protein. D 2004 Elsevier B.V. All rights reserved. Keywords: Neuronal degeneration; RNAi rescue; TRP channel; Unregulated Ca2+ influx; Ion channel hypothesis; Drosophila
1. Introduction Calcium homeostasis is vital for cell function and survival. Both genetic and environmental factors may disrupt cellular Ca2 + regulation. For example, pathological peptides arising from mutations or oxidative stress may be incorporated into the plasma membrane causing unregulated ion flow. The hypothesis that cell death can be caused by such unregulated ion flow through channels formed from the pathological peptides is referred to as ‘‘ion channel hypothesis’’ (reviewed in Ref. [1]). The nervous system appears to be particularly susceptible to damage by cellular ion imbalance [2]. Indeed, a large body of experimental evidence supports the ‘‘ion channel hypothesis’’ in certain forms of human neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases [3,4]. Furthermore, it has been recently shown that sustained increase in the cytosolic Ca2 + concentration causes production of intraneuronal amyloidh1– 42 (Abeta1 – 42) and induces neuronal death [5]. In spite of intense search for cures, effective prevention * Corresponding author. Tel.: +1-765-494-8202; fax: +1-765-4940876. E-mail address: [email protected] (W.L. Pak). 1 Present address: Cambria Biosciences, 8A Henshaw St., Woburn, MA 01801, USA. 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.002
and management strategies are lacking for age-related human neurodegenerative diseases [6]. In Drosophila melanogaster, it has been demonstrated that excessive Ca2 + influx can cause massive neuronal degeneration [7]. The Drosophila trp gene [8,9], predominantly expressed in photoreceptors in the eye, encodes a subunit of a highly Ca2 +-permeable TRP channel, which is primarily localized in the rhabdomeres, a specialized structure for phototransduction consisting of densely packed microvilli. Photostimulation of rhodopsin, a G-protein-coupled receptor, triggers a cascade of reactions that lead to the opening of the TRP channels on the photoreceptor plasma membrane. An autosomal dominant mutation TrpP365 (Phe550Ile) in the trp gene causes the TRP channels to be constitutively active resulting in excessive Ca2 + influx into the photoreceptor cells, and this excessive Ca2 + influx causes photoreceptor degeneration [7,10]. Degeneration caused by TrpP365 is greatly ameliorated by another mutation, inaF [11]. The inaF gene encodes a highly eye-enriched protein, INAF, of unknown function. Several lines of evidence [11] suggest that INAF protein might have a regulatory role on the TRP channel function. However, it was also found [11] that the inaF mutation causes a significant reduction in quantity of the TRP protein. Thus, it was unclear as to how much of the amelioration of the TrpP365-caused degeneration was attributable to the
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reduction of the TRP channel protein and how much to shutting down the channel by affecting channel regulation. In particular, we wanted to determine whether reduction in the quantity of the TRP channel alone was sufficient to suppress degeneration. It has been recently reported that anoxia-induced cell death of cultured murine cortical neurons is mediated by the activation of TRPM7 [12], a Ca2 +-permeable cation channel of the TRP superfamily, demonstrating that cell death by overactivation of TRP channels can also occur in mammalian neurons. These investigators also showed that suppression of TRPM7 by RNAi inhibits Ca2 + uptake and partially rescues the neurons from anoxia-induced cell death [12]. Here we show, using whole organisms, complete rescue of neuronal degeneration caused by persistent and excessive Ca2 + influx in Drosophila by RNAi that directly targets the native transcript for the Ca2 + channel protein TRP.
2. Experimental procedures 2.1. RNAi construct To make the RNAi construct, the 5V portion of the trp gene that contains the first intron (834 bp) was ligated tailto-tail with the corresponding 5Vportion of trp cDNA (826 bp) (Fig. 1A). The native upstream regulatory region of the trp gene was placed at the two ends of the RNAi construct in opposite orientations so that transcription can start from both ends. Such a construct is advantageous because double-stranded RNA molecules can form either from complementary RNA strands transcribed by the two oppositely oriented promoters or by snapping back of a single strand transcribed by either of the two promoters. The intron was included to break the perfect palindrome because long perfect palindromes are usually eliminated from host bacteria [13], rendering low yields of plasmid DNA. The RNAi construct was subcloned into a P element vector (pCaSpeR4) for germline transformation. It should be noted that the RNAi construct does not specifically target the Trp365 mutant allele. Instead, it is intended to target mutant and wild-type alleles equally because the construct utilizes the first exon and a part of the second exon sequences (Fig. 1A) while the mutation is in the eighth exon of the trp gene. 2.2. Electroretinogram (ERG) recording ERGs were recorded as previously described [14], using glass electrodes filled with Hoyle’s saline. The light stimuli originating from a tungsten halogen lamp (Bausch & Lomb) were delivered to the flies with a fiber optic light guide. The unattenuated intensity at the level of the fly eye was approximately 47 mW/cm2. The light was attenuated with neutral density filters as needed and filtered with a sharp cut
Fig. 1. (A) Schematic diagram of the RNAi construct. The arrows indicate the directions of transcription. (B, C) Western analyses showing reduction in quantity of the TRP protein by RNA interference (RNAi). Twenty-fourto thirty-six-hour-old flies (day 1 post-eclosion) were used in these experiments. (B) Lanes 1 – 5 were loaded with protein extracts from wildtype heads and diluted to 100%, 40%, 20%, 10%, and 5% wild-type equivalents, respectively. Lane 6, Ri/Ri; lane 7, Ri/+; lane 8, wild type at 100% equivalent. Note the reduction in the quantity of TRP protein by RNAi construct is dose dependent. (C) Lanes 1 – 7 were loaded with protein extracts from wild-type heads at 10%, 15%, 20%, 25%, 30%, 35%, and 40% equivalents, respectively; lane 8, Ri/Ri. One hundred percent wildtype and RNAi lanes were loaded with extracts from two heads per lane.
orange filter (Corning CS 2 –73) for orange stimuli. The flies were dark adapted for two min before each recording. 2.3. Western blot analysis Ten fly heads were homogenized in 40 Al of SDS-PAGE sample buffer containing 50 mM DTT and proteinase inhibitors of recommended concentrations (CompleteR, Roche Molecular Biochemicals). The homogenate was boiled for 5 min and centrifuged (12,000 g for 3 min), and 8 Al of the supernatant were loaded onto each lane of 8% SDS-polyacrylamide gels. For quantity estimation, protein extracts from wild-type fly heads were diluted in the sample buffer by known amounts to achieve various fractional concentrations of the wild-type amount. Western blots were generated using a standard protocol and the primary anti-TRP antibody (monoclonal, the Benzer Laboratory, California Institute of Technology) was used at 1:3000 dilution.
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2.4. Deep pseudopupil (dpp) measurement Flies were reared and aged in a 12-h light/12-h dark cycle. Newly emerging adult flies collected within 24 h of eclosion were considered 0 day post-eclosion. FlyNap (Carolina Biological) was used to immobilize the flies, and the dpp [15] was observed under a dissecting microscope. At least 30 flies were examined for each data point, and the flies were discarded after each observation. 2.5. Confocal microscopy Fly eyes were dissected in a fixative (4% paraformaldehyde in phosphate-buffered saline with 0.3% Triton X-100), and the dissected retinas (photoreceptor layers) were kept in the same fixative for 1 h. After incubation in phosphatebuffered saline that contained 4% normal goat serum, the fixed retinas were stained with phalloidin-tetramethylrhodamine B isothiocyanate (Sigma) to label filamentous actin in the rhabdomeres. Transverse optical sections of f 1-Am thickness were taken f 6 Am from the distal tips of the rhabdomeres for observation.
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stimulus of 4-s duration, the ERG waveform of the flies harboring RNAi was indistinguishable from that of wild type (Fig. 2). In chemically induced trp mutants with no detectable amount of TRP, on the other hand, the same stimulus would have generated a strong mutant phenotype, described above. With a bright stimulus of 30-s duration, a small but noticeable decay of the photoreceptor potential was observed during light stimulus in flies homozygous for RNAi (RNAi/RNAi) (Fig. 2, second trace). Increasing the number of RNAi copies to four, by generating flies that carry the RNAi harboring P element on both the second and third chromosomes (RNAi/RNAi;RNAi/RNAi), resulted in a further reduction in the TRP protein and a further decay of the ERG. The quantity of the TRP protein was approximately 18% of the wild-type amount (not shown) in these flies, and the ERG decayed, during 30-s stimuli, to a level much closer to baseline than with two RNAi copies (Fig. 2, cf. second and third traces). Under the same conditions, wild type maintained a sustained response (Fig. 2, first trace). Thus, both the reduction in the TRP amount and the level of ERG decay were RNAi copy-number-dependent, suggesting that RNAi is indeed responsible for both these effects. The ERG phenotype induced by RNAi, on the other hand, was relatively mild.
3. Results 3.1. RNAi causes a reduction in the quantity of the TRP protein By P-element-mediated germline transformation, five Drosophila transformant lines carrying the RNAi transgene were obtained. Of the five, two lines had P element insertion on the second chromosome, and the remaining three, on the third chromosome. No obvious adverse effects of transgenes on fitness of the flies were observed in any of the lines. To determine whether the RNAi transgene causes degradation of the trp transcripts, and thereby reduces the amount of the TRP protein, we analyzed the relative quantities of the TRP protein in the RNAi flies by Western blot analysis. The quantity of the TRP protein in flies heterozygous for the RNAi transgene (RNAi/+) was reduced to approximately 60% of that in wild type (Fig. 1B, lane 7), and in flies homozygous for the RNAi transgene (RNAi/RNAi), it was further reduced to approximately 30% of the wild-type amount (Fig. 1B,C). We next tested whether the reduction in quantity of the TRP protein by RNAi would cause an alteration in lightinduced responses of the eye. In most of the chemically induced trp mutants previously reported, no detectable amount of TRP is present [8,9]. Light-induced responses of these trp mutants are characterized by smaller than normal amplitudes and a rapid decay of the photoreceptor potential to baseline even during a brief but strong stimulus, whereas, in wild type, a sustained component is maintained throughout the stimulus. We used the ERG to measure lightinduced mass responses of the eye extracellularly. With a
Fig. 2. ERG showing a mild but distinctly noticeable decay (arrow) of the photoreceptor potential caused by the RNAi transgene during strong, prolonged illumination. Ri/Ri designates flies carrying two copies of the RNAi transgene on the second chromosome, whereas Ri/Ri;Ri/Ri designates flies carrying four copies of the RNAi transgene on the second and third chromosomes. Note the rate of decay of the photoreceptor potentials during stimulus is correlated with the copy number of the RNAi transgene. The decrease in ERG amplitudes during 30-s illumination was quantified by taking the ratio of the amplitude at light offset to that at light onset. These ratios are: wild type, 79 F 8% (n = 4); Ri/Ri, 41 F 6% (n = 7); and Ri/Ri;Ri/Ri, 28 F 6% (n = 4). The numbers below the stimulus protocol trace at the bottom refer to stimulus intensities. 0: unattenuated intensity; 2 and 4: attenuated by 2 and 4 log units, respectively. Or: unattenuated orange stimulus, obtained by interposing a Corning orange filter in the light path; 4/Or: orange stimulus attenuated by 4 log units, obtained by interposing both an orange filter and a neutral density filter. See Experimental procedures for filters used.
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3.2. RNAi completely rescues photoreceptor cell degeneration in TrpP365/+ To test whether a reduction of TRP by RNAi can rescue photoreceptor degeneration caused by TrpP365, the RNAi construct was introduced into a heterozygous TrpP365 background by crossing RNAi to TrpP365 flies. The integrity of the rhabdomeres of photoreceptor cells was monitored both by using confocal microscopy and by examining the dpp. Because actin filaments thread the microvilli comprising the rhabdomeres, the rhabdomeres can be visualized in confocal microscopy by staining the filamentous actin molecules with rhodamine-phalloidin. The dpp is a virtual image of superposed neighboring rhabdomeres. When the superposition fails either because of disappearance or disarrangement of the rhabdomeres, the dpp disappears. On day 0 post-eclosion, all flies heterozygous for TrpP365 have apparently normal dpp. However, by day 6 post-eclosion, all of these flies become dpp-negative (Fig. 3). In contrast, nearly 100% of flies carrying the RNAi construct in a TrpP365/+ background are dpp-positive at least up to 28 days post-eclosion. Some of the very young RNAi flies (0 to 2 days posteclosion), however, appear to be dpp-negative. This phenomenon is probably related to delayed development of the rhabdomeres or misalignment of some of the rhabdomeres at an early age because the flies become dpp-positive as they become older (Fig. 3). Consistent with the dpp analysis, confocal microscopy also demonstrated that photoreceptor degeneration in TrpP365/+ was completely rescued by the RNAi transgene (Fig. 4). In TrpP365/+, all seven rhabdomeres were present at day 0 post-eclosion, but no intact rhabdomeres were detectable by 8 days post-eclosion. By contrast, in TrpP365/+ flies carrying the RNAi transgene, all rhabdomeres were still intact even at 28 days post-eclosion, just as in control flies that do not carry the mutation TrpP365 (Ri/Ri;Ri/+).
Fig. 4. Confocal microscopic analyses showing rapid degeneration of photoreceptor cells in P365/+ (bottom row) and preservation of rhabdomere structures with the introduction of the RNAi construct (top row). Ri/Ri;Ri/+ (middle row) was included as a control. These flies carry three copies of RNAi transgene as in the test flies (top row). Unlike the test flies, they have no P365, but carry instead two copies of trp+.
3.3. RNAi substantially rescues the ERG phenotype In addition to maintaining the integrity of the rhabdomere structure, the RNAi transgene substantially rescued the photoreceptor physiology (Fig. 5) of TrpP365/+ flies, as monitored by ERG recording. Compared with those of wild type, the ERGs of TrpP365/+ (Fig. 5) are characterized by (1) a significantly reduced amplitude, (2) markedly slower termination kinetics at stimulus off, and (3) nearly or completely absent on-transients. Very small transients are sometimes present in young TrpP365/+ flies, but they disappear by day 7 post-eclosion. The ERG transients arise in L1/L2 cells in the lamina as a result of synaptic inputs from the majority class of photoreceptors, R1 – 6. Photoreceptors of the minority class, R7/8, bypass the lamina and
Fig. 3. Deep pseudopupil (dpp) analysis to assess the integrity of rhabdomeres of photoreceptor cells. Note the rapid disappearance of dpp in flies heterozygous for TrpP365 (P365/+) whereas nearly 100% of the flies with RNAi transgene in a P365/+ background were dpp-positive even at day 28 post-eclosion. Ri/Ri;Ri/P365 flies carry one copy of wild-type trp and one copy of TrpP365, whereas Ri/Ri;Ri/+ flies carry two copies of trp+ on the third chromosome.
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Fig. 5. ERG analyses showing functional rescue of the photoreceptor cells in P365/+ by an RNAi transgene. The wild-type and P365/+ flies were 7 days old, while the RNAi/RNAi;RNAi/P365 flies were 28 days old. Note the presence of the on- and off-transients (arrow heads) and the improved amplitude of the receptor potential component of the ERG in flies carrying the RNAi transgene. The labels for stimulus protocol at the bottom of the figure are as explained in Fig. 2.
synapse in the medulla, and thus do not contribute to the transients. Therefore, the absence of transients is an indication of disrupted synaptic communication between R1 –6 photoreceptors and their postsynaptic target neurons in the lamina [16]. The small ERG amplitudes and the absence of the transients in TrpP365/+ together indicate that R1 – 6 photoreceptors are almost completely nonfunctional in these mutants (Fig. 5, third trace). The residual responses are almost entirely from R7/8 photoreceptors because they do not contribute to the transients. Introducing the RNAi construct into TrpP365/+ restored the ERG amplitudes to nearly the wild-type levels and, in addition, brought back both the on- and off-transients even at 28 days post-eclosion (Fig. 5). The results indicate that R1 – 6 photoreceptors, which were previously nonfunctional, are now functional and contribute to the near-normal ERG amplitudes and to the generation of the transients. However, the termination time course of the responses at stimulus offset was still slower than in wild type, indicating that rescue is not complete.
4. Discussion We have shown in this work that RNAi targeting the native trp transcript completely rescues the photoreceptor degeneration in TrpP365 heterozygotes at least up to 28-day post-eclosion. Because the TrpP365 mutation causes the TRP channels to be constitutively active, leading to persistent and excessive Ca2 + influx, the above results suggest that the Ca2 + influx is brought back to a tolerable range [17] by RNAi, thereby protecting the photoreceptors from degeneration.
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The expression of RNAi transgene in this study was under the control of the native trp promoter and cisregulatory sequences. Therefore, expression of RNAi and that of mutant TrpP365 were synchronized and colocalized. Whenever and wherever TrpP365 was transcribed, so also was the RNAi transgene to permit effective degradation of TrpP365 transcripts. Significantly, total elimination of the TRP protein is not necessary to achieve complete rescue of degeneration. Even with four copies of RNAi, a significant amount (18%) of TRP remains, and the ERG phenotype caused by the protein reduction is relatively mild, becoming apparent only with very prolonged, bright illuminations. It thus appears that RNAi does not completely prevent expression of TRP although the native trp promoter was used in the RNAi construct (see Experimental procedures). The reason might be that some of the endogenous transcripts reach the translation complex without being degraded because of the concentration-dependent interaction between double-stranded interfering RNA and native target RNA. Complete or near-complete elimination of the TRP protein would cause a strong mutant phenotype [8,9,11,17], which would have undesirable side effects on the fly. Thus, the results demonstrate that complete rescue of degeneration can be achieved by RNAi without significant adverse side effects on the organism. Rescue of the ERG phenotype by the RNAi transgene is also substantial, although not complete (Fig. 5). It may be that the amount of the mutant TRPP365 protein in these flies is still sufficiently large to cause a larger-than-normal Ca2 + influx. Furthermore, because the TRPP365 channel is constitutively active, the Ca2 + influx would persist even in the absence of light stimulus. Nevertheless, the level of Ca2 + influx in these flies appears to be in a near-normal range so that the ERG is substantially rescued and the integrity of the neurons is preserved. Conceivably, one can lower the quantity of the TRPP365 protein further to an optimal range so that both the photoreceptor degeneration and ERG phenotypes are completely rescued. One might achieve this goal, for example, by genetically increasing the copy number of the RNAi transgene and/or by using an inducible promoter to control the RNAi transgene expression in time, space, and quantity. Photoreceptor degeneration in TrpP365 proceeds in total darkness because the mutant channels are constitutively active [7]. In TrpP365 homozygotes, any additional effects of light cannot be determined because degeneration is already nearly complete at eclosion with or without light present. However, using TrpP365 heterozygotes, in which degeneration proceeds more slowly, Yoon et al. [7] showed that light exacerbates the degeneration probably by activating the TRP channels formed from wild-type subunits, causing an additional increase in Ca2 + influx into photoreceptor cells. Light-accelerated degeneration in TrpP365/+ flies is an example of synergistic interactions of genetic and environmental factor(s) that cause alterations in Ca2 +
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homeostasis to contribute to the altered onset and progression of neurodegeneration. Imbalance of cellular ions has been considered an etiological basis of many diseases [2,18,19]. This imbalance may arise from genetically altered endogenous ion channels, channels formed from pathological peptides [1,20 –22], disrupted plasma membrane [2], altered ion pump activity, or altered internal stores. Cellular ion imbalance triggers disturbances in cell physiology, including altered enzymatic reactions and gene expression, leading to neurodegeneration. Thus, degeneration in TrpP365 mutants may be but one manifestation of more commonly and widely occurring forms of neurodegeneration. As has been previously discussed by Yoon et al. [7], even that subclass of such degenerations caused by overactive TRP-related channels may not be limited to Drosophila but occurs much more widely. For example, a large number of mammalian TRPrelated channels has been identified. Many of them are abundantly expressed in the brain, and some are highly Ca2 +-permeable (reviewed in Ref. [23]). A certain class of mutations in the genes encoding these channels [7] or certain perturbations in environmental conditions could overactivate the channels to cause neurodegeneration, as in TrpP365. The recent demonstration by Aarts et al. [12] that anoxic cell death of cultured murine cortical neurons is mediated by the activation of TRPM7, a member of the TRP cation channel superfamily, suggests that neuronal cell death by overactivation of TRP channels does, indeed, occur in mammalian neurons. By showing that photoreceptor degeneration in TrpP365 can be rescued through RNAi targeting of a class of highly Ca2 +-permeable channels, TRP, we have provided a feasible model system for controlling neuronal Ca2 + influx and degeneration in cases where the disease-causing protein is either overexpressed or overactive. Once fine tuned, the system described here may be useful for studying the consequences of perturbed Ca2 + homeostasis in neurons and its implications on the progression of certain forms of age-related human neurodegenerative diseases. Furthermore, it may be useful for examining the interplay of genetic determinants and environmental factors in the development of such diseases. For such studies, mammalian mutations implicated in neurodegeneration could be expressed in the Drosophila eye to study their consequences, thus taking advantage of genetic tractability of Drosophila [24 – 26].
Acknowledgements The authors wish to thank the reviewers for their constructive comments and suggestions. This work was supported by a grant from the National Eye Institute, NIH (EY00033) to W.L.P.
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