Increased/Reduced Levels of Ataxin-2 Enhance/Suppress Expanded Ataxin-1 Toxicity in the Drosophila Eye
Expression of Ataxin-1[82Q] in the eye of SCA182Q
flies causes external and internal abnormal phenotypes [25
]. Externally, the eyes of these animals show severe ommatidial disorganization as well as interommatidial bristle loss when compared with control eyes (, compare A and A′ with B and B′). Internally, examination of the retina reveals tissue loss and shortened and curved photoreceptor neurons (, compare F with G). In a screen for genetic modifiers of Ataxin-1[82Q]-induced toxicity we recovered EP(3)3145
as an enhancer of the eye phenotype (data not shown). This is an insertion of an EP transposable element [37
] in the 5′ end of dAtx2
, the Drosophila
orthologue of human Ataxin-2. The Drosophila
and human proteins share 23% amino acid identity and 36% amino-acid similarity over the entire protein with the most conserved sequences corresponding to the ATX2-N and ATX2-C domains (43% and 62% identity, respectively) [33
]. Molecular analysis revealed that the EP element is inserted 3121 bp upstream of the ATG and in the same orientation as the dAtx2
transcription unit (data not shown and [33
]). These data suggested that EP(3)3145
over-expresses the dAtx2
transcription unit to enhance the SCA182Q
eye phenotype. As described below, this possibility was confirmed using a transgene that over-expresses the dAtx2
dAtx2 Levels Modulate Ataxin-1[82Q]-Induced Eye Neurotoxicity
Co-expression of a wild-type dAtx2 transgene (dAtx2OE
) at low levels enhances the Ataxin-1[82Q]-induced eye phenotype. Externally, the eyes of SCA182Q/dAtx2OE
animals show no bristles and increased ommatidial disorganization when compared with the eyes of SCA182Q
controls (compare D and D′ with B and B′). Internally, photoreceptor cells are considerably shorter (compare I with G). Expression of the same low levels of dAtx2 alone in the eye causes relatively mild external disorganization and reduction of the retinal width (E, E′ and J). Overexpression of dAtx2 from EP(3)3145
also aggravates the phenotypes of other fly models of neurodegenerative diseases besides SCA1 [38
]. However, since overexpression of dAtx2 causes an eye phenotype by itself (E, E′ and J) and it is toxic in many other tissues [33
], it is difficult to make strong conclusions about the specificity of these genetic interactions.
To test the specificity of the genetic interaction, we investigated if decreasing the levels of endogenous dAtx2 modifies expanded Ataxin-1-induced toxicity. For this, we used a 1.4 kb deletion in the dAtx2
) that removes part of the dAtx2
promoter, the ATG codon and extends into the first intron [33
]. We find that flies expressing Ataxin-1[82Q] and heterozygous for the dAtx2X1
mutant allele show a strong suppression of the eye phenotype, with much improved arrangement of the ommatidia and bristles compared to eyes from flies expressing Ataxin-1[82Q] with normal dAtx2 levels (compare C and C′ with B and B′). This suppression is also evident in the retinas of SCA182Q/dAtx2X1
flies that show elongated photoreceptors and very little tissue loss (compare H with G). To further test the specificity of this interaction, and to exclude potential genetic background artefacts, we asked whether adding back dAtx2 to SCA182Q/dAtx2X1
flies eliminates the suppression effect. Figure S1
shows that SCA182Q/dAtx2X1
flies show an eye phenotype that is very similar to the phenotype of SCA182Q
flies. The effects of the dAtx2X1
alleles decreasing/increasing dAtx2 levels are demonstrated in Figure S2
Since dAtx2 is an RNA binding protein, we investigated if the observed suppression of Ataxin-1[82Q] toxicity was the result of dAtx2 affecting the levels of the SCA182Q mRNA transcript or the levels of Ataxin-1 [82Q] protein. As shown in K and L neither the levels of SCA182Q mRNA nor the levels of the Ataxin-1[82Q] protein are affected by changing the levels of dAtx2.
Reduced Levels of Ataxin-2 Suppress Expanded Ataxin-1-Induced Neuronal Dysfunction in Drosophila
To verify that the genetic interaction between Ataxin-1 and dAtx2 is not limited to the eye, we analyzed the effect of altering dAtx2 levels on expanded Ataxin-1-induced neuronal dysfunction.
The motor performance of flies as a function of age can be quantified using a climbing assay [40
]. This assay has been used to analyze the effects of toxic proteins on neurons in other Drosophila
models of neurodegenerative diseases [41
]. Control flies show no significant decrease in their motor performance until late in life. A shows that ~74% of control flies still climb after thirty-six days (black triangles). Flies expressing Ataxin-1[82Q] specifically in the nervous system (using nrv2-GAL4
) display a progressive impairment of their motor performance (A, blue circles). In the context of the Drosophila
life span, this is a late onset and progressive phenotype as compared to the performance of control flies in the same period of time. We then analyzed the effect of decreased levels of dAtx2 on the climbing phenotype caused by Ataxin-1[82Q]. As shown in A (red squares) in SCA182Q
flies also heterozygous for the dAtx2X1
mutation climbing performance is significantly improved compared to flies with normal dAtx2 levels (p<0.0001 for r
nova (rma) between genotypes). A show that while all SCA182Q
animals fail to climb after 26 days, SCA182Q/ dAtx2X1
flies continue to climb until later in life. Thus the impairments in motor performance caused by neuronal expression of Ataxin-1[82Q] are suppressed by decreased dAtx2
Specificity of the dAtx2/ Ataxin-1 Interaction
We also studied the effect of Ataxin-1[82Q] expression in a life span assay. B shows that expression of Ataxin-1[82Q] in the nervous system leads to premature death in SCA182Q flies in comparison to GFP controls (B, compare blue circles with black triangles). While SCA182Q animals do not survive past 30 days, this early lethality phenotype is suppressed in SCA182Q/ dAtx2X1animals (B, red squares).
Decreased Levels of dAtx2 Do Not Suppress Neurodegeneration Caused by Expanded Huntingtin
To investigate whether dAtx-2 also modulates neurodegeneration in other models of polyglutamine disease, we tested the effect of altering the dAtx2 levels in a Drosophila
model of Huntington's disease [7
]. Adult flies expressing an expanded N-terminal fragment of human huntingtin (N-Htt128Q
) in the eye show a progressive retinal degeneration which becomes obvious at day 5 after eclosion [7
retinas show disorganized and missing photoreceptors (D, compare with control in 2C). N-Htt128Q
flies also overexpressing dAtx2 present a more degenerated retina than flies expressing N-Htt128Q
with normal levels of dAtx2 (data not shown). However, since overexpression of dAtx2 is sufficient to cause retinal degeneration (J), this result is not conclusive by itself. Therefore, we tested the effect of decreasing dAtx2 levels on the N-Htt128Q
induced retinal degeneration. As shown in D-E, decreasing the levels of dAtx2 (N-Htt128Q/dAtx2X1
) does not obviously alter degeneration induced by N-Htt128Q
in the Drosophila
We also investigated a possible genetic interaction between dAtx2 and N-Htt128Q in the motor performance assay described above. Like with Ataxin-1[82Q], expression of N-Htt128Q in the nervous system leads to motor performance impairments (F) where N-Htt128Q animals stop climbing before day 20. The climbing performance of animals expressing N-Htt128Q with decreased levels of dAtx2 (N-Htt128Q/dAtx2X1) is not significantly different from that of animals expressing N-Htt128Q with normal levels of dAtx2 (F). Therefore decreasing the levels of dAtx2 fails to suppress N-Htt128Q induced degeneration both in the retina and in the nervous system.
Ataxin-2 Levels Modulate Expanded Ataxin-1-Induced Loss of Mechanoreceptors Caused by Loss of Senseless Protein
Senseless (Sens) is a proneural factor that is expressed and required in the sensory organ precursor (SOP) cells of the peripheral nervous system [20
]. Expression of high levels of Ataxin-1[82Q] in the thoracic SOPs using scabrous-GAL4
) leads to a reduction in Sens protein levels in these cells and loss of large mechanoreceptors (macrochaetae) in the thorax of adult flies [18
]. Therefore, scoring adult thoracic macrochaetae in SCA182Q
animals provides a quantitative phenotype with a known molecular foundation.
We analyzed the effect of altering Ataxin-2 levels on Ataxin-1[82Q]-induced loss of mechanoreceptors. This was performed by quantifying the number of macrochaetae in the adult thorax of SCA182Q animals with different levels of dAtx2. Using a relatively low expressing Ataxin-1[82Q] line, we defined conditions in which sca-GAL4-mediated expression in the SOP cells causes only 9% of macrochaetae loss (A-column-3 and C, compare to controls A-column-1 and B). Expression of wild-type dAtx2 alone (dAtx2OE) leads to no loss of machrochaetae (A-column-2 and D), but co-expression of dAtx2 and Ataxin-1[82Q] (SCA182Q/dAtx2OE) leads to a severe loss of macrochaetae compared to Ataxin-1[82Q] alone (E, compare with C). Quantification shows an ~80% decrease in the number of macrochaetae when both proteins are co-expressed compared to controls (A-columns 1–4, p<0.0001, Tukey-Kramer HSD).
dAtx2 Levels Modulate Ataxin-1[82Q]-Induced Loss of Mechanoreceptors
In the wing imaginal disc, Sens expression includes the precursors of the large thoracic mechanoreceptor bristles, and the two parallel rows of bristles at each side of the wing margin (F-G). We quantified the amount of fluorescence detected after anti-Sens immunostaining in wing margin cells expressing Ataxin-1[82Q] with normal or increased levels of dAtx2. Expression of either low levels of Ataxin-1[82Q] or dAtx2 with sca-Gal4 (at 25C) produces no detectable decrease in the levels of Sens in the wing margin (H and I compare with G and quantification in K). However co-expression of both Ataxin-1[82Q] and dAtx2 in the same conditions induces a strong decrease in the levels of Sens (J). Quantification of the Sens signal in SCA182Q/dAtx2OE animals (K, fourth column) reveals a decrease of ~50% in the amount of Sens signal when compared to either wild type, SCA182Q or dAtx2OE controls (p<0.001, Tukey-Kramer HSD).
In addition, we investigated the consequences of decreasing the amount of dAtx2 (using the dAtx2X1 allele) in conditions where Ataxin-1[82Q] reduces Sens levels in the wing margin (i.e. at 27°C and 29°C). However, we did not detect a significant modification (data not shown). Since it is possible that we are not able to detect small changes on Sens levels in wing discs from late third instar larvae, or this changes might happen later in the SOP development, we also investigated the effect of decreasing the amount of Ataxin-2 on Ataxin-1[82Q]-induced mechanoreceptor loss. Expression of Ataxin-1[82Q] at high levels (27°C) in the SOP cells results in the loss of ~20% of thoracic macrochaetae when compared to controls (L compare columns 1 and 2 p<0.0001, Tukey-Kramer HSD; see M). Reducing the levels of endogenous dAtx2 with the heterozygous dAtx2X1 mutation partially rescues the Ataxin-1[82Q]-induced bristle phenotype. Loss of macrochaetae in SCA182Q/ dAtx2X1 animals is approximately half of that seen in animals with normal levels Ataxin-2 (L, compare columns 2 and 3 p<0.005, Tukey-Kramer HSD, and see N).
Physical Interaction between Human Expanded Ataxin-1 and Ataxin-2 Proteins
To further characterize the interactions between Ataxin-1 and Ataxin-2, we investigated possible protein-protein interactions. Lysates from cells expressing Drosophila or human Ataxin-2 and GST-Ataxin-1 [82Q] were subjected to co-affinity purification (co-AP) glutathione-S-transferase (GST) pull-down assays. As shown in A (lanes 1 and 2) and B (lanes 1 and 4), GST-Ataxin-1[82Q] is able to pull down the Drosophila and human Ataxin-2 proteins, which indicates that both proteins are able to physically interact. We also asked whether this interaction is polyglutamine dependent. As shown in B lanes 2–4 we did not detect significant differences in the interactions between GST-Ataxin-1 with 2, 30 or 82 glutamines and human Ataxin-2 in this co-AP assay.
The Ataxin-1[82Q] and Ataxin-2 Proteins Physically Interact
Ataxin-1[82Q] is phosphorylated at Serine residue 776 on the C-terminal portion of the protein, and a (Ser776Ala) mutation inhibits the toxicity of Ataxin-1[82Q] in mice[17
]. We investigated the importance of Ser776 for the Ataxin-1[82Q]-dAtx2 interaction. As shown in (A lane 3) the interaction of dAtx2 with Ataxin-1[82Q]S776A
is weaker than with normal Ataxin-1[82Q], suggesting that this interaction is phosphorylation dependent.
In addition, we investigated if Ataxin-1 can pull down endogenous hATX2. We find that unexpanded Ataxin-1 is able to precipitate endogenous hATX2 from human cells, suggesting that the two proteins may be functional interactors in vivo (C).
We also investigated whether the interaction between Ataxin-1 and Ataxin-2 is cytoplasmic or nuclear. We carried out co-AP assays with Ataxin-1 and dAtx2 after nuclear/cytoplasmic fractionation of cultured cells. D shows that we were not able to detect differences in protein interactions between these cellular compartments using this assay.
Lastly, we investigated whether specific domains of the hAtaxin-1 protein are responsible for the interaction with hAtaxin-2. Co-AP experiments were carried out with lysates from cells expressing Myc-hAtaxin-2 and one of the following hAtaxin-1 fragments tagged with GST: polyglutamine expanded N-terminal (aa# 1–575, E lane-3), C-terminus Ataxin-1 containing the AXH domain (aa# 529–816; E lane-4) or the AXH domain alone (aa# 558–700; E lane-5). All three fragments pull-down Myc-hAtaxin-2, indicating that each Ataxin-1 fragment can interact independently with hAtaxin-2 (E lanes 3–5). However, the interaction of hAtaxin-2 is stronger with the N-terminal Ataxin-1 fragment (E, lane-3) as compared to the C-terminal or AXH portions (E lanes 4 and 5 respectively), since less N-terminal peptide pulls down more hAtaxin-2.
Ataxin-2 Accumulates in the Nucleus of Expanded Ataxin-1-Expressing Drosophila Cells and Human Neurons
The co-AP assays with expanded hAtaxin-1 and hAtaxin-2 indicate that the two proteins are able to interact. However, Ataxin-1 normally localizes to the nucleus in Drosophila
and many human cell types, while Ataxin-2 is a cytoplasmic protein. To address whether the interaction observed in cultured cells is relevant in vivo, we monitored the localization of Ataxin-2 in Drosophila
cells expressing Ataxin-1[82Q]. Since Ataxin-1[82Q]-induced toxicity is suppressed by dAtx2
loss of function in the Drosophila
eye; we first analyzed the localization of dAtx2 in retinal cells. dAtx2 is not normally detected in the nuclei of retinal cells from either control eyes or eyes overexpressing dAtx2 (A and C respectively). In contrast, we find that endogenous dAtx2 localizes to the nuclei of retinal cells expressing Ataxin-1[82Q]. Furthermore, nuclear dAtx2 signal is detected both diffusely in the nucleoplasm as well as in nuclear inclusions (NIs) (B). To confirm this unexpected result, we examined the localization of dAtx2 in other Ataxin-1[82Q]-expressing neurons. Similar to the results obtained in the retina, endogenous dAtx2 normally localizes to the cytoplasm and is not detected in the nuclei of neurons from the v
ord ( E-E′′, ok107-GAL4
pattern shown in D). However, Ataxin-1[82Q]-expressing VNC neurons show nuclear accumulation of dAtx2 ( F-F′′). Next we investigated whether dAtx2 and Ataxin-1 colocalize. Co-staining of Ataxin-1 and dAtx2 is not possible since the available antibodies for both proteins were raised in rabbit; however, Ataxin-1 NIs in Drosophila
neurons are positive for Ubiquitin [25
], so we performed a double staining for dAtx2 and Ubiquitin. G-G′′ shows that the dAtx2 NIs present in the neurons of SCA182Q
flies are positive for Ubiquitin. These results indicate that expanded Ataxin-1 causes dAtx2 to localize to the nucleus and suggest that both proteins co-aggregate in NIs.
Expanded Ataxin-1 Induces Nuclear Accumulation of Ataxin-2 in Drosophila and Human Neurons
To validate these results and investigate their relevance for SCA1 pathogenesis, we analyzed the localization of hAtaxin-2 in SCA1 neurons from human postmortem brain samples by anti-Ataxin-2 immunohistochemistry. Pontine neurons from control samples consistently show cytoplasmic localization of hAtaxin-2 (H). Pontine neurons from SCA1 brain samples display frequent Ataxin-1 NIs that are clearly visible with Hematoxylin staining. We found that approximately twenty percent of these NIs are positive for hAtaxin-2 (I); see also[10
]. These findings, together with the co-AP data using human Ataxin-1 and Ataxin-2 proteins, suggest that the Ataxin-1-Ataxin-2 interactions observed in Drosophila
may be relevant for SCA1 pathology.
Nuclear Accumulation of Ataxin-2 Is Induced by Pathogenic Ataxin-1[82Q], but Not Unexpanded Ataxin-1[2Q] and Ataxin-1[30Q]
SCA1 pathogenesis is triggered by polyglutamine expansion in Ataxin-1 beyond 39–44 residues [5
]. To investigate if nuclear accumulation of Ataxin-2 is specific to the pathogenic Ataxin-1 form, we analyzed dAtx2 localization in Drosophila
VNC neurons expressing human Ataxin-1 with different polyglutamine lengths: Ataxin-1[2Q], Ataxin-1[30Q] and Ataxin-1[82Q]. The Ataxin-1[2Q] line used has higher levels of protein expression than the Ataxin-1[30Q] and [82Q] lines, both of which have comparable expression levels (Western blot data not shown). A-D shows that no nuclear dAtx2 signal is detected in control neurons (A), or neurons expressing Ataxin-1[2Q] or [30Q] (B and C). In contrast, accumulation of dAtx2 is detected in the nucleus of Ataxin-1[82Q]-expressing neurons (D). Although this result does not rule out an interaction between wild-type Ataxin-1 and dAtx2, it indicates that abundant nuclear accumulation of Ataxin-2 is specific to the expanded Ataxin-1 form responsible for SCA1 pathogenesis.
Pathogenic Ataxin-1[82Q], but Not Unexpanded Ataxin-1, Induces Nuclear Accumulation of dAtx2 In Vivo
Nuclear Accumulation of Ataxin-2 Causes Severe Eye Toxicity
The observations that expanded Ataxin-1 induces nuclear accumulation of Ataxin-2 and that decreasing the levels of endogenous Ataxin-2 suppresses toxicity in SCA182Q flies, suggest that nuclear accumulation of Ataxin-2 may lead to neurotoxicity. To test this hypothesis, we generated dAtx2 transgenic flies with an exogenous nuclear localization signal (NLS) engineered on its C-terminus end (dAtx2NLS) ( compare A-A′ with C). Although wild-type Ataxin-2 is only detected in the cytoplasm in Drosophila and human neurons, it is difficult to rule out the possibility that some Ataxin-2 may be present in the nucleus. Therefore, we also generated flies carrying a dAtx2 construct with an exogenous nuclear export signal (NES) (dAtx2NES) to use as an additional control (B-B′). Ten transgenic lines were recovered for each dAtx2 construct, with a wide range of expression levels for each transgene. We selected transgenic lines expressing wild-type dAtx2 (dAtx2OE), dAtx2NES or dAtx2NLS at similar levels (D compare lanes 2–4), and compared their toxicity in the eye. As shown in E-H, dAtx2NLS is more toxic than wild-type dAtx2 or dAtx2NES. While both wild-type dAtx2 and dAtx2NES induce a relatively mild eye phenotype compared to controls ( E-G), expression of dAtx2NLS results in strong eye toxicity (H). Eyes of dAtx2NLS flies show a severely disorganized ommatidial lattice and a complete absence of interommatidial bristles (H). These observations were consistent in several lines for each of the dAtx2 constructs at similar levels of expression (data not shown).
Nuclear Accumulation of dAtx2 Increases Its Toxicity In Vivo
In summary, expression of dAtx2NES
and wild-type dAtx2 in the eye cause similar mild phenotypes. This is consistent with dAtx2 being mainly cytoplasmic, and with observations of SCA2 pathogenesis in the cytoplasm[44
]. Interestingly, increasing the levels of Ataxin-2 in the nucleus is sufficient to cause a much more severe eye phenotype. These observations suggest that the toxicity of expanded Ataxin-1 is mediated in part, by the nuclear accumulation of Ataxin-2.
Nuclear Ataxin-2 Decreases Sens Protein Levels and Induces Loss of Mechanoreceptor Bristles
To further test the hypothesis that nuclear Ataxin-2 contributes to expanded Ataxin-1-induced toxicity, we analyzed the effect of expressing the different dAtx2 transgenes on Sens distribution in the wing margin SOPs. Expression of Ataxin-1[82Q] in the antero-posterior compartment boundary (using dpp-GAL4) induces a cell autonomous decrease of Sens only in the dpp-GAL4 expressing area of the wing margin (A, A′ and 8B, B′, arrowhead). This provides a molecular readout of Ataxin-1[82Q]-induced neurotoxicity.
Nuclear dAtx2 Induces Decreased Levels of Senseless (Sens) Protein and Loss of Mechanoreceptors
Expression of dAtx2NES
does not reduce the levels of Sens, whose distribution in imaginal wing discs is unchanged (C and C′, arrowhead). Next, we tested the effect of dAtx2 with a nuclear localization signal (dAtx2NLS
). Like Ataxin-1[82Q], expression of dAtx2NLS
induces loss of Sens in the wing margin SOPs (D and D′, arrowhead). dAtx2NLS
-induced loss of Sens is also observed in other cell types. Salivary gland cells express Sens at high levels [20
], which localizes to the nucleus (E-H). Salivary gland cells expressing dAtx2NES
show no detectable change in Sens levels or distribution (I-L). In contrast, expression of dAtx2NLS
causes a dramatic decrease in the amount of Sens (M-P). The nuclei of dAtx2NLS
cells are still present and their morphology is similar to controls, indicating that Sens loss is unlikely a consequence of cell death. Therefore, nuclear dAtx2 mimics Ataxin-1[82Q] in causing loss of Sens protein accumulation.
Expression of Ataxin-1 in the thoracic SOPs (using sca-GAL4
) leads to loss of macrochaetae in the adult thorax ( and ref.[18
]). Therefore, we also investigated the consequences of nuclear or cytoplasmic Ataxin-2 accumulation on the development of adult macrochaetae. As with wild-type dAtx2 (), expression of dAtx2NES
in the thoracic SOP cells causes no visible change in the number of macrochaetae in the adult thorax (Q and S column-1). In contrast, expression of dAtx2NLS
induces a significant decrease in the number of macrochaetae, with an approximate twenty percent reduction in comparison to control animals (R and S column-2). These results indicate that increasing the levels of nuclear dAtx2 mimics expanded Ataxin-1 in inducing loss of mechanoreceptors and reducing the levels of Sens protein.