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Vol. 19, Issue 12, 5517-5528, December 2008
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Department of Biosciences and Nutrition, Karolinska Institute, S-14157 Huddinge, Sweden; and School of Life Sciences, Södertörn University College, S-14189 Huddinge, Sweden
Submitted April 23, 2008;
Revised August 15, 2008;
Accepted September 30, 2008
Monitoring Editor: Marcos Gonzalez-Gaitan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Huang et al., 1998; Otsuki et al., 2004), brain development and neuronal functions (Ma et al., 2006; Zhang et al., 2006), and ciliogenesis. Cilia develop as specialized subcellular structures with sensory or motile functions that project off many different cell types. Their structure and function have been investigated in mammals, Drosophila, and C. elegans. Initially, the characterization of the single C. elegans RFX transcription factor, DAF-19, had established for the first time a connection between RFX transcription factors and cilia development (Swoboda et al., 2000) and provided a basis for subsequent studies in other species. Drosophila dRFX is expressed in the peripheral nervous system, where it is essential for the proper function of ciliated type I sensory organs (Dubruille et al., 2002; Laurencon et al., 2007). Mammalian RFX3 is responsible for nodal cilia development, the specification of left-right asymmetry and the differentiation of ciliated ependymal cells in the brain (Bonnafe et al., 2004; Baas et al., 2006). Thus, the role of RFX transcriptions factors in ciliogenesis is conserved across species. In C. elegans, the gene daf-19 is expressed in ciliated sensory neurons mostly located in the head and tail of the worm (Swoboda et al., 2000). These neurons are the major source of input for environmental signals for the worm. daf-19 mutant worms are completely devoid of ciliated structures and are consequently unable to respond to environmental signals such as food, dauer pheromone, or nose touch (Perkins et al., 1986). Nevertheless, in the laboratory daf-19 mutants are viable and thus a suitable model to study ciliogenesis. We and others have identified a large number of direct DAF-19 target genes based on the presence of the x-box promoter sequence motif, the binding site for DAF-19. Their expression in ciliated sensory neurons was dependent on both daf-19 and the promoter x-box, and many of them are required for cilia structure and function (Blacque et al., 2005; Efimenko et al., 2005; Chen et al., 2006).
In the present study, we show that DAF-19 not only regulates the formation of cilia in sensory neurons but also is required for the maintenance of synaptic functions in the remainder of the nervous system. The hermaphrodite C. elegans nervous system consists of 302 neurons (60 of which are ciliated) that are connected via 
7000 chemical synapses and 700 gap junctions (White et al., 1986). Chemical synapses are established either between neurons or between neurons and muscle cells, at the so-called neuromuscular junctions. Each synapse consists of three major areas: 1) the synaptic vesicle pool, made up of vesicles at various stages of the recycling process or ready for neurotransmitter release; 2) the presynaptic terminal, where synaptic vesicles fuse in a multistep process and release neurotransmitters into the synaptic cleft; and 3) the postsynaptic target area in the receiving neuron, the receptive field, in which neurotransmitter receptors cluster. The isolation of a large number of C. elegans synapse mutants has provided us with detailed knowledge about the function of the synapse, especially the life cycle of synaptic vesicles. Recent work addressed the hierarchical assembly of the presynaptic terminal, providing detailed insight into the interdependence of assembly steps at a molecular level (Dai et al., 2006; Patel et al., 2006). However, how the expression of individual synaptic components is regulated after their initial establishment and how their constant supplies are maintained, remains largely unknown.
Here, we present a detailed analysis of three different daf-19 transcripts. We show that the short isoform daf-19c is expressed in all ciliated sensory neurons and is sufficient to rescue ciliogenesis phenotypes of daf-19 mutants. The two long isoforms daf-19a/b are expressed in basically all nonciliated neurons. We describe novel behavioral and cellular phenotypes of daf-19. In particular, we demonstrate that DAF-19A/B are necessary to maintain expression levels of several synaptic proteins, which assigns DAF-19 a function in neurotransmission. Surprisingly, this reduced synaptic protein expression is rather mild at larval stages but declines progressively as adult daf-19 mutants age. Therefore, our study for the first time establishes a member of the RFX transcription factor family as a regulator of synaptic maintenance. Intriguingly, the synaptic defects in daf-19 mutants display strong parallels to the synaptic decline observed in human neurodegenerative disorders, suggesting that similar mechanisms may be affected.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The strains and transgenes used in this work are summarized in Supplemental Table 4. All strains were grown at 20°C. At this temperature, daf-19 mutants display a highly penetrant Daf-c phenotype. However, 10% of the population does not activate the dauer formation program and can be used for experiments (Swoboda et al., 2000). Worms were picked singly at larval stage 4 (L4) before behavioral and paralysis assays that required a small number of worms (<50 animals/assay). Antibody stainings of mixed stage populations were performed on large batches of daf-19 worms. For all experiments that required large populations of staged worms (Western blot, quantitative polymerase chain reaction [PCR], and antibody stainings) or that involved the analysis of nonrescuing transgenes (transcriptional gfp fusions of x-box candidate genes, intron-gfp fusions, translational gfp fusions of synaptic genes), we used the daf-12 (sa204) background. The daf-12 mutation suppresses the Daf-c phenotype of daf-19 and prevents dauer formation.
Injection Constructs, Germ Line Transformation, and Green Fluorescent Protein (GFP) Expression Analyses
pGG20 and pGG21 contain the last 250 base pairs of daf-19 intron 3 and daf-19 intron 4 fused to gfp, respectively. The daf-19 rescue and deletion constructs pTJ803, pGG14, and pGG18 (see Figure 2) were derived from pTJ786 (daf-19 genomic plus 2.9-kb promoter). pGG67 is a genomic/cDNA fusion rescue construct specific for daf-19a (see Figure 4). Transcriptional gfp fusions of daf-19 were injected at 100 ng/µl and daf-19 rescue constructs were injected at 10 ng/µl. Synaptic markers and promoter gfp fusions were injected at 50–70 ng/µl. Adult hermaphrodites were transformed using standard techniques (Mello et al., 1991).
Behavioral Assays
Paralysis assays were performed on nematode growth medium agar plates containing 500 µM aldicarb or 100 µM levamisole. In addition, the resistance of daf-19 mutants to levamisole was confirmed at concentrations up to 1 mM (data not shown). At least 25–30 1-d-old adult worms were examined for each strain. Worms were classified as paralyzed when they did not move upon prodding with a pick three times in a row.
For dwelling/roaming assays, 1-d-old adult worms were transferred singly to fresh plates with a bacterial lawn of standardized size. After 1 h, worms were removed, each plate was put on a transparency with a grid (5 x 5 mm), and the number of squares that were filled with worm tracks was counted (Figure 4A). Each assay was repeated at least twice, with two independent lines for each transgene. More than 30 worms were examined in paralysis and dwelling/roaming assays.
DiI Staining, Microscopy, and Fluorescence Imaging
Fluorescent dye-filling assays with DiI were performed as described previously (Starich et al., 1995). For live imaging of GFP expression, worms were anesthetized in 0.1% sodium azide in M9 buffer and immobilized on a 2% agar pad. Differential interference contrast and fluorescence pictures were taken on an Axioplan 2 microscope (Carl Zeiss, Jena, Germany). We also used the microscope together with the OpenLab software (Improvision, Coventry, United Kingdom) for the analysis of expression levels of synaptic proteins (antibody stainings). Pictures of the comarker UNC-10 (unchanged between wild type and daf-19) and the synaptic protein under investigation were taken at fixed exposure times (optimized for the UNC-10 staining intensity). The intensity of the signal for the synaptic protein under these conditions was classified as "strong" when the picture was overexposed and as "weak" when the picture was underexposed (cf. Figures 6 and 7 and Supplemental Table 2).
Northern Blot Analysis and RNase Protection Assay
Embryos were isolated from gravid wild-type adults grown on egg medium by hypochlorite treatment. Embryonic total RNA was extracted using TRIzol (Invitrogen, Paisley, United Kingdom). For Northern blot, radioactive probes were prepared using the Prime-It random labeling kit (Stratagene, La Jolla, CA) and purified over ProbeQant G50 columns (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). RNase protection assay: Radioactive probes were prepared according to the manufacturer's instructions (MAXIscript kit; Ambion, Austin, TX), and hybridization to 20 µg total RNA was carried out according to the instructions in the manual for the RPA III kit (Ambion).
Quantitative Real-Time PCR
We used TRIzol and the RNeasy kit (QIAGEN, Dorking, Surrey, United Kingdom) to extract total RNA from staged 2-d-old adult worms. All samples were checked for RNA integrity (Agilent 2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA) and subjected to DNase digestion and single-strand cDNA synthesis (iScript; Bio-Rad, Hemel Hempstead, United Kingdom). Expression levels of selected genes were analyzed in an Applied Biosystems 7300 thermocycler (Applied Biosystems, Foster City, CA) by using actin as a reference gene. Each reaction was run in triplicates on two independent biological samples for each strain. All primers had a melting temperature of 58–60°C and produced a single amplicon. Data were analyzed using the Fast SDS software 1.3.1 (Applied Biosystems).
Antibody Production
cDNA fragments corresponding to DAF-19 amino acids 2–212 (for AbDAF19N) and 340–513 (for AbDAF19C), respectively, were expressed in BL21 (DE3) bacterial cells. Immunization of rabbits was carried out at Gramsch Laboratories (Schwabhausen, Germany). On Western blots, AbDAF19N detected a specific band of 120 kDa, by using wild-type protein extracts, corresponding to DAF-19A/B. This band was absent from protein extracts from daf-19 mutant worms (Supplemental Figure 1A). AbDAF19C was not suitable for Western blot analysis. On worm whole-mount stainings, both antibodies detected a signal in neuronal nuclei at all stages (Supplemental Figure 1, B and D). Aside from that, DAF-19 was also detectable in hypodermal cells at larval stages (data not shown).
Western Blot Analysis
Worms were staged by hypochlorite treatment of gravid adults. Western blots were incubated with AbDAF19N (1:250), anti-tubulin (YOL 1/34; 1:100), anti-UNC-17 (1:200), anti-SNB-1 (SN1; 1:200), horseradish peroxidase (HRP) anti-rat (1:10,000), and HRP anti-mouse (1:5000).
Antibody Staining
Staining with antibodies against UNC-29 and UNC-49 required permeabilization through freeze-fracture (Gally and Bessereau, 2003). For all other antibodies, whole-mount fixation and permeabilization were carried out as described previously (Finney et al., 1988). Worms were incubated with a 1:400 dilution of affinity-purified anti-DAF-19 antibodies. Other antibodies used were anti-OSM-5 (1:200), anti-SNB-1 (Ab1092; 1:2000), anti-SNB-1 (SN1; 1:200), anti-SNT-1 (R558; 1:100), anti-UNC-10 (RIM; 1:200), anti-UNC-13 (1:800), anti-UNC-17 (1:1000), anti-UNC-18 (G247; 1:100), anti-UNC-29 (1:200), anti-UNC-31 (1:200), anti-UNC-49 (1:800), anti-UNC-64 (Ab940; 1:5000), Alexa488 and Alexa546 (1:250: Jackson ImmunoResearch Laboratories, West Grove, PA), and Cy5 (1:1000; Rockland Immunochemicals, Gilbertsville, PA). The SN1 and RIM antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). For all antibodies, we investigated the entire nervous system. However, for reasons of equal comparisons, we mainly focused on the head region/nerve ring. Confocal pictures of antibody stainings were taken on a TCS SP microscope (Leica, Wetzlar, Germany).
DNA Sequence Motif Searches
DNA sequences of C. elegans and Caenorhabditis briggsae synapse genes (3-kb promoter, the entire coding region and 1-kb downstream of the stop codon) were scanned for possible matches to an x-box consensus sequence RYYNYY(N)1-3RRNRRY with VectorNTI (Invitrogen). Candidate motifs were analyzed for 1) motifs that are conserved between both species and occur in several genes, or 2) motifs that occur in several C. elegans genes, in case the candidate motif lacked conservation in other nematodes species. Candidate motifs conserved between C. elegans and C. briggsae were not found.
To identify conserved motifs unrelated to the x-box, we searched 1.5 kb upstream of the ATG of the same genes. We scanned for motifs of 5–10, 8–14, and 10–16 nucleotides length using MEME (http://meme.sdsc.edu).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In addition to the 2.9-kb band, probes specific for the remaining exons also detected a 2.4-kb transcript, which we termed short isoform daf-19c. To visualize all three isoforms daf-19a/b/c in one experiment, we conducted an RNase protection assay. A cDNA probe against exons 3–4 protected fragments of three different sizes, corresponding to the transcripts daf-19a (containing only exon 3), daf-19b (containing exons 3 and 4), and daf-19c (containing only exon 4) (Figure 1, B and C). Using 5'-rapid amplification of cDNA ends, we amplified a fragment that includes daf-19c exon 4 fused to the SL1 splice leader (data not shown). This is consistent with a trans-splice at the beginning of exon 4 followed by an ATG at position +9, in-frame with the remainder of the daf-19 transcript. In summary, these results show that in addition to the known long isoforms daf-19a/b, a third short isoform daf-19c exists that comprises SL1-spliced exons 4–12.
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). The identification of daf-19c raised the question about the functional significance of each transcript. To test for isoform-specific functions, we generated genomic deletion constructs and introduced them into a daf-19 mutant background (Figure 2A). Fragments of daf-19 lacking the promoter and the region up to intron 3 were still able to express DAF-19, as determined by staining with AbDAF19C (Supplemental Figure 2F). The expression was restricted to a small set of neurons in the head and the tail, a pattern reminiscent of ciliated sensory neurons. Consistent with the expression pattern, these constructs were sufficient to activate the expression of osm-5 and bbs-7, two well characterized, direct daf-19 target genes that are expressed in ciliated sensory neurons and function in cilia formation (Figure 2B and Supplemental Figure 2, A' and A'') (Haycraft et al., 2001; Blacque et al., 2004). As expected, these daf-19 deletion constructs also rescued the Dyf and Daf-c phenotypes of daf-19 mutants (Figure 2, A and C; data not shown). By contrast, DNA constructs starting downstream of exon 4 failed to rescue (Figure 2, A, D, and E). We also expressed either daf-19a or daf-19c cDNAs from the gpa-13 promoter in a daf-19 mutant background. gpa-13 drives expression in five ciliated sensory neurons: ADF, ASH, AWC, PHA, and PHB (Jansen et al., 1999), out of which ASH (in the head) and PHA and PHB (in the tail) can be stained with the fluorescent dye DiI (Hedgecock et al., 1985). We found that gpa-13(p)::daf-19c, but not gpa-13(p)::daf-19a was sufficient to rescue cilia formation in sensory neurons as visualized by fluorescent dye DiI filling (Figure 2, F–I).
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DAF-19A/B Are Expressed in Nonciliated Neurons
To elucidate the functions of DAF-19A/B, we analyzed their expression patterns in detail, by using antibodies against the N- and C-terminal regions of DAF-19, AbDAF19N, and AbDAF19C, respectively. Although the N-terminal antibody AbDAF19N recognizes epitopes unique to the long isoforms DAF-19A/B, the C-terminal antibody AbDAF19C recognizes the same epitopes common to isoforms DAF-19A/B/C (Figure 3A). To prove that AbDAF19N specifically detects DAF-19A/B and not C, we compared both antibodies on transgenic rescue lines expressing only DAF-19A or DAF-19C, respectively. As expected, we could detect DAF-19A with the N- and C-terminal antibodies, but DAF-19C only with the C-terminal antibody (Supplemental Figure 2, A–F). Stainings of wild-type worms with both antibodies detected DAF-19 in the majority of neuronal nuclei in the head and tail ganglia and in the ventral nerve cord. This signal was absent in all daf-19 mutant alleles tested (m86, m334, m407, rh1024, sa190, and sa232 affect all three isoforms equally; Swoboda et al., 2000), proving the specificity of both antibodies (Supplemental Figure 1, B–E). Although the AbDAF19N and AbDAF19C staining patterns overlapped in large parts, they were not identical. Posterior to the nerve ring, where the cell bodies of the amphid ciliated sensory neurons are located, we observed a group of cells, which stained only with AbDAF19C, but not with AbDAF19N (Supplemental Figure 1, B and D).
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200–240 neurons (data not shown). To understand where DAF-19A/B may exert their functions, we determined their expression patterns in detail. We stained gfp reporter lines, which mark subgroups of neurons, with anti-GFP and with AbDAF19N antibodies to determine whether they label the same neurons. Using nine different markers, we tested nearly half of all 302 neurons in the adult hermaphrodite, corresponding to 60 different classes of neurons (Figure 3H and Supplemental Table 1). We found that DAF-19A/B were expressed only in nonciliated neurons and not in ciliated sensory neurons (Figure 3). For example, ceh-23::gfp is expressed in many ciliated sensory neurons and the nonciliated neurons AIY and CAN. DAF-19A/B were detected in AIY and CAN but not in ciliated sensory neurons (Figure 3B; data not shown). Similarly, the nonciliated neurons marked with nmr-1::gfp stained with AbDAF-19N and therefore express DAF-19A/B (Figure 3, D and E). Thus, DAF-19C is specific for ciliated sensory neurons, and DAF-19A/B are specific for nonciliated neurons. In total, we found in 86 of 92 tested nonciliated neurons expression of DAF-19A/B, representing many different neuronal classes. In summary, DAF-19A/B are expressed in 200–240 nonciliated neurons and DAF-19C is expressed in 60 ciliated sensory neurons, which adds up to a basically pan-neuronal expression pattern of DAF-19 in the C. elegans hermaphrodite.
Dwelling/Roaming Behavior Depends on Multiple daf-19 Isoforms
Mutations in genes with broad neuronal expression often lead to the impaired movement of worms (UNCoordinated phenotype). daf-19 mutants move in a wild-type like manner and show no obvious Unc phenotype. We also tested daf-19 mutants in body bend assays to determine their movement speed, and we found that they can move as fast as wild type (data not shown). More specific aspects of C. elegans behavior (mating, feeding, egg laying, or patterns of movement) are usually dependent on or influenced by sensory abilities of the worm and thus depend on daf-19c. We did not identify a specific behavior that exclusively required nonciliated neurons or DAF-19A/B (data not shown). However, when performing body bend assays, we observed in daf-19 mutants severe defects in their dwelling/roaming behavior, which was dependent on all three DAF-19 isoforms. When put on a fresh plate seeded with bacteria, a single wild-type worm covers the entire bacterial lawn with tracks within a short time (dwelling/roaming) (Figure 4, A and B). In contrast, daf-19 mutants (we tested m86, rh1024, and sa232) move only for a short time and then start feeding locally (Figure 4D; data not shown). A similar behavior is observed in many cilia mutants, in which genes that are expressed exclusively in ciliated sensory neurons are mutated (e.g., che-13; Haycraft et al., 2003) and che-11 (Bell et al., 2006; Figure 4C; data not shown). To test the functions of the different DAF-19 isoforms in the dwelling/roaming behavior, we generated isoform-specific rescue constructs (Figure 4I). The dwelling/roaming phenotype of daf-19 mutants could partially be rescued by daf-19a or daf-19c (Figure 4, F and G). Complete rescue occurs only when all isoforms were present via a full-length genomic daf-19 construct (Figure 4, E and H). From these behavioral experiments, we conclude that the dwelling/roaming phenotype of daf-19 mutants is not merely caused by the lack of cilia, because the function of both the long and the short daf-19 isoforms are required.
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DAF-19A/B Regulate Synaptic Protein Expression Indirectly
DAF-19C regulates target cilia genes directly through a conserved promoter motif, the x-box. Are synaptic genes regulated by DAF-19A/B in a similar manner? Because all DAF-19 isoforms contain the same DNA binding domain (Figures 1C and 3A), we reasoned that they could bind to overall very similar DNA sequence motifs and that direct target genes for DAF-19A/B are included in published lists of predicted x-box genes (Blacque et al., 2005; Efimenko et al., 2005; Chen et al., 2006). We filtered those lists for all genes with functions at synapses or in vesicle formation/transport (Supplemental Table 3). ida-1, snb-1, snt-1, unc-17, and unc-64 were not among them. In addition, we searched those five genes for degenerated, x-box-like or other conserved sequence motifs. None of these searches revealed any common motifs (data not shown), suggesting that they do not harbor a binding site for DAF-19A/B. To search for other possible direct DAF-19A/B targets, we checked the expression of multiple candidates from the above-mentioned lists for their dependence on the transcription factor. None of them was affected in daf-19 mutants (Supplemental Table 3). To finally test whether snb-1, unc-17, and unc-64 are directly or indirectly regulated at the transcript level, we compared their expression levels by quantitative real-time PCR. We did not detect any difference between wild type and daf-19 mutants in transcript levels of these three genes (Figure 7D). Thus, we conclude that DAF-19A/B do not regulate synaptic genes at the transcriptional level. We speculate that DAF-19A/B maintain synaptic protein expression in nonciliated neurons via an indirect mechanism, yet to be discovered (Figure 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Here, we identified a novel short transcript daf-19c that lacks exons 1-3. This short isoform DAF-19C is specifically expressed in ciliated sensory neurons from an internal promoter, and it is sufficient to rescue all cilia-related phenotypes of daf-19 mutants (Dyf, Daf-c, expression of cilia-specific, direct target genes). In contrast, the long isoforms DAF-19A/B are expressed from a different promoter in almost all nonciliated neurons, resulting in a basically pan-neuronal expression pattern of DAF-19. This expression of multiple isoforms via the so-called two-promoter system is common to many genes in C. elegans and crucial for the execution of their isoform-specific functions (Choi and Newman, 2006). We discovered that daf-19 mutants are resistant to the pharmacological substances aldicarb and levamisole, both of which modulate cholinergic synaptic transmission and lead to paralysis. The reason for this resistance was found in strongly reduced levels of synaptic vesicle proteins that were observed in adult but not juvenile animals. In addition, the lack of DAF-19 results in impaired dwelling/roaming behavior of the worm. These phenotypes can be rescued by the long isoform DAF-19A and therefore implicate a novel role of DAF-19 in the maintenance of synaptic neurotransmission.
How Do the Different DAF-19 Isoforms Activate Different Groups of Target Genes?
A large number of direct target genes has been identified for the cilia-specific short isoform DAF-19C. All those genes have in common that they 1) are expressed and function in ciliated sensory neurons and 2) contain an x-box promoter motif. Direct target genes of DAF-19A/B in nonciliated neurons currently remain unidentified. Furthermore, DAF-19A is not sufficient to replace DAF-19C in ciliated sensory neurons, indicating that these isoforms activate different target genes. Therefore, what determines the respective functions of the different isoforms?
First, the x-box DNA sequence motifs bound by DAF-19A/B could vary slightly but significantly from the motifs bound by DAF-19C. In C. elegans, the consensus in cilia-specific x-box genes contains a defined spacer of two central nucleotides (Efimenko et al., 2005), whereas the consensus sequence for hRFX has a variable spacer of zero to three nucleotides (Emery et al., 1996; Gajiwala et al., 2000). It is possible that the larger DAF-19A/B also could bind a consensus sequence with no or three spacer nucleotides, like hRFX proteins do. Alternatively, DAF-19A/B could act on x-box motifs in positions different from hitherto proven x-box motifs (i.e., >250 base pairs upstream of the ATG or within introns).
In another scenario, DAF-19–interacting proteins could decide which genes can be transcribed. DAF-19A/B contain an N-terminal part encoded by exons 1-3 lacking in DAF-19C. This N-terminal extension might serve as a site for protein interactions through which isoform-specific binding partners regulate the affinity to synaptic x-box genes instead of cilia x-box genes. Interestingly, RFX genes in all eukaryotes encode proteins of a size similar to the long isoforms DAF-19A/B, having a long N-terminal part upstream of the DNA binding domain. In addition, for some RFX genes, such as daf-19, alternative splicing of different isoforms has been demonstrated (e.g., Zhang et al., 2006). However, the protein part encoded by daf-19 exons 1-3 is not highly conserved at the amino acid level across species. Conservation between RFX proteins of different organisms could thus exist at a structural level. We assume that the N-terminal part of the protein, despite the lack of any assigned conserved domains, is important for the specific function of DAF-19A/B and other RFX proteins. It will thus be essential to characterize the function of the protein domains encoded by exons 1-3.
DAF-19A/B Are Required for Pre- and Postsynaptic Functions in Neurons
We discovered novel daf-19 mutant phenotypes that are caused by the lack of DAF-19A/B and suggest pre- and postsynaptic maintenance defects in neurotransmission. In agreement with these defects, we found that the abundance of several synaptic proteins, especially SNB-1, UNC-17, and UNC-64, was gradually reduced during adulthood. Three characteristics set the synaptic defects of daf-19 apart from all other synapse mutants identified so far: 1) Intriguingly, the decline of synaptic protein levels was most prominently seen in adult worms, whereas larval stages were hardly affected. 2) In neurons both pre- and postsynaptic functions are affected. 3) Because DAF-19A/B are expressed in neurons but not in muscles, it is likely that muscular postsynaptic terminals are intact. The absence of DAF-19 in muscular tissue indicates that the protein does not have a function in muscle cells. This explains why ectopic expression of daf-19 in body wall muscles does not rescue the levamisole-induced paralysis phenotype of daf-19 mutants. The facts listed above also help explain why daf-19 mutants do not have a severe Unc phenotype and are only moderately resistant to paralyzing substances such as aldicarb and levamisole as opposed to the complete resistance seen, for example, in the Unc mutants unc-29, unc-64, or snb-1 (Nonet et al., 1998; Saifee et al., 1998).
Although our paralysis experiments using levamisole revealed deficiencies at postsynaptic terminals in daf-19 mutants, we currently do not know their cause. All postsynaptic proteins checked were unchanged in daf-19 mutants. It is unlikely that the presynaptic effects found induce an indirect postsynaptic defect (resistance to levamisole) through a feedback mechanism. In that case, daf-19 mutants should on levamisole phenocopy other presynaptic mutants, such as snb-1. We therefore hypothesize that in addition to the presynaptic proteins we describe, so far unidentified postsynaptic molecules are also affected by the lack of DAF-19.
Maintaining Synaptic Protein Expression: A Novel Role for DAF-19A/B
Several screens have been performed that used SNB-1::GFP as synaptic vesicle marker (Zhen and Jin, 1999; Schaefer et al., 2000; Zhen et al., 2000; Crump et al., 2001; Shen and Bargmann, 2003). Others investigated genes with predicted roles in synaptic functions (Sieburth et al., 2005), synaptic vesicle recycling and transport (Koushika et al., 2004; Dittman and Kaplan, 2006). These screens uncovered genes required for the localization of SNB-1::GFP at the synapse but not for the maintenance of SNB-1 function. Therefore, daf-19 is the first C. elegans mutant that shows a strong reduction of several synaptic proteins, especially during the later phases of adulthood. This suggests that DAF-19A/B are required for the maintenance of synaptic components rather than for their expression during development.
We identified several synaptic vesicle proteins that are reduced upon loss of daf-19. Two possible scenarios could explain these findings: 1) DAF-19A/B have an influence on synaptic vesicle biogenesis/recycling, or 2) DAF-19A/B regulate a neuronal gene or process that is required for synaptic vesicle protein expression or maintenance. If a general reduction of synaptic vesicles was taking place, one would expect all vesicle proteins to be reduced to similar extents. Although we formally cannot rule out this possibility, the various degrees of reduction between different vesicle proteins (strong reduction of SNB-1 and UNC-64, mild reduction of UNC-17, and no reduction of SNG-1) argue against a general vesicle problem and indicate that these proteins are regulated differentially. Work from mammalian systems supports the notion of individual regulation of synapse components (Shimohama et al., 1998). Furthermore, the increase of synaptic proteins during neuronal development is not due to the increase of the transcriptional rate, but it is regulated at the level of protein stability (Daly and Ziff, 1997). Because most synaptic proteins are highly conserved, it is very likely that also in C. elegans the expression, maintenance, or both of synaptic proteins is individually regulated. We hypothesize that if DAF-19A/B regulate synaptic protein expression, they execute this function indirectly at a posttranscriptional level, because transcript abundance of the corresponding genes in daf-19 mutants were similar to wild type (Figure 8).
Cilia development is an essential process regulated by RFX transcription factors across species. Is it similar with regard to the functional maintenance of synapses? Although brain defects have been reported for Rfx3- and Rfx4_v3-deficient mice (Baas et al., 2006; Zhang et al., 2006), embryonic lethality precluded the analysis of late brain defects. Our analysis of daf-19 mutants suggests that the specific investigation of synapse-related functions of RFX transcription factors in other organisms is relevant to synaptic maintenance.
The C. elegans daf-19 Mutant: A New Disease Model for Functional Synaptic Decline?
Deregulation of synaptic proteins has been described for several neurological diseases, such as Huntington's disease (Morton et al., 2001) or Alzheimer's disease (Sze et al., 2000; Reddy et al., 2005). Research concerning neurodegeneration nowadays increasingly focuses on the loss of synaptic proteins, which is thought to trigger synaptic loss (Selkoe, 2002). The phenotypes seen in daf-19 mutants show parallels to the loss of synaptic proteins described for neurodegenerative diseases. RFX transcription factors as well as the majority of synaptic proteins in C. elegans are highly conserved, which suggests that synaptic protein stability in different organisms may be similarly regulated. Therefore, C. elegans and the daf-19 mutant in particular may in the future prove to be a useful model system to experimentally dissect the mechanisms that maintain synaptic function.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Peter Swoboda (peter.swoboda{at}ki.se).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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