InteractiveFly: GeneBrief

Ataxin-2: Biological Overview | References

BIOLOGICAL OVERVIEW

Spinocerebellar ataxia type 2 (SCA-2) is an autosomal dominantly inherited neurodegenerative disease caused by trinucleotide (CAG) expansion in the ATXN2 gene resulting in the lengthening of polyglutamine stretch in the encoded protein ataxin-2 (Lastres-Becker, 2008). Ataxin-2 is a large conserved protein that carries an Sm-domain and a PAM-2 motif on the C-terminal side of the Sm-domain. The PAM-2 motif mediates interaction with the C-terminal helical domain of the poly(A) binding protein (PABP; see Drosophila pAbp) and is present in several PABP-interacting proteins (Albrecht, 2004). Levels of mutant ataxin-2 (with expanded polyglutamine stretch) are higher in brain tissue of patients with SCA-2 compared with that of wild-type ataxin-2 in normal individuals (Huynh, 1999; Koyano, 1999). Further, abnormal expression of Ataxin-2 has been shown to be deleterious in Drosophila (Satterfield, 2002). However, the biological function of ataxin-2 and the mechanism by which lengthening of the polyglutamine stretch in ataxin-2 leads to the disease are not clear (Tharun, 2008 and references therein).

Several studies implicate ataxin-2 in mRNA decay and regulation of translation suggesting that deregulation of these processes could be related to the disease. Ataxin-2 homologs from multiple organisms have been shown to interact with PABP consistent with the presence of PAM-2 motif in Ataxin-2 (Albrecht, 2004; Ciosk, 2004; Ralser, 2005; Satterfield, 2006). Depletion of Ataxin-2 homolog in C. elegans affects germline development and this seems to be due to the deregulation of translational repression by GLD-1 and MEX-3 of their mRNA targets (Ciosk, 2004). Reducing the expression of ATXN2 in mammalian cells using siRNAs impairs the formation of stress granules which are the sites where untranslated mRNAs are localized during stress (Nonhoff, 2007). Finally, both in human cells and in Drosophila, Ataxin-2 is associated with the polysomes (Satterfield, 2006). These observations suggest that Ataxin-2 may have a role in translational repression in vivo. Interestingly, enhancement and suppression of Ataxin-2 expression in human cells leads to decrease and increase in the levels of PABP, respectively, without affecting the levels of PABP mRNA (Nonhoff, 2007). Given that PABP is a key translation factor, it remains to be seen if the effects on translation caused by alterations in Ataxin-2 expression are related to the changes in the levels of PABP. Ataxin-2 overexpression also leads to decrease in the number of P-bodies in human cells. This seems to be related to the ability of Ataxin-2 to interact with Dhh1/RCK/p54, which is one of the decay factors important for P-body assembly in human cells (Andrei, 2005; Nonhoff, 2007). Importantly, reduction in P-bodies was caused by overexpression of Ataxin-2 irrespective of whether the polyglutamine stretch in Ataxin-2 was normal or long (Nonhoff, 2007). These observations together suggest that deregulation of mRNA decay and/or translational repression resulting from abnormal expression of Ataxin-2 may be one of the reasons for the disease phenotype and the expanded polyglutamine stretch could contribute to increased expression of the mutant Ataxin-2 in the diseased individuals (Tharun, 2008 and references therein).

Studies in Drosophila suggest that Ataxin-2 is required for microRNA function and synapse-specific long-term olfactory habituation. Local control of mRNA translation has been proposed as a mechanism for regulating synapse-specific plasticity associated with long-term memory. Glomerulus-selective plasticity of Drosophila multiglomerular local interneurons observed during long-term olfactory habituation (LTH) requires the Ataxin-2 protein (Atx2) to function in uniglomerular projection neurons (PNs) postsynaptic to local interneurons (LNs). PN-selective knockdown of Atx2 selectively blocks LTH to odorants to which the PN responds and in addition selectively blocks LTH-associated structural and functional plasticity in odorant-responsive glomeruli. Atx2 has been shown previously to bind DEAD box helicases of the Me31B family, proteins associated with Argonaute (Ago) and microRNA (miRNA) function. Robust transdominant interactions of atx2 with me31B and ago1 indicate that Atx2 functions with miRNA-pathway components for LTH and associated synaptic plasticity. Further direct experiments show that Atx2 is required for miRNA-mediated repression of several translational reporters in vivo. Together, these observations (1) show that Atx2 and miRNA components regulate synapse-specific long-term plasticity in vivo; (2) identify Atx2 as a component of the miRNA pathway; and (3) provide insight into the biological function of Atx2 that is of potential relevance to spinocerebellar ataxia and neurodegenerative disease (McCann, 2011).

In the mammalian brain, single neurons form up to 100,000 different synapses whose weights may be regulated independently during learning. In principle, the synapse-specificity of short-term plasticity may be explained simply by the restriction of signaling events to active synapses. However, synapse-specific long-term plasticity, which depends on products of nuclear gene expression that would be available in a cell-wide manner, clearly depends on distinct synaptic tags that mark only active synapses (McCann, 2011).

Several lines of evidence suggest that activity-regulated local translation of synaptic mRNAs normally stored in a repressed state contributes to the synapse-specificity of long-term plasticity (Richter, 2009; Martin, 2002). Consistent with this idea, several translational control molecules, such as fragile X mental retardation 1 protein, Staufen, cytoplasmic polyadenylation element binding protein/Orb2, and the Gld2 polyA polymerase, are required in Drosophila for long-term but not short-term memory. In the context of identified synapses, local protein synthesis is required for cAMP response element-binding protein (CREB)-dependent, synapse-specific long-term plasticity in cultured Aplysia sensorimotor synapses: In this system, postsynaptic translation may trigger a retrograde signal, which in turn stimulates local translation at presynaptic terminals. However, in vivo, the requirement for and regulation of local protein synthesis at synapses remains poorly understood, in part because of the paucity of preparations in which behavioral learning arises from plasticity within a defined, experimentally convenient, neural circuit (McCann, 2011).

Recent work has shown that long-term olfactory habituation (LTH), a phenomenon in which sustained exposure to an odorant results in a decreased behavioral response, arises through plasticity of synapses between local interneurons (LNs) and projection neurons (PNs) in the Drosophila antennal lobe (Das, 2011; Sachse, 2007). Although LTH requires the transcription factor CREB2 to function (globally) in a multiglomerular class of LNs, LTH is odorant selective and associated with glomerulus-selective (and hence local) structural and physiological plasticity. In screening candidate RNA-binding proteins for potential roles in PNs during LTH, Ataxin-2 (Atx2), a molecule of considerable interest for its known involvement in the human neurodegenerative disease spinocerebellar ataxia-2 (SCA2), was identified. Expansion of a polyglutamine tract in human Atx2 from about 22 (normal) to >32 (pathogenic) glutamines causes degeneration of cerebellar Purkinje cells (Lastres-Becker, 2008). While Atx2 has been implicated in many different biological functions (Lastres-Becker 2008; Satterfield, 2002), it is generally believed to function in RNA regulation (Nonhoff, 2007; Satterfield, 2006; Ciosk, 2004; Buchan, 2008). Evidence for this role comes from biochemical and cell biological studies of the protein or its evolutionarily conserved orthologs in Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila melanogaster (McCann, 2011).

In C. elegans, Atx2 is required in postembryonic germline cells for appropriate translational control of GLD-1- and MEX-3-target mRNAs (Ciosk, 2004). Atx2 binds the RNA regulatory proteins, polyA-binding protein (PABP) and Me31B/RCK/ Dhh1p/CGH-1, through domains also required for its observed assembly with polyribosomes (Nonhoff, 2007; Satterfield, 2006; Ciosk, 2004). At a cell biological level, Atx2 function has been shown to regulate the assembly of P-bodies and stress granules, distinct cytoplasmic messenger ribonucleoprotein particles that contain translationally repressed mRNAs, together with the translational repressor Me31B/RCK/Dhh1p (McCann, 2011 and references therein).

Significantly, both the proteins and cytoplasmic structures with which Atx2 associates have been linked to translation repression by microRNAs (miRNAs), a class of small, noncoding RNAs that bind complementary sequences in mRNA 3'UTRs and repress translation via the RNA-induced silencing complex (RISC). Furthermore, miRNAs and miRNA components have been linked either to long-term memory in Drosophila or to sensorimotor synapses (McCann, 2011).

This study shows that (1) Atx2 functions in olfactory projection neurons for LTH as well as associated glomerulus-selective physiological and structural plasticity; (2) Atx2 functions in LTH with the known miRNA-pathway proteins Argonaute 1 (Ago1) and Me31B; and (3) Atx2 is part of a general machinery required for efficient miRNA-mediated translational repression (McCann, 2011).

When tested in a Y-maze apparatus, flies exposed to either 15% CO2 or 5% ethyl butyrate (EB) for 30 min show a reduced aversive response that lasts less than 1 h (short-term habituation, STH). In contrast, flies exposed to 5% CO2 or 20% EB for 4 d show reduced aversion for days (LTH) and reduced odor-evoked responses in respective odor-responsive PNs, together with CREB-dependent growth of odor-responsive glomeruli (V and DM2/DM5, respectively). In this well-defined behavioral and synaptic context, it was asked whether PNs require Atx2 for LTH and associated synapse-specific structural plasticity. Expression of a UAS-Atx2RNAi construct in GH146-expressing neurons responsive to EB but not to CO2 (GH146Gal4/UASAtx2RNAi) completely blocked LTH to EB without altering LTH to CO2. Atx2 knockdown in GH146-expressing PNs blocked LTH to EB but had no effect on either STH to EB or the EB-avoidance response. Similarly, knockdown of Atx2 in the CO2-responsive VPN (VPNGal4;UASAtx2RNAi/+) selectively blocked LTH to CO2 without altering either STH to CO2 or the naive olfactory response to CO2. Thus, Atx2 is selectively required in glomerulus-specific PNs for odorant-selective LTH (McCann, 2011).

Two observations argue that Atx2 functions in adult neurons for LTH. First, both baseline behavioral responses to odorants and STH are normal in animals after Atx2 knockdown in PNs, indicating relatively normal development of the olfactory system. Second and more direct evidence is the selective block in LTH to EB seen following adult-specific knockdown of Atx2 in EB-responsive PNs using the TubGal80ts system (McCann, 2011).

Atx2 knockdown in odor-responsive PNs blocks not only olfactory LTH but also the LTH-associated increase in the volume of behaviorally relevant glomeruli. Thus, following 4 d of EB exposure, GH146Gal4/UAS-Atx2RNAi flies, which do not show LTH, also show no increase in the volume of either the DM5 glomerulus, previously shown to mediate the aversive response to this odorant, or the EB-responsive DM2 glomerulus. In contrast, the same GH146Gal4/UAS-Atx2RNAi flies show normal LTH to CO2 and robust increases in the volume of the VPN glomerulus in response to 4-d CO2 exposure as observed in control flies. Similarly, VPNGal4;UAS-Atx2RNAi/+ flies do not show LTH to CO2 or associated growth of the V glomerulus but display normal EB-induced LTH and EB-associated growth of DM5. Thus, Atx2 is required in specific PNs for the glomerulus-selective structural plasticity that accompanies odorant-selective LTH (McCann, 2011).

Normal LTH to EB is associated with an experience-dependent reduction in the EB-evoked physiological response of DM2 and DM5 PNs. This reduction can be measured in vivo by imaging odor-evoked calcium transients in PNs of flies expressing the genetically encoded calcium sensor, GCaMP3 (McCann, 2011).

To test whether this LTH-associated physiological plasticity requires Atx2 function in PNs, EB-evoked calcium fluxes were imaged and quantified in PN dendrites of 4-d EB-exposed and mock-exposed GH146Gal4, UAS-GCaMP3/UAS-Atx2RNAi flies (which do not show LTH to EB), and these results were compared with normally habituating GH146Gal4,UAS-GCaMP3/+ controls. In DM2 and DM5 of GH146Gal4, UAS-GCaMP3/UAS-Atx2RNAi flies, 4-d EB exposure caused significantly less change in EB (McCann, 2011).

Biochemical interactions of Atx2 orthologs in Drosophila and other organisms point to an interesting potential mechanism through which Atx2 regulates synapse-specific long-term plasticity required for LTH. In particular, Atx2 binding to Me31B and PABP orthologs, which in turn interact with other core miRNA-pathway proteins, GW182 and Argonaute, suggests that Atx2 may regulate miRNA-mediated translational repression directly. Could the function of Atx2 in LTH reflect a role in the miRNA pathway (McCann, 2011)?

To address this question, the possibility of strong dominant genetic interactions between atx2^X1 and mutations affecting core components of the miRNA pathway was examined. First, LTH and STH were examined in ago1^K08121/+; atx2^X1/+ double-heterozygote animals, and these behaviors were compared with those of single-heterozygote controls. The results were striking. Although STH to EB and CO2 was normal in double heterozygote ago1^K08121/+; atx2^X1/+ animals, LTH to both EB and CO2 was completely abolished. In contrast, control +/+; atx2^X1/+ and ago1^K08121/P[atx2+]; atx2^X1/+ animals showed normal LTH to both odorants (McCann, 2011).

The observation that the atx2 genomic rescue construct P[atx2+] restored normal LTH to ago1^K08121/+; atx2^X1/+ flies also shows that altered LTH in the double-heterozygote flies is caused specifically by a defect in atx2. In a similar experiment LTH and STH were examined in me31B^δ2/+; atx2^X1/+ double-heterozygote animals exposed to EB or CO2. Again, these double heterozygotes showed no LTH but normal STH. The defects in LTH were not observed in +/+; atx2^X1/+ or me31B^δ2 /P[atx2+]; atx2^X1/+ animals, further confirming the involvement of atx2. LTH-associated structural plasticity also was blocked in ago1^K08121/+; atx2^X1/+ and me31B^δ2/+; atx2^X1/+ double heterozygotes. Thus, although the V and DM5 glomeruli of +/+; atx2^X1/+ flies showed the expected growth following 4 d of CO2 or EB exposure, respectively, both the EB-evoked increase in DM5 volume and the CO2-induced increase in V was abolished in ago1^K08121/+; atx2^X1/+ and me31B^δ2/+; atx2^X1/+ double heterozygotes. In every instance, the defect in structural plasticity was restored by a wild-type genomic atx2+ transgene: Both ago1^K08121/ P[atx2+]; atx2^X1/+ flies and me31B^δ2/P[atx2+]; atx2^X1/+ flies showed normal odor-induced structural plasticity (McCann, 2011).

These data indicate that Atx2 functions in odorant-selective LTHas well as in glomerulus-selective structural plasticity through a pathway that depends on Ago1 and on Me31B, two proteins previously linked with miRNA-driven translational control. Consistent with this hypothesis, RNAi-based knockdown of Me31B in EB-responsive PNs mimics the effects of Atx2 knockdown, causing a specific defect in LTH to EB (McCann, 2011).

The observed genetic interactions of atx2 mutations with me31B and ago1 mutations point to a likely role for the Atx2 protein in regulating Ago1- and Me31B-dependent, miRNA-mediated translational repression in vivo. To examine this possibility, it was asked if Drosophila Atx2 is required for miRNA-mediated translational repression in wing imaginal discs, a tissue in which the function and activities of endogenous miRNAs can be analyzed conveniently (McCann, 2011).

To reduce levels of endogenous Atx2 in identified subpopulations of wing imaginal disc cells, either a patched Gal4-driven RNAi construct (UAS-Atx2RNAi) was used against atx2 or the Flippase recognition target-Flippase (FRT-FLP) recombinase system to generate genetic-mosaic animals carrying clusters of homozygous atx2^X1/atx2^X1 mutant cells in the wing imaginal discs of hs-flp;+/+; FRT82B, atx2^X1/FRT82B, arm-lacZ. Homozygous mutant atx2^X1/atx2^X1 cells were identified using either an Atx2 antibody or a surrogate, anti-lacZ staining, which here labels all cells except the atx2^X1/atx2^X1 mutant clones generated by mitotic recombination (McCann, 2011).

To examine Atx2 function in the miRNA pathway, such clones were generated in a genetic background that included any one of a number of transgenically encoded, miRNA-dependent translational reporters, and how loss of Atx2 affected GFP expression of these reporters was assessed. Reporters for head involution defective (hid), bantam, mir-12, costal-2, and sickle were used. The hid, sickle, and costal-2 reporters consist of the 3' UTR of hid, sickle, or costal-2, respectively, placed downstream of GFP-coding sequences under the control of a tubulin promoter (McCann, 2011).

The 3' UTR of hid is repressed by endogenous bantam miRNA and that of sickle by miR-2b. The bantam and miR-12 reporters consist of two copies of the bantam target recognition sequence or four copies of the miR-12 target recognition sequence, respectively, also downstream of GFP-coding sequences (McCann, 2011).

Atx2-deficient cells had a noticeable reduction in the intensity of Me31B and Ago1 staining, suggesting that in vivo Drosophila Atx2 is necessary for maintaining Me31B particles potentially involved in miRNA-mediated translational repression. In addition, and consistent with a defect in miRNA function in vivo, cells lacking Atx2 showed distinctly elevated expression of specific miRNA reporters (McCann, 2011).

Clones of atx2^X1/atx2^X1 mutant cells showed clear up-regulation of the hid reporter compared with flanking atx2^X1/+ or +/+ cells. The increased hid reporter levels in atx2^X1/atx2^X1 mutant cells were not observed if similar clones also expressed a wild-type atx2 genomic transgene). This observed genetic rescue confirms that the increase in hid-reporter expression in atx2^X1/atx2^X1 cells is caused by the absence of atx2 and not by any other unknown potential mutation on the FRT82B,atx2^X1 chromosome. Thus, as predicted by its biochemical and genetic interactions with various miRNA-pathway components, Atx2 is required for optimal repression of a miRNA reporter in vivo (McCann, 2011).

Further experiments confirmed that this requirement reflects a broad requirement for Atx2 function for the repression of many different miRNA-target genes. Clones of atx2^X1/atx2^X1 cells also show increased expression of the sickle, bantam BandBŒ), and miR12 reporters. Given that the latter two reporters are regulated by artificial UTRs engineered to be repressed solely through the miRNA pathway, these data strongly argue that Atx2 is broadly required for miRNA function. In contrast to the other four miRNA reporters, costal-2 reporter expression was not increased detectably in atx2^X1/atx2^X1 cells. This result was surprising, because the costal-2 reporter is similar to the other reporters in being repressed by miRNAs through a mechanism that requires Dicer-1 (Dcr-1), the endonuclease involved in miRNA biogenesis. Therefore, Atx2 is required only for repression of a subset of miRNA targets (McCann, 2011).

A model is considered in which Atx2 functions in only one of two miRNA-repression pathways recently distinguished in Drosophila. Although produced by Dcr-1, miRNAs may repress translation by one of two alternative pathways: either through an Ago1-RISC that requires GW182 or through an Ago2 RISC via a poorly understood GW182-independent mechanism. To test the possibility that Atx2 would be required exclusively for the Ago1/GW182-dependent pathway, previously shown to be required for bantam miRNA function, how the various reporters were affected by knockdown of GW182 was examined (McCann, 2011).

To knock down GW182 in identified groups of cells, the FLP-out technique was used to overexpress a transgenic RNAi construct against GW182 in wing imaginal discs expressing hid, miR12, or costal-2 reporters. Cells expressing a GW182RNAi construct (labeled by anti-lacZ staining) showed visibly increased expression of the Atx2-sensitive hid and miR12 reporters but no up-regulation of the Atx2-insenstive costal-2 reporter (McCann, 2011).

To understand why the costal-2 reporter could be insensitive to GW182 (or Atx2) knockdown, the sequence of its 3' UTR was examined and it was found to contain not only target sites for miR12 and miR283, but also two binding sites for miR277. This finding is significant because, unlike the majority of Dcr1-dependent miRNAs, miR277 is loaded preferentially onto Ago2-RISC complexes because of the extensive base-pairing between its miRNA and miRNA* strands. Thus, these observations suggest that Atx2, although necessary for GW182-dependent repression through Ago1-RISC, may not be necessary for Ago2-RISC.dependent repression (McCann, 2011).

This tentative model is supported by the observation that RNAi-induced, Ago-2-dependent eye phenotypes also are not sensitive to knockdown of Atx2. Knockdown of the caspase inhibitor Drosophila inhibitor of apoptosis (DIAP) by GMRGal4- driven expression of UAS-DIAPRNAi results in significantly smaller eyes because of increased apoptosis. The cell-death phenotype is suppressed if Ago2 levels are reduced by simultaneous expression of UAS-Ago2RNAi. However, similar coexpression of a functional UAS-Atx2RNAi (or UAS-GFPRNAi) does not alter phenotypes of GMRGal4;UASDIAPRNAi flies (McCann, 2011).

Taken together with prior evidence that Atx2 associates physically with Me31B and PABP, two proteins required for the Ago1-RISC pathway, these data indicate that Atx2 is part of a core pathway required for miRNA-mediated translational repression. However, Atx2 may be dispensable for repression by the Ago2-RISC pathway (McCann, 2011).

The circuit that underlies LTH has allowed experience-induced, synapse-specific plasticity to be examined in the context of behavioral memory. Previous pioneering work in cultured Aplysia sensorimotor synapses has led to a model in which CREB-dependent nuclear gene expression provides global (cell-wide) control of long-term facilitation. This facilitation can be restricted to specific synapses, in part through the synapse-specific local translation of stored mRNAs, which also is required for long-term plasticity. In the context of olfactory LTH, which is driven by the plasticity of inhibitory LN-PN synapses in the antennal lobe, previous work has shown that CREB function is required globally in a multiglomerular class of LNs for LTH to CO2 and EB. This study now shows that LTH additionally requires Atx2 in postsynaptic PNs for the glomerulus-restricted plasticity necessary for odorant-selective LTH. Knockdown of Atx2 in adult-stage PNs selectively blocks LTH without affecting either basal odorant response or STH. This distinctive phenotype also is shown following Me31B knockdown in PNs or in animals doubly heterozygote for atx2 and ago1 or atx2 and me31B. When taken together with independent observations that Atx2 is required for efficient miRNA function, these very strong genetic interactions point to a role for Atx2 in miRNA-mediated translational control in the regulation of long-term memory (McCann, 2011).

It is hypothesized that, under appropriate circumstances, NMDA receptor activation in PN dendrites may trigger local protein synthesis, perhaps through RISC degradation, giving rise to a retrograde signal from PNs to LNs that in turn stimulates or synergizes with the cell biological changes required for glomerulus- limited, long-term plasticity. The data do not demonstrate that Atx2 and Me31B function in local translation of synaptic mRNAs, but they do show a specific requirement for Atx2 and Me31B for miRNA function and synapse-specific LTH (but not STH) and thus provide a strong argument for local translation of synaptic mRNAs being the underlying mechanism by which these proteins regulate synapse-specific long-term plasticity in vivo (McCann, 2011).

The proposed need for postsynaptic translation and postulate of retrograde signaling are consistent with recent observations and models explaining long-term synaptic facilitation in Aplysia (Wang, 2009; Cai, 2008). In Drosophila, these models may be tested and elaborated through further genetic and in vivo studies and may lead to an understanding of the local and global mechanisms and their interactions that regulate long-term synaptic plasticity (McCann, 2011).

An important finding is that Atx2 is required for translational repression of four different miRNA reporters. Taken together with prior evidence, in particular that Atx2 binds two known components of the miRNA pathway, this finding indicates a wide and general requirement for Atx2 in miRNA-mediated translational repression (McCann, 2011).

However, Atx2 is not required for silencing of the costal-2 reporter, an observation that may be may be explained by costal-2 reporter's repression by a possibly Atx2-independent RISC complex. Previous work has shown that miRNAs partition between two different silencing complexes, Ago1-RISC and Ago2-RISC; in contrast, siRNAs associate almost exclusively with Ago2-RISC. Ago1-RISC and Ago2-RISC silence mRNAs by different mechanisms: Ago1-RISC is characterized by its dependence on GW182. The specific pathway that produces an miRNA or siRNA does not require that small RNA to associate with a particular Ago protein. Thus, although bantam and miR-277 miRNAs are produced by Dcr1, bantam associates exclusively with Ago1-RISC, whereas miR-277 is loaded into the Ago2 pathway. This loading of miR-227 occurs because, in contrast to the bantam microRNA, which has several bulges and mismatches, the duplex precursor to miR-277 strongly resembles an siRNA precursor with a high degree of perfect matching. By demonstrating that loss of Atx2 causes up-regulation of GW182- or Ago1-dependent miRNA reporters, the results identify Atx2 as a frequently used component of the Ago1-GW182 RISC pathway. Loss of Atx2 does not affect repression of the GW182-insensitive costal-2 reporter, possibly repressed via the Ago2-RISC pathway. This observation, combined with the insensitivity of the RNAi pathway to Atx2 knockdown in the Drosophila eye, suggests that Atx2 may not be required for Ago2-RISC function (McCann, 2011).

Atx2's role in the Ago1-miRNA pathway raises the question as to how Atx2 influences miRNA-mediated translational repression. Uncovering Atx2's molecular mechanism of action is complicated by lack of consensus as to how miRNAs regulate gene expression. However, Atx2 is likely to function through its interactions with PABP [mediated by its PABP-interacting motif 2 (PAM2) domain] or Me31B [via its Like Sm (Lsm) and Like-Sm-associated domain (LsmAD) domains] (McCann, 2011).

Three possible models for Atx2 actions are considered. Under appropriate conditions, Atx2 interactions with PABP could help break eukaryotic initiation factor (eIF) 4G eIF4g-PABP interactions required for efficient translational initiation. In addition, directly or through interactions with Me31B, Atx2 may help recruit either of two deadenylase complexes that promote mRNA deadenylation and consequent repression (McCann, 2011).

The identification of Atx2 as a core component of the neuronal and nonneuronal miRNA repression machinery has implications for understanding spinocerebellar ataxias and some forms of amyotrophic lateral sclerosis. Several studies underline the importance of functional components of the miRNA repression machinery in the mammalian brain. Bilen (2006) demonstrated that miRNA-regulated activities play a role in polyglutamine-induced neurodegeneration. In addition, prior work has shown that Atx2 is required for pathogenic forms of Atx1 and Atx3 to induce neurodegeneration in Drosophila, suggesting a potentially common pathway for neuronal loss in different ataxias (Lessing, 2008). Loss of Dcr1 function results in microcephaly and progressive neurodegeneration, consistent with a model in which miRNA function is required for maintaining the adult nervous system (Saba, 2010). Given Atx2's involvement in human neurodegenerative disease, the current findings may help illuminate some of the phenotypes and symptoms of SCA2 patients and also may illuminate possibly common pathways for neuronal loss in different neurodegenerative conditions. If altered miRNA function contributes to neurodegeneration in SCA2 or related diseases, then it is possible that these diseases arise because of altered regulation of a subset of Atx2-target mRNAs in neurons. The identification and study of such target mRNAs may contribute to further understanding and potential therapeutic strategies (McCann, 2011).

FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control

Fragile X mental retardation protein (FMRP) and Ataxin-2 (Atx2) are triplet expansion disease- and stress granule-associated proteins implicated in neuronal translational control and microRNA function. This study shows that Drosophila FMRP (dFMR1) is required for long-term olfactory habituation (LTH), a phenomenon dependent on Atx2-dependent potentiation of inhibitory transmission from local interneurons (LNs) to projection neurons (PNs) in the antennal lobe. dFMR1 is also required for LTH-associated depression of odor-evoked calcium transients in PNs. Strong transdominant genetic interactions among dFMR1, atx2, the deadbox helicase me31B, and argonaute1 (ago1) mutants, as well as coimmunoprecitation of dFMR1 with Atx2, indicate that dFMR1 and Atx2 function together in a microRNA-dependent process necessary for LTH. Consistently, PN or LN knockdown of dFMR1, Atx2, Me31B, or the miRNA-pathway protein GW182 increases expression of a Ca2+/calmodulin-dependent protein kinase II (CaMKII) translational reporter. Moreover, brain immunoprecipitates of dFMR1 and Atx2 proteins include CaMKII mRNA, indicating respective physical interactions with this mRNA. Because CaMKII is necessary for LTH, these data indicate that fragile X mental retardation protein and Atx2 act via at least one common target RNA for memory-associated long-term synaptic plasticity. The observed requirement in LNs and PNs supports an emerging view that both presynaptic and postsynaptic translation are necessary for long-term synaptic plasticity. However, whereas Atx2 is necessary for the integrity of dendritic and somatic Me31B-containing particles, dFmr1 is not. Together, these data indicate that dFmr1 and Atx2 function in long-term but not short-term memory, regulating translation of at least some common presynaptic and postsynaptic target mRNAs in the same cells (Sudhakaran, 2013).

Observations presented in this study lead to three significant insights into the endogenous functions of dFmr1 and Atx2 in the nervous system and their contribution to long-term synaptic plasticity. First, the data strongly indicate that both proteins function in the same pathway, namely translational control, to mediate the form of long-term memory analyzed in this study. Second, the remarkably similar effects of knocking down these proteins in LNs and PNs provide in vivo support for an emerging idea that translational control of mRNAs in both presynaptic and postsynaptic compartments of participating synapses is necessary for long-term synaptic plasticity. Finally, although both dFmr1 and Atx2 have isoforms containing prion-like, Q/N domains, the different effects of loss of Atx2 and dFmr1 on neuronal Me31B aggregates indicate important differences in the mechanisms by which the two proteins function in translational control (Sudhakaran, 2013).

The different molecular and clinical consequence of pathogenic mutations in FMRP and Atx2 encoding genes has led to largely different perspectives on their functions. Fragile X causative mutations cause reduced levels of the encoding mRNA and lower levels of FMRP, leading to increased protein synthesis and a range of pathologies evident in children and young adults. These pathologies importantly do not include the formation of inclusion bodies. In contrast, SCA-2 and amyotrophic laterosclerosis causative mutations in Atx2 result in the dominant formation of inclusion body pathologies and age-dependent degeneration of the affected neuronal types. Observations made in this article indicate that the distinctive pathologies of the two diseases have obscured common molecular functions for the two proteins in vivo (Sudhakaran, 2013).

The genetic, behavioral, and biochemical observations show (1) shared roles of the two proteins in olfactory neurons for long-term but not short-term habituation, and (2) striking transdominant genetic interactions of dfrm1 and atx2 mutations with each other as well as with miRNA pathway proteins, which is not only consistent with prior genetic and behavioral studies of the two respective proteins but also strongly indicative of a common role for the two proteins in translational repression of neuronal mRNAs. This conclusion is supported at a mechanistic level by (3) the finding that both proteins are required for efficient repression mediated by the 3' UTR of CaMKII, a 3' UTR that this study shows to be repressed by the miRNA pathway, and (4) strong evidence for in vivo biochemical interaction among dFmr1 and Atx2 and for binding of these regulatory proteins with the UTR of the CaMKII transcript that they jointly regulate. Thus, dFMR1 and Atx2 function with miRNA pathway proteins for the regulation of a dendritically localized mRNA in identified olfactory neurons (Sudhakaran, 2013).

An unexpected observation was that dFMR1 and Atx2 seemed to be necessary for LTH as well as for CaMKII reporter regulation in both inhibitory LNs and excitatory PNs of the antennal lobe (Sudhakaran, 2013).

Until recently mammalian FMRP was regarded as a postsynaptic protein, consistent with the view that translational control of mRNAs essential for long-term plasticity occurs exclusively in postsynaptic dendrites. In contrast, work in Aplysia indicated that translational control of mRNAs is required in presynaptic terminals for long-term synaptic plasticity. This conflict between vertebrate and invertebrate perspectives is beginning to be resolved by findings that (1) mammalian FMRP is present in axons and presynaptic terminals; and that (2) translational control of both presynaptic and postsynaptic mRNAs is essential for long-term plasticity of cultured Aplysia sensorimotor synapses (Sudhakaran, 2013 and references therein).

Prior studies at the Drosophila neuromuscular junction have strongly indicated presynaptic functions for dFmr1 and translational control but have also pointed to their significant postsynaptic involvement in neuromuscular junction maturation, growth, and plasticity. More direct studies of experience-induced long-term plasticity have been performed in the context of Drosophila olfactory associative memory, wherein a specific dFmr1 isoform in particular and translational control in general are necessary for long-term forms of memory. However, the incomplete understanding of the underlying circuit mechanism has made it difficult to conclude presynaptic, postsynaptic, or dual locations for dFmr1 function in long-term memory. In contrast, recent work showing an essential role for Atx2 and Me31B in PNs for LTH more strongly indicate a postsynaptic requirement for translational control mediated by these proteins; however, this did not address a potential additional presynaptic function (Sudhakaran, 2013).

The finding that dFmr1 and Atx2 are necessary in both LNs and PNs for LTH, a process driven by changes in the strength of LN–PN synapses, provides powerful in vivo support for a consensus model in which translational control on both sides of the synapse is necessary for long-term plasticity. A formal caveat is that the anatomy of LN–PN synapses in Drosophila antennal lobes remains to be clarified at the EM level. If it emerges that these are reciprocal, dendrodendritic synapses, similar to those between granule and mitral cells in the mammalian olfactory bulb, then a clear assignment of the terms 'presynaptic' and 'postsynaptic' to the deduced activities of dFmr1 and Atx2 in this context may require further experiments (Sudhakaran, 2013).

Previous studies in Drosophila have indicated a broader role for Atx2 than dFmr1 in miRNA function in nonneuronal cells. Although Atx2 is necessary for optimal repression of four miRNA sensors examined in wing imaginal disk cells, dFmr1 is not necessary for repression of any of these sensors. The resulting conclusion that dFmr1 is required only for a subset of miRNAs to function in context of specific UTRs is consistent with the observation that only a subset of neuronal miRNAs associate with mammalian FMRP and that the protein shows poor colocalization with miRNA pathway and P-body components in mammalian cells. Parallel studies have shown that Atx2 in cells from yeast to man is required for the formation of mRNP aggregates termed stress granules, which in mammalian cells also contain Me31B/RCK and FMRP. In addition, biochemical interactions between these proteins and their mammalian homologs with each other as well as with other components of the miRNA pathway have been reported. However, neither the mechanisms of Atx2-driven mRNP assembly, nor the potential role for FMRP in such assembly, have been tested in molecular detail (Sudhakaran, 2013).

The demonstration that loss of Atx2 in neurons results in a substantial depletion of Me31B-positive foci in PN cell bodies and in dendrites is consistent with Atx2 being required for the assembly of these two different (somatic and synaptic) in vivo mRNP assemblies. Thus, the mechanisms that govern their assembly, particularly of synaptic mRNPs in vivo, overlap with mechanisms used in P-body and stress granule assembly in nonneuronal cells (Sudhakaran, 2013).

The finding that loss of dFmr1 has no visible effect on these Me31B-positive foci can be explained using either of two models. A simple model is that dFmr1 is not required for mRNP assembly, a function mediated exclusively by Atx2. This would suggest that Atx2 contains one or more functional domains missing in dFmr1 that allow the multivalent interactions necessary for mRNP assembly. This is most consistent with the observation that that although dFMR1 is a component of stress granules in Drosophila nonneuronal cells, it is not required for their assembly. An alternative model would allow both dFmr1 and Atx2 to mediate mRNP assembly but posit that dFmr1 is only present on a small subset of mRNPs, in contrast to Atx2, which is present on the majority. In such a scenario, loss of dFmr1 would only affect a very small number of mRNPs, too low to detect using the microscopic methods used in this study. In the context of these models, it is interesting that both dFmr1 and Atx2 contain prion-like Q/N domains, potentially capable of mediating mRNP assembly. It is to be noted here that the dFmr1 Q/N domain, although lacking prion-forming properties, is capable of serving as a protein interaction domain enabling the assembly of dFmr1 into RNP complexes. This observation would support the view that dFmr1 may be involved in the formation of only a subset of cellular mRNP complexes. Future studies that probe the potential distinctive properties of these assembly domains may help discriminate between these models. In addition, potential interaction of Atx2 with other proteins that are involved in mRNP formation across species, like Staufen, could help to understand the mechanisms behind Atx2-dependent function in mRNP assembly (Sudhakaran, 2013).

However, the observations presented in this study clearly show that despite the remarkable similarities in the roles of dFmr1 and Atx2 for repression of CaMKII expression at synapses and the control of synaptic plasticity that underlies long-term olfactory habituation, both proteins also have distinctive molecular functions in vivo (Sudhakaran, 2013).

Mutations that affect neuronal translational control are frequently associated with neurological disease, particularly with autism and neurodegeneration. Although these clinical conditions differ substantially in their presentation, a broadly common element is the reduced ability to adapt dynamically to changing environments, a process that may require activity-regulated translational control at synapses. Taken together with others, the observations of this study suggest that there may be two routes to defective activity-regulated translation. First, as in dFmr1 mutants, the key mRNAs are no longer sequestered and repressed, leading to a reduced ability to induce a necessary activity-induced increase in their translation. Second, it is suggested that increased aggregation of neuronal mRNPs (indicated by the frequent occurrence of TDP-43 and Atx2-positive mRNP aggregates in neurodegenerative disease) may result in a pathologically hyperrepressed state from which key mRNAs cannot be recruited for activity-induced translation. Thus, altered activity-regulated translation may provide a partial explanation not only for defects in memory consolidation associated with early-stage neurodegenerative disease but also for defects in adaptive ability seen in autism spectrum disorders (Sudhakaran, 2013).

Ataxin-2 and its Drosophila homolog, ATX2, interact with PABP and physically assemble with polyribosomes

Mutations resulting in the expansion of a polyglutamine tract in the protein ataxin-2 give rise to the neurodegenerative disorders spinocerebellar ataxia type 2 and Parkinson's disease. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration are unknown. This study demonstrates that ataxin-2 and its Drosophila homolog, ATX2, assemble with polyribosomes and poly(A)-binding protein (PABP), a key regulator of mRNA translation. The assembly of ATX2 with polyribosomes is mediated independently by two distinct evolutionarily conserved regions of ATX2: an N-terminal Lsm/Lsm-associated domain (LsmAD), found in proteins that function in nuclear RNA processing and mRNA decay, and a PAM2 motif, found in proteins that interact physically with PABP. The PAM2 motif mediates a physical interaction of ATX2 with PABP in addition to promoting ATX2 assembly with polyribosomes. These results suggest a model in which ATX2 binds mRNA directly through its Lsm/LsmAD domain and indirectly via binding PABP that is itself directly bound to mRNA. These findings, coupled with work on other ataxin-2 family members, suggest that ATX2 plays a direct role in translational regulation. These results raise the possibility that polyglutamine expansions within ataxin-2 cause neurodegeneration by interfering with the translational regulation of particular mRNAs (Satterfield, 2006; full text of article).

Given the evidence supporting a role for ataxin-2 in translational regulation, the question arises as to the mechanism by which ataxin-2 imposes this regulation. One possibility is that ataxin-2 directly influences the activity of PABP. PABP promotes translation by facilitating the interaction between the 5' and 3' ends of the mRNA, a process thought to promote the re-initiation of translation of terminating ribosomes (Kahvejian, 2001). PABP accomplishes this task by simultaneously binding to the poly(A) tail and to the PAM2 motif of eIF4G, a component of the 5' cap-binding translation initiation complex (see A model depicting how ATX2 might influence translation). Another PAM2 protein, Paip1, mimics the activity of eIF4G by simultaneously binding poly(A)-bound PABP and eIF4A, another component of the 5' cap-binding translation initiation complex. In contrast to eIF4G and Paip1, another PAM2 protein, Paip2, inhibits translation. Paip2 accomplishes this task by binding PABP and preventing its assembly onto the poly(A) tail (Kahvejian, 2001; Khaleghpour, 2001; Roy, 2004). The finding that ATX2 is capable of assembling with poly(A)-bound dPABP indicates that, unlike Paip2, ATX2 does not prevent dPABP from assembling with the poly(A) tail. Assuming that ATX2 influences dPABP activity, it appears to do so while dPABP is assembled with the poly(A) tail, possibly by promoting or preventing the interaction between dPABP and the 5' cap-binding translation initiation complex. Further work will be required to elucidate the functional significance of the ATX2-dPABP interaction (Satterfield, 2006).

Although previous evidence indicates that ataxin-2 family members interact functionally with PABP, several observations indicate that ataxin-2 does not act solely through PABP. For example, in yeast, Pbp1 deletions suppress the lethality caused by deletion of Pab1, indicating that Pbp1p can perform a functional role in the complete absence of Pab1p (Mangus, 1998). Moreover, an ATX2 transgenic construct that encodes a protein lacking the PAM2 motif significantly extends the lethal phase of ATX2 mutant flies. Although ATX2 null mutants do not develop beyond the second instar larval stage, these mutants can be rescued to the adult stage of development using a wild-type ATX2 transgene. Use of ATX2 transgenes bearing a PAM2 deletion can also extend the lethal phase of ATX2 mutants to the pupal stage of development, although none of the partially rescued offspring survives to the adult stage of development. Given that the PAM2 motif is required for ATX2 to interact with dPABP, this observation indicates that ATX2 possesses a biological activity that is independent of its physical interaction with dPABP. The finding that the Lsm/LsmAD domain of ATX2 is capable of promoting its assembly with polyribosomes independently of the PAM2 motif, together with the observation that the Lsm/LsmAD domain represents the only other evolutionarily conserved sequence in ATX2, suggests that this domain is the source of the dPABP-independent activity of ATX2 (Satterfield, 2006).

Assuming that the Lsm/LsmAD domain is responsible for the dPABP-independent activity of ATX2 and that this activity serves a translational regulatory role, the question arises as to the precise mechanism by which this domain regulates translation. Although eukaryotic Lsm and the related Sm proteins are not currently known to regulate translation, one well-studied bacterial Sm protein, Hfq, does appear to regulate translation. Hfq functions as an RNA chaperone and regulates translation by stabilizing basepairing interactions between small non-coding RNAs (sRNAs) and their mRNA targets. These sRNA-mRNA interactions influence translation by altering the physical structure of the mRNA target. Although only limited sequence homology exists between Hfq and other Sm and Lsm proteins, the crystal structures of Staphylococcus aureus Hfq and eukaryotic Sm proteins are nearly identical, indicating that these proteins may function in a similar fashion. Furthermore, studies of eukaryotic Sm and Lsm proteins suggest that these proteins also function by mediating RNA-RNA interactions. The structural similarity of Sm and Lsm proteins raises the possibility that the Lsm/LsmAD domain of ATX2 might function, like Hfq, to regulate translation by mediating RNA-RNA interactions. An attractive potential target of ataxin-2 regulation in eukaryotes is the group of sRNAs known as microRNAs. MicroRNAs are known to play translational regulatory roles by basepairing with particular target mRNAs on polyribosomes. Studies are currently underway to investigate this possible mode of ATX2 regulation (Satterfield, 2006).

Previous work on several other polyglutamine disorders indicates that pathogenesis results from a reduction in transcriptional efficiency. Although the cytoplasmic localization of ataxin-2 indicates that this protein does not directly influence transcription, the finding that ATX2 physically assembles with polyribosomes, coupled with other work on ataxin-2 homologs, raises the possibility of a conserved mechanism of polyglutamine pathogenesis involving dysfunctional gene expression. In contrast to the transcriptional alterations associated with other polyglutamine diseases, polyglutamine expansions within ataxin-2 may adversely affect gene expression by impairing translation. Although polyglutamine expansion of human ataxin-2 does not detectably influence the binding of ataxin-2 to polyribosomes, it remains conceivable that polyglutamine expansions within ataxin-2 influence a function of ataxin-2 in translational regulation that lies downstream of polyribosome binding. The finding that polyglutamine expansions within ataxin-2 also cause a heritable form of Parkinsonism further suggests that altered translational regulation of particular targets might trigger the degeneration of dopaminergic neurons in the substantia nigra. As increasing evidence indicates that an overabundance of the protein alpha-synuclein plays an important role in the pathogenesis of Parkinson's disease, the current findings raise the interesting possibility that polyglutamine expansions within ataxin-2 lead to increased translation of alpha-synuclein. Future studies aimed at a better understanding of ataxin-2 function and the effects of polyglutamine expansions on that function will be required to directly address the hypothesis that translational dysregulation underlies ataxin-2-mediated neurodegeneration (Satterfield, 2006).

Drosophila Ataxin-2 mediates expanded Ataxin-1-induced neurodegeneration in a Drosophila model of SCA1

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1) and Ataxin-2 (ATXN2), respectively, two proteins that are otherwise unrelated. This study used a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. Wild-type Drosophila Ataxin-2 (dAtx2) is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q]) toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, it was shown that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. This hypothesis was tested by engineering dAtx2 transgenes with nuclear localization signal (NLS) and nuclear export signal (NES). NLS-dAtx2, but not NES-dAtx2, was found to mimic the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology (Al-Ramahi, 2007).

This study reports functional interactions between the proteins causing two distinct Spinocerebellar ataxias. A Drosophila model of SCA1 was used to show that wild-type dAtx2 (the fly homolog of the protein that when expanded causes SCA2) mediates, at least in part, neuronal degeneration caused by expanded Ataxin-1 (the protein triggering SCA1). Ataxin-1[82Q]-induced toxicity is worsened by increasing the levels of dAtx2. More significantly, decreasing the levels of dAtx2 suppresses expanded Ataxin-1-induced neuronal degeneration as shown in several independent assays. The suppression of Ataxin-1[82Q] phenotypes by partial loss of function of dAtx2 argues against a possible mechanism by which sequestration and depletion of Ataxin-2 contributes to expanded Ataxin-1-induced neurodegeneration. This is further supported by lack of cerebellar or other neuronal abnormalities in mice that are deficient for Ataxin-2 (Al-Ramahi, 2007).

The human expanded Ataxin-1 interacts with the dAtx2 and human Ataxin-2 proteins in co-AP assays. Furthermore, overexpressed Ataxin-1 pulls down endogenous hAtaxin-2 in cultured cells. These results suggest that Ataxin-1 and Ataxin-2 may be functional interactors in vivo. Consistent with this, it was found that expanded Ataxin-1 induces accumulation of Ataxin-2 in the nucleus, where the two proteins localize in nuclear inclusions (NIs) both in Drosophila neurons and SCA1 human brain tissue. These are surprising observations since Ataxin-2 is normally a cytoplasmic protein both in humans and Drosophila. Interestingly, wild-type Ataxin-1 can cause neurotoxicity when overexpressed, although to a much lesser extent than expanded Ataxin-1. However, nuclear accumulation of dAtx2 is triggered by pathogenic but not wild-type forms of Ataxin-1, at least in detectable amounts. Taken together these data suggested that accumulation of Ataxin-2 in the nucleus contributes to the exacerbated toxicity of expanded Ataxin-1, and is an important mechanism of pathogenesis in SCA1. To investigate this hypothesis dAtx2 was targeted to the nucleus by means of an exogenous NLS signal. It was found that dAtx2^NLS is sufficient to cause a dramatic increase of its toxicity, when compared to either wild-type dAtx2 or dAtx2 with an exogenous nuclear export signal (dAtx2^NES) expressed at similar levels (Al-Ramahi, 2007).

To further test the hypothesis that nuclear accumulation of Ataxin-2 contributes to neurodegeneration caused by expanded Ataxin-1 Sens levels were investigated. Sens and its murine orthologue Gfi1 are proneural factors whose levels are decreased in the presence of expanded Ataxin-1; thus providing a molecular readout for the neurotoxicity of Ataxin-1. In Drosophila, reduction of Sens levels leads to the loss of mechanoreceptors, so Sens was monitored in the context of flies expressing either dAtx2^NLS or dAtx2^NES but not carrying the Ataxin-1[82Q] transgene. It was found that nuclear targeted, but not cytoplasmic, dAtx2 mimics both the Sens reduction and mechanoreceptor loss phenotypes caused by Ataxin-1[82Q] (Al-Ramahi, 2007).

Expanded Ataxin-2 accumulates both in the cytoplasm and the nuclei of SCA2 postmortem brains. In mouse and cell culture models of SCA2, expanded Ataxin-2 accumulates in the cytoplasm and its nuclear accumulation is not necessary to induce toxicity. However, nuclear accumulation of expanded Ataxin-2 also occurs in cultured cells, and is consistently observed in human SCA2 postmortem brainstem neurons. These observations suggest that both nuclear and cytoplasmic mechanisms of pathogenesis contribute to neurodegeneration in SCA2, as it is known to occur in other polyglutamine diseases like HD and SCA3. One possibility is that Ataxin-2 shuttles between the nucleus and the cytoplasm although the protein is normally detected only in the cytoplasm. The data show that accumulation of dAtx2 in the nucleus is more harmful than in the cytoplasm. Thus, neurons with nuclear Ataxin-2 in SCA2 patients may be relatively more compromised than neurons where Ataxin-2 accumulates in the cytoplasm. In agreement with this possibility, expanded Ataxin-2 is found in the nuclei of pontine neurons of SCA2 brains, one of the neuronal groups and brain regions with prominent degeneration in SCA2 (Al-Ramahi, 2007 and references therein).

Reducing Ataxin-2 levels suppresses expanded Ataxin-1 toxicity, strongly arguing against a mechanism of pathogenesis by loss of function of Ataxin-2 in the cytoplasm. Studies of the normal function of Ataxin-2 and its yeast, C. elegans, and Drosophila homologs suggest a role in translational regulation. Thus, an attractive possibility is that Ataxin-1 [82Q] requires dAtx2 to impair Sens translation and induce the loss of mechanoreceptors. Consistent with this hypothesis is the finding that partial loss of function of dAtx2 suppresses the loss of mechanoreceptors phenotype caused by expanded Ataxin-1 (Al-Ramahi, 2007).

The data described in this study uncover unexpected functional interactions between proteins involved in two different SCAs. Nuclear accumulation of Ataxin-2, normally a cytoplasmic protein, is a common denominator of SCA1 and SCA2, and leads to reduced levels of at least one important proneural factor; i.e. Sens, whose mammalian orthologue Gfi1 is required for Purkinje cell survival. Thus neuronal degeneration may take place through common mechanisms in different ataxias, and one of these mechanisms may involve the abnormal accumulation of Ataxin-2 in neuronal nuclei (Al-Ramahi, 2007).

Polyglutamine genes interact to modulate the severity and progression of neurodegeneration in Drosophila

The expansion of polyglutamine tracts in a variety of proteins causes devastating, dominantly inherited neurodegenerative diseases, including six forms of spinal cerebellar ataxia (SCA). Although a polyglutamine expansion encoded in a single allele of each of the responsible genes is sufficient for the onset of each disease, clinical observations suggest that interactions between these genes may affect disease progression. In a screen for modifiers of neurodegeneration due to SCA3 in Drosophila, atx2, the fly ortholog of the human gene that causes a related ataxia, SCA2, was isolated. The normal activity of Ataxin-2 (Atx2), also called Sca2 in the literature, is critical for SCA3 degeneration, and Atx2 activity hastens the onset of nuclear inclusions associated with SCA3. These activities depend on a conserved protein interaction domain of Atx2, the PAM2 motif, which mediates binding of cytoplasmic poly(A)-binding protein (PABP). PABP also influences SCA3-associated neurodegeneration. These studies indicate that the toxicity of one polyglutamine disease protein can be dramatically modulated by the normal activity of another. It is proposed that functional links between these genes are critical to disease severity and progression, such that therapeutics for one disease may be applicable to others (Lessing, 2008; full text of article).

PABP is the only known protein to date that interacts directly with Atx2 through the PAM2 motif (Kozlov, 2001; Ralser, 2005; Satterfield, 2006); therefore, given the important role of the PAM2 motif, it was asked if PABP played a role in SCA3 neurodegeneration. Heterozygosity for the available pabp allele had no effect on Atx3 toxicity, although this allele is unlikely to be a complete loss of function (Sigrist, 2000). A deletion chromosome that removed the pabp gene was tested, comparing to appropriate control lines. Flies expressing pathogenic Atx3 that were heterozygous for this deletion showed dramatically enhanced photoreceptor loss. Control experiments confirmed that the deletion alone, in the absence of pathogenic Atx3, did not cause neurodegeneration. In contrast to the loss-of-function situation, overexpression of PABP significantly suppressed neurodegeneration. These observations indicated that PABP has the opposite activity as Atx2 with respect to Atx3-dependent neurodegeneration: whereas Atx2 enhances the toxicity of Atx3, PABP is protective (Lessing, 2008).

Whether PABP could modulate the degeneration induced by strong expression of Atx2 was tested. Decreased PABP function enhanced Atx2-dependent photoreceptor loss; likewise, up-regulation of PABP protected against photoreceptor degeneration. These studies suggest that the toxicity of Atx2 is mitigated by physical association with PABP, and they are consistent with PABP also playing a crucial role in the Atx2-Atx3 interaction. Together with results demonstrating the crucial role of the PAM2 motif, these data highlight the importance of the normal biological activity of Atx2 and of PABP in modulating the toxicity of pathogenic Atx3 (Lessing, 2008).

Thus the toxicity of pathogenic human Atx3 is critically dependent on Atx2 activity. Reduction of endogenous Atx2 function mitigated Atx3-induced neurodegeneration, and up-regulation of Atx2 synergistically enhanced degeneration. This study also revealed the roles in neural integrity played by the non-polyglutamine PAM2 motif of Atx2 and by PABP, which binds to Atx2 via this motif. These data are consistent with and expand upon clinical findings suggesting interactions between Atx2 and Atx3 in human disease. In the fly, endogenous Atx2 colocalized with pathogenic Atx3 in inclusions, as seen in human patients, with up-regulation of Atx2 enhancing Atx3 toxicity concomitant with a faster onset of inclusions and of SDS-insoluble complexes. These findings suggest that therapeutic approaches to modulate Atx2 activity may be effective against multiple disease situations, including SCA2 and SCA3 (Lessing, 2008).

Interestingly, normal Atx2 is toxic, causing degeneration when up-regulated. Previous animal models have demonstrated that normal protein products associated with SCA1 and Parkinson's disease - Ataxin-1 (see Drosophila Ataxin-1) and alpha-Synuclein, respectively - are also toxic when expressed at sufficiently high levels. Expansion of the polyglutamine domain in Ataxin-1 or Parkinson disease-associated missense mutations of alpha-Synuclein presumably lead to increased levels of the respective proteins, sufficiently high to elicit disease. Up-regulation of Drosophila Atx2 may cause degeneration for similar reasons. These studies further reveal that neuronal toxicity of Atx2 depends on its PAM2 motif, an observation with an interesting parallel to Ataxin-1, the protein that causes SCA1 -- an expanded polyglutamine repeat in Ataxin-1 is not sufficient to cause neurodegeneration in mouse models for SCA1, but rather pathogenic Ataxin-1 also requires its AXH domain to cause disease (Lessing, 2008).

The importance of the PAM2 motif for Atx2's toxicity and for the enhancement of Atx3 toxicity suggests a clue to the mechanism of the interaction. The PAM2 motif has been shown to bind specifically to the PABC domain, with PABP being currently the only known PABC-containing protein that interacts with Atx2. PABP is a ubiquitously expressed and essential protein that binds to the polyadenylated tails of mRNAs and is required for their translation. Furthermore, biochemical and genetic data support an interaction between Atx2 and PABP across many species (Ciosk, 2004; Satterfield, 2006; Mangus, 1998). Data from C. elegans indicate that loss of Atx2 can result in misregulated translation, and in yeast Atx2 negatively regulates PABP. Consistent with these findings, this study has shown that Atx2 and PABP have opposing activities in modulating the progression of SCA3 toxicity in flies (Lessing, 2008).

Protein interaction studies indicate that Atx2 and Atx3 do not interact directly; in a survey of the interaction network of ataxia-associated proteins, Atx2 and Atx3 were separated by four nodes. However, the known function of PABP and the role of the PAM2 motif in localizing Atx2 to polyribosomes (Satterfield, 2006) together indicate that Atx2 and PABP modulate translation of specific transcripts. Since Atx2 is sufficient to cause neurodegeneration in the absence of pathogenic Atx3, Atx3 mRNAs cannot be the sole target of Atx2-PABP interactions, and additional transcript targets must be critical to normal neuronal integrity (Lessing, 2008).

Experiments in Drosophila demonstrate that the fly provides an outstanding complement to clinical observations and to vertebrate disease models. In this case, the fly has highlighted the significance of intriguing interactions between the genes that cause SCA2 and SCA3 diseases that can be supported by molecular and genetic findings. More specifically, these data indicate striking crosstalk between the pathways of normal Atx2 function and pathogenic Atx3 activity. Further understanding of both the Atx2 and Atx3 pathways may reveal insight into maintenance of neuronal integrity in a number of distinct disease situations (Lessing, 2008).

A Drosophila homolog of the polyglutamine disease gene SCA2 is a dosage-sensitive regulator of actin filament formation

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by the expansion of a CAG repeat encoding a polyglutamine tract in ataxin-2, the SCA2 gene product. The normal cellular function of ataxin-2 and the mechanism by which polyglutamine expansion of ataxin-2 causes neurodegeneration remain unknown. This study used genetic and molecular approaches to investigate the function of a Drosophila homolog of the SCA2 gene (Datx2). Like human ataxin-2, Datx2 is found throughout development in a variety of tissue types and localizes to the cytoplasm. Mutations that reduce Datx2 activity or transgenic overexpression of Datx2 result in female sterility, aberrant sensory bristle morphology, loss or degeneration of tissues, and lethality. These phenotypes appear to result from actin filament formation defects occurring downstream of actin synthesis. Further studies demonstrate that Datx2 does not assemble with actin filaments, suggesting that the role of Datx2 in actin filament formation is indirect. These results indicate that Datx2 is a dosage-sensitive regulator of actin filament formation. Given that loss of cytoskeleton-dependent dendritic structure defines an early event in SCA2 pathogenesis, these findings suggest the possibility that dysregulation of actin cytoskeletal structure resulting from altered ataxin-2 activity is responsible for neurodegeneration in SCA2 (Satterfield, 2002; full text of article).

Functions of Ataxin-2 orthologs in other species

ATX-2, the C. elegans ortholog of human Ataxin-2, regulates centrosome size and microtubule dynamics

Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. This study reports a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. ATX-2 influences MT behavior through γ-Tubulin (see Drosophila γ-Tubulin) at the centrosome. These data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics (Stubenvoll, 2016).

Search PubMed for articles about Drosophila Atx2

Albrecht, M. and Lengauer, T. (2004). Survey on the PABC recognition motif PAM2. Biochem. Biophys. Res. Commun. 316: 129-138. PubMed ID: 15003521

Al-Ramahi, I., et al. (2007). dAtaxin-2 mediates expanded Ataxin-1-induced neurodegeneration in a Drosophila model of SCA1. PLoS Genet. 3(12): e234. PubMed ID: 18166084

Andrei, M. A., et al. (2005). A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA 11: 717-727. PubMed ID: 15840819

Bilen, J., Liu, N., Burnett, B. G., Pittman, R. N. and Bonini, N. M. (2006). MicroRNA pathways modulate polyglutamine-induced neurodegeneration. Mol. Cell 24: 157-163. PubMed ID: 17018300

Buchan, J. R., Muhlrad, D. and Parker, R. (2008). P bodies promote stress granule assembly in Saccharomyces cerevisiae. J. Cell Biol. 183: 441-455. PubMed ID: 18981231

Cai, D., Chen, S. and Glanzman, D. L. (2008). Postsynaptic regulation of long-term facilitation in Aplysia. Curr. Biol. 18: 920-925. PubMed ID: 18571411

Ciosk, R., DePalma, M. and Priess, J. R. (2004). ATX-2, the C. elegans ortholog of ataxin 2, functions in translational regulation in the germline. Development 131(19): 4831-41. PubMed ID: 15342467

Das, S., et al. (2011). Plasticity of local GABAergic interneurons drives olfactory habituation. Proc. Natl. Acad. Sci. 108(36): E646-54. PubMed ID: 21795607

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date revised: 2 January 2023

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