The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span (original) (raw)
Mutants of Caz, the Drosophila homolog of FUS, have defective development. Drosophila has a single FUS homolog with 53% amino acid identity to human FUS (hFUS) (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI57883DS1) encoded by the cabeza (caz) gene on the X chromosome, which previously identified as an RRM domain–encoding gene expressed in neurons (17). We generated a transgenic Drosophila Caz construct, Caz genomic, that included 5′ sequence including the predicted caz promoter, full coding region, and 3′ sequences. Into this construct, we additionally inserted a Flag epitope in frame in the _caz_-coding region. With this transgene, we found that Caz protein was expressed both in neurons (Figure 1A) and nonneuronal cells including glia and muscles (data not shown). Caz protein was localized in the nucleus in both neurons and other cell types (Figure 1B and data not shown) and had a homogenous distribution throughout the nucleus similar to the neuronal RNA-binding protein Elav (Figure 1B). In order to generate a caz mutant, we mobilized an existing transposable element (EP1564) inserted near caz to generate a small deletion, Df[1]383, that removed 58% of the caz gene in addition to 5′ sequence including the promoter (Figure 1C). This small deletion also disrupted a nearby gene (CG32576), so we generated a rescuing genomic transgene for CG32576 and inserted this on the Df[1]383 chromosome. This combination of Df[1]383 and CG32576 rescuing transgene we named caz1 (Figure 1C).
Characterization of Cabeza, the Drosophila homolog of human FUS/TLS. (A) Expression pattern of a caz transgene under the control of the endogenous promoter in the adult brain detected using a FLAG epitope introduced immediately after the start codon. (B) Larval neuronal nuclei expressing genomic Caz detected with FLAG alone (upper panel) or colabeled with the neuronal RNA binding protein Elav. (C) Schematic of caz1 mutant construction. The transposon EP1564 was mobilized to create a small deletion Df[1]383 that removes 58% of the caz gene, caz promoter sequences, and disrupts the nearby gene CG32576. A rescuing transgene for CG32576 was inserted onto the Df[1]383 chromosome to create caz1 mutants. (D) Percentage of male larva of the indicated genotypes that eclosed to produce adults (n > 100). Pan-neuronal expression of Caz, human FUS, or ALS mutant FUS (FUSR522G and FUSP525L) transgenes rescue eclosion equally (genotype: caz1, C155-Gal4/Y; UAS transgene). (E) Representative image of 10 superimposed paths of 60 seconds of adult locomotion for control (precise excision) of 1-day-old adult male flies. (F) Representative image of 10 superimposed paths of 60 seconds of adult locomotion of caz1 mutant 1-day-old adult male flies. (G) Walking speed of 1-day-old adult male flies of the indicated genotypes in a 60-second trial (n > 30). (H) Percentage survival of adult male flies of the indicated genotypes (n > 68). Error bars represent SEM. Scale bars: 100 μm (A); 5 μm (B). *P < 0.05; ***P < 0.001.
caz1 mutant male larva appeared morphologically normal; however, only 14.0% (P < 0.001) successfully completed pupation and eclosed to produce viable adults compared with controls (Figure 1D). This defect was fully rescued by the Caz genomic transgene. We then used the GAL4 system (18) to drive expression of a UAS Caz cDNA transgene in neurons alone. When Caz was restored only in the nervous system by neuronal (C155-GAL4) expression of transgenic UAS Caz, adult viability was also restored to 83.7% (P < 0.001) of control levels, suggesting Caz is predominately but not exclusively required in the nervous system for normal adult eclosion (Figure 1D). We next tested the ability of human FUS to rescue caz mutants by generating a UAS human FUS transgene. Transgenic hFUS was localized in the nucleus of Drosophila motor neurons similar to Caz protein (Supplemental Figure 1B). Expression of hFUS in the nervous system of caz mutants restored adult viability to levels similar to those seen with the expression of Caz, indicating conservation of protein function (Figure 1D). We also generated hFUSR522G and hFUSP525L transgenes based on fALS mutations (6). Using phiC31 transgenesis (19), mutant hFUS transgenes were inserted into the same locus as the wild-type hFUS transgene, and we confirmed they were expressed at identical levels (Supplemental Figure 1C). Both mutant hFUS proteins also localized to the nucleus of Drosophila motor neurons (Supplemental Figure 1B). Expression of hFUSR522G or hFUSP525L in caz mutants rescued eclosion to a degree similar to that seen with the expression of Caz, suggesting these mutations do not disrupt the activities of hFUS required to restore Drosophila eclosion (Figure 1D).
caz mutant adults have reduced locomotion and longevity that is not rescued by fALS FUS. We next examined caz1 mutant males that survived to adulthood. Externally, these mutants had a mild rough eye defect, abnormal genitalia, and defects in both bristles and wing vein organization, all of which were rescued by the caz genomic transgene (data not shown). Prominently, they also exhibited defects in locomotion. The majority of caz mutants were unable to fly, and compared with controls, they walked slowly, fell over frequently, and had difficulty righting themselves (see Supplemental Videos 1 and 2). We quantified this defect with 2 assays. First, we tested climbing ability using a negative geotaxis assay. In this paradigm, caz mutants had a 55.9% decrease (P < 0.001) in climbing ability compared with controls, which was fully rescued by the Caz genomic transgene (Supplemental Figure 2A). Second, we used quantitative videotracking to measure the locomotor speed of caz mutants and controls (Figure 1, E and G) and observed a 67.6% relative reduction (P < 0.001) in locomotor speed in caz mutants that was also fully rescued by the Caz genomic transgene (Figure 1G). When we restored Caz only in the neurons of caz mutants we could increase locomotor speed by 82% (P < 0.001) over caz mutants alone (Figure 1G), but these animals were still significantly slower than controls or animals rescued with genomic Caz, suggesting nonneuronal expression of Caz also contributes to locomotor speed. We next tested to determine whether hFUS could rescue caz mutant locomotion. Expression of wild-type hFUS in caz mutant neurons increased locomotor speed by 75% (P < 0.001) and was not significantly different from rescue with transgenic Caz (Figure 1G). In contrast, however, expression of hFUSR522G in caz mutant neurons showed no significant improvement of the locomotor speed of caz mutants (Figure 1G). Expression of hFUSP525L did increase locomotor speed of caz mutants significantly, but these animals were still 23.6% (P < 0.05) slower than mutants rescued with wild-type hFUS (Figure 1G). Overexpression of wild-type or mutant hFUS in the neurons of control animals had no significant effect on locomotion (Supplemental Figure 2B). These results reveal that, unlike the requirements for FUS during eclosion, fALS mutant isoforms of human FUS are defective in an activity that is essential for normal locomotion in Drosophila.
We further observed that caz mutant males had a shorter life span than control male flies (Figure 1H). The average life span of control males was 53 days, whereas caz mutants lived an average of 23 days, a 57% decrease (P < 0.001). The Caz genomic transgene fully restored the average life span of caz mutants to control levels (Supplemental Figure 2C), as did neuronal expression of transgenic Caz or hFUS (Figure 1H). In contrast, expression of hFUSR522G or hFUSP525L in caz mutants did not significantly restore median life span, although maximum life span was increased (Figure 1H). Overexpression of Caz or hFUS transgenes in control animals had no effect on longevity (Supplemental Figure 2D). Therefore, as was found for locomotion, fALS mutant FUS proteins were deficient in an activity required for longevity in Drosophila. We examined the brain tissue of 25-day-old caz mutants and controls for evidence of neuronal loss (Supplemental Figure 2E) and did not observe vacuolization or other evidence of extensive neuronal death. Interestingly, overexpression of fALS mutant human SOD1 in Drosophila can also inhibit locomotion without inducing neuronal loss (20).
tbph mutants have phenotypes similar to those of caz mutants. The Drosophila homolog of TDP-43, TBPH, is only expressed in neurons (15, 16), and protein-null mutants of tbph can survive to adulthood (ref. 21 and Supplemental Figure 3G). We examined the phenotype of adult transallelic null mutants of tbph to compare them with caz mutants. Unlike in caz mutants, we did not observe defects in eye, wing, or genital development in adult tbph mutants. When we examined the eclosion rate of tbph mutants, we found that only 19.4% (P < 0.001) of tpbh mutants survived to adulthood compared with controls (Figure 2A). This defect was rescued completely by expression of either transgenic Drosophila TPBH or human TDP-43 (hTDP-43) in the neurons of tbph mutants. We also examined adult locomotor speed in adult tbph mutants (Figure 2, B and C). Compared with controls, tbph mutants had a dramatic 88.4% (P < 0.001) reduction of locomotor speed that was also fully rescued by neuronal expression of TBPH or hTDP-43. Finally, we examined the longevity of these mutants (Figure 2D). We found that the marked reduction in the survival of tbph mutants to an average of 4 days (P < 0.001) was also restored to control levels by neuronal expression of TBPH or hTDP-43. Therefore, consistent with previous studies (21), we find that tbph mutants, in comparison with caz mutants, have similar, though quantitatively more severe, eclosion, adult locomotion, and longevity phenotypes.
caz and tbph are members of a genetic pathway. (A) Percentage of male larva of the indicated genotypes that eclosed to produce adults (n > 100). The _tbph_–/– genotype is _tbph_∆23/Df[2R]BSC660. + indicates neuronal expression of UAS-TBPH, UAS-TDP-43, or UAS-Caz with C155-Gal4. (B) Representative images of 10 superimposed paths of 60 seconds of adult locomotion of 1-day-old adult male flies of control, tbph mutant, or caz1 mutants either alone or expressing UAS-Caz or UAS-TBPH. (C) Walking speed of 1-day-old adult male flies of the indicated genotypes in a 60-second trial (n > 30). (D) Percentage survival of adult male flies of the indicated genotypes (n > 100). Error bars represent SEM. ***P < 0.001.
caz and tbph are components of a common genetic pathway in neurons. To determine whether a genetic relationship existed between caz and tbph, we attempted to cross-rescue tbph or caz mutants by overexpression of the other gene. First, we expressed transgenic TPBH in the nervous system of caz mutants and examined eclosion rate, adult locomotor velocity, and longevity. Expression of TBPH in caz mutants resulted in no alteration in any of these measures when compared with caz mutants alone (Figure 2, A–D). We next overexpressed Caz in tbph mutants. Surprisingly, overexpression of Caz in tbph mutants restored their eclosion frequency to levels not significantly different from those achieved by expression of TBPH (Figure 2A). Caz overexpression in tpbh mutants also increased adult locomotion velocity by 229.8% (P < 0.001) compared with tbph mutants alone, although these animals were still significantly slower than mutants rescued with TBPH (Figure 2, B and C). Most dramatically, Caz overexpression had a dramatic effect on tbph mutant longevity, increasing the mean life span of tbph mutants 13-fold (P < 0.001) to levels not significantly different from animals rescued with TBPH expression (Figure 2D). These data indicated that caz and tbph might function in a common genetic pathway, necessary for neuronal development, locomotion, and longevity, in which Caz was epistatic to TBPH.
To test this model further, we searched for gain-of-function phenotypes for Caz and TBPH. Mutants of tbph have been reported to have defects in larval neuromuscular junction (NMJ) synapse morphology (21); however, we were unable to replicate this finding in transallelic combinations of tbph (Figure 3, K and M). In contrast, however, when TBPH or TDP-43 were overexpressed in motor neurons, we did observe a dramatic expansion of NMJ terminal size with a 68.4% and 65.5% (P < 0.001) increase in the number of synaptic boutons (Figure 3, B, C, and M), respectively. We observed no change when we overexpressed a fALS mutant, TDP-43M337V, at identical levels (Figure 3, D and M, and Supplemental Figure 3H). Similarly, caz1 mutants had normal NMJ morphology (Figure 3, I and M); however, overexpression of wild-type Caz or hFUS did induce an expansion of NMJ size with a 35.3% or 35.2% (P < 0.001) increase in synaptic bouton numbers, respectively (Figure 3, E, F, and M). In contrast, overexpression of hFUSR522G or hFUSP525L did not significantly increase NMJ terminal size (Figure 3, G, H, and M). Thus, NMJ terminal expansion is a common phenotype induced by overexpression of both Caz/hFUS and TBPH/hTDP-43 in motor neurons that is inhibited by fALS mutations. We used this assay to further test the genetic interaction between caz and tbph. We first overexpressed TBPH in the motor neurons of caz mutants. When we did this, the NMJ expansion induced by TBPH overexpression was completely suppressed to control levels (Figure 3, I and M), suggesting that TBPH requires the presence of Caz to induce NMJ terminal expansion. In contrast, when we overexpressed Caz in a tbph mutant, it induced a level of NMJ terminal expansion similar to that observed when Caz was overexpressed in a control background (Figure 3, J and M). This indicates that Caz does not require TBPH to induce NMJ expansion and further supports a model in which Caz functions in a genetic pathway downstream of TBPH.
Caz is epistatic to tbph. (A–L) Third instar NMJ terminals stained with anti-CSP (green) to label the presynapse and anti-HRP (red) to label the neuronal membrane at muscle 4, segment A3 for motor neuron overexpressing (OE) transgenic Caz, FUS, TBPH, TDP-43, and FUS or TDP-43 ALS mutants in wild-type (B–H) or tbph and caz1 mutants (J and L) driven in motor neurons by OK6-Gal4. The _tbph_–/– genotype is tbph∆23/Df[2R]BSC660. Overexpression of wild-type TBPH, TDP-43, Caz, or FUS proteins induces NMJ expansion while ALS mutant FUS or TDP-43M337V does not (B–H). Mutants of caz or tbph have normal NMJ morphology (I and K). The NMJ expansion induced by expression of wild-type TDP-43 is completely suppressed in caz mutants (J); however, the NMJ expansion induced by Caz overexpression is not suppressed in tbph mutants (L). (M) Quantification of synapse terminal bouton number divided by muscle surface area for muscle 4 segment A3 normalized to control. Error bars represent SEM. ***P < 0.001.
Caz and Tbph proteins interact in neurons. Human FUS and TDP-43 proteins have been reported to physically interact in mammalian cell culture (12, 14, 22), so we next determined whether this interaction also occurred in Drosophila neurons. We generated a YFP-tagged TBPH transgene and used it to test for interaction with Flag-tagged Caz in extracts from Drosophila adult brains. We found that when Caz was pulled down, TPBH was coimmunoprecipitated, confirming this protein interaction also occurred in Drosophila neurons (Figure 4A). We next determined whether this interaction was dependent upon RNA by treating brain extracts with RNase prior to coimmunoprecipitation. We found that this dramatically reduced the coimmunoprecipitation of TBPH by Caz, indicating that the interaction of Caz with TBPH is at least partially dependent upon RNA (Figure 4A). We next asked whether Caz and TBPH are required to regulate the expression or stability of each other. To do this, we examined the protein levels of TBPH in caz mutants and vice versa. We found no difference in the level of TBPH in caz mutant brain extracts using an anti-TBPH antibody (Figure 4B). Similarly, when we introduced the genomic flag-tagged Caz transgene into tbph mutants, we also saw no change in Caz protein levels (Figure 4B). Therefore, while Caz and TPBH proteins can interact with each other, neither gene is required to maintain normal levels of the other protein.
Caz and TBPH proteins interact. (A) YFP-tagged TBPH expressed in Drosophila adult brains with C155-Gal4 coimmunoprecipitates when flag-tagged Caz is used for pulldown. Treatment of brain extracts with RNaseA inhibits this interaction. (B) Endogenous TBPH protein levels were not changed in 1-day-old caz mutant males compared with precise excision controls, and genomic flag-Caz protein is similar in 1-day-old tbph mutants compared with controls. (C) 4 of the 8 C-terminal amino acids of human FUS and Drosophila Caz are identical (green), and 2 (red) were mutated in Caz cDNA transgenes. (D) Drosophila motor neuron cell bodies expressing UAS-Caz, UAS-CazR395G, and UAS-CazP398L (green), histone-YFP (red), and cytoplasmic β-galactosidase (blue) driven by the motor neuron driver OK319-Gal4 or single channel images of Caz or Caz mutants (gray). Unlike wild-type Caz, both Caz C-terminal mutants are found extensively in the cytoplasm. (E) YFP-TBPH coimmunoprecipitates when flag-tagged CazR395G or CazP398L is used for pulldown from adult brain extracts.
The C terminus of FUS has been shown to act as a noncanonical nuclear localization sequence (9). To determine whether homologous amino acids are required for nucleus localization in Caz, we generated UAS Caz transgenes with point mutations that change the conserved C-terminal amino acid arginine 395 to glutamate (CazR395G) or proline 398 to leucine (CazP398L) (Figure 4C). These transgenes were inserted in the same locus as wild-type Caz, and expression levels of these mutant transgenes were similar (Supplemental Figure 3I). Like wild-type Caz, overexpression of either mutant protein in neurons did not alter adult eclosion frequency or locomotor velocity (data not shown). However, in contrast to wild-type Caz, both mutant Caz proteins were found to be distributed extensively throughout the cytoplasm in addition to the nucleus when expressed in motor neurons (Figure 4D). Thus, in context of Caz, these C-terminal amino acids are also required to correctly localize the protein only to the nucleus. We then asked whether these mutant Caz proteins could still biochemically interact with TBPH. We found that TBPH could be coimmunoprecipitated by pull-down of either CazR395G and CazP398L, indicating that these proteins retained their ability to interact with TBPH (Figure 4E).