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Human Molecular Genetics Advance Access originally published online on October 18, 2007
Human Molecular Genetics 2008 17(2):293-302; doi:10.1093/hmg/ddm305
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

ubiquilin antagonizes presenilin and promotes neurodegeneration in Drosophila

Atish Ganguly1,3,{dagger}, R.M. Renny Feldman1,3,{dagger},{ddagger} and Ming Guo1,2,3,*

1 Department of Neurology 2 Department of Pharmacology 3 Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA

* To whom correspondence should be addressed. Tel: +1 3102069406; Fax: +1 3102069406; Email: mingfly{at}ucla.edu

Received August 24, 2007; Revised September 24, 2007; Accepted October 10, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The majority of familial Alzheimer's disease (AD) cases are caused by mutations in presenilins, therefore, identifying regulators of presenilins is crucial for understanding AD pathogenesis. Ubiquilin 1 (UBQLN1) binds Presenilins in mammalian cells; however, the functional significance of this interaction in vivo remains unclear. Moreover, while genetic variants in UBQLN1 have recently been reported to associate with an increased risk for AD, whether these variants have altered function is unknown. Here, we show that Drosophila Ubiquilin (Ubqn) binds to Drosophila Presenilin (Psn), and that loss of ubqn function suppresses phenotypes that arise from loss of psn function in vivo. In addition, overexpression of ubqn in the eye results in adult-onset, age-dependent retinal degeneration, which is at least partially apoptotic in nature. The degeneration associated with ubqn overexpression can also be suppressed by psn overexpression and enhanced by expression of a dominant negative version of Psn. Remarkably, expression of the human AD-associated variant of UBQLN1 leads to more severe degeneration than does comparable expression of the human wildtype UBQLN1. Together, these data identify Ubqn as a regulator of Psn, support an important role for UBQLN1 in AD pathogenesis, and suggest the possibility that expression of a human AD-associated variant can cause neurodegeneration independent of amyloid production.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
One critical pathogenic event leading to Alzheimer's disease (AD) is the progressive accumulation of amyloid plaques in the brain. These plaques consist primarily of the Amyloid β (Aβ) peptides, Aβ40 and Aβ42, which are generated by sequential cleavage of the Amyloid Precursor Protein (APP) by two proteases known as β-secretase (BACE) and {gamma}-secretase (1). {gamma}-Secretase activity resides in a membrane-spanning multi-protein complex, which minimally contains four components, of which Presenilin 1 (PS1) or Presenilin 2 (PS2) constitute the catalytic core (2,3). Mutations in three genes, APP, PS-1 and PS-2 lead to early onset familial forms of AD (4–7), and a polymorphism in Apolipoprotein E (8,9) is associated with increased susceptibility to late onset AD. These genes are believed to account for as little as 30% of the genetic variance in AD susceptibility, suggesting that important genes remain to be identified (10).

UBQLN1, also known as PLIC1, belongs to a family of evolutionarily conserved proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains (11) (Fig. 1A). UBQLN1 also has a central region that contains Sti1 repeats (Fig. 1A), which are associated with chaperone activity in other proteins (12). Several observations suggest links between UBQLN1 and AD. First, the genomic region containing UBQLN1, 9q22, has been identified as containing one major candidate gene for conferring a predisposition to late onset AD (13,14). Some reports (15,16), but not others (17–20), suggest that genetic variants in the UBQLN1 gene, including one known as UBQ-8i that deletes one Sti1 repeat (Fig. 1A), are associated with increased risk for the more prevalent late-onset forms of AD. Secondly, in post-mortem AD brains, UBQLN1 is found in neurofibrillary tangles (11), a pathological hallmark of AD along with amyloid plaques (1). Thirdly, UBQLN1 interacts with APP in cultured cells, and downregulation of UBQLN1 expression alters APP levels and Aβ secretion through modulation of APP trafficking (21). Finally, UBQLN1 was found to bind PS1 and PS2 in a yeast two-hybrid screen (11). UBQLN1 and Presenilins partially co-localize in cultured cells (11,22), and are found to be localized in close physical proximity by a photobleach dequenching FRET assay in human brain sections (22). However, the biological significance of this physical interaction is unclear. On the one hand, overexpression of UBQLN1 in cultured cells inhibits the degradation of ubiquitinated forms of PS (11,23), suggesting that UBQLN1 positively regulates PS levels. On the other hand, observations by one group (24), but not a second group (21), suggest that UBQLN1 negatively regulates PS endoproteolysis. Since endoproteolysis is required to generate fragments of PS, which heterodimerize and become key components of the active {gamma}-secretase complex, this result could be interpreted as UBQLN1 negatively regulating PS function. With these interesting yet conflicting links between UBQLN1 and PS, it is important to determine if UBQLN1 interacts with PS in vivo and, if so, whether UBQLN1 promotes or antagonizes PS function. In addition, the implication of UBQLN1 variants as risk factors for AD requires functional studies of the gene and its disease associated alleles to determine if and how UBQLN1 contributes to AD pathogenesis.


Figure 1
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  		Figure 1. Human and fly Ubiquilin share a conserved domain structure and physically interact with Presenilin. (A) Schematic depicting human UBQLN1, UBQ-8i and fly Ubqn. (B) Western blots of lysates from larvae ubiquitously expressing indicated transgenes. ubqn-RNAi leads to a loss of Ubqn signal. The expression levels of UBQLN1 and UBQ-8i are comparable, and Ubqn levels are unaffected by the presence of either human variant (also see Fig.  3H). A non-specific band (*) serves as an internal loading control. (C) GST pulldown assay. The input lane shows Myc-Psn levels from 1% of the S2 cell lysates used for each pulldown. Compared with control (GST alone), Ubqn, but not Ubqn{Delta}UBA, binds to full length Psn (asterisk). Arrowhead marks higher molecular weight forms of Psn. Both human UBQLN1 and the UBQ-8i variant show similar binding to Psn. Coomassie blue staining reveals equivalent amounts of proteins used in the pulldown.

 
Drosophila is a useful system to study AD-related processes. Drosophila contains homologs of APP and all four components of the {gamma}-secretase complex, and expression of human APP in Drosophila results in cleavage by endogenous {gamma}-secretase activity (25,26). Drosophila is also useful for studying presenilin functions (27–29) and neurodegeneration (30,31). Here, we explore roles for ubiquilin in AD-related processes using Drosophila as a model. Our data, together with a related study (32), suggest that ubiquilin antagonizes presenilin function in vivo. In addition, we show that the expression of a human AD associated variant in Drosophila causes an earlier onset, and more severe adult-onset eye degeneration when compared with wild-type human UBQLN1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Drosophila homolog of UBQLN1 binds to Psn via its UBA domain in vitro
The Drosophila melanogaster genome contains a single UBQLN1 homolog (CG14224, hereafter called ubqn). Ubqn shares 46% amino acid identity and 63% similarity with human UBQLN1 (Supplementary Material, Fig. S1). Both proteins share predicted N-terminal UBL and C-terminal UBA domains, as well as four Sti1 repeats (Fig. 1A). To facilitate the characterization of ubqn, we generated polyclonal antibodies against Ubqn that are highly specific (Fig. 1B). Furthermore, we generated two RNAi constructs targeted to two independent regions of ubqn to carry out general and tissue-specific silencing of ubqn using the UAS-GAL4 system (33). Ubiquitous expression of ubqn-RNAi using the daughterless (da)-Gal4 driver resulted in a loss of detectable Ubqn (Fig. 1B). Both ubqn RNAi transgenes silenced ubqn expression to similar levels (data not shown) and gave identical phenotypes in all experiments. In addition, we also generated a transgene for overexpressing ubqn (UAS-ubqn), and the effect of ubqn overexpression was confirmed with anti-Ubqn antibodies (Fig. 1B and 3H).

UBQLN1 has been shown to directly bind PS in vitro (11). To determine if Ubqn could bind Psn, we mixed purified GST and GST-Ubqn fusion proteins with lysates from Schneider 2 (S2) cells transfected with Myc-tagged Psn. Indeed, Myc-Psn specifically bound GST-Ubqn, but not GST alone (Fig. 1C). Deletion of the UBA domain abrogated the interaction between Ubqn and Psn, indicating that the UBA domain is required for this interaction (Fig. 1C). Moreover, human UBQLN1 also bound to fly Psn (Fig. 1C), indicating that the physical interaction of Ubiquilin and Presenilin is conserved across species.

Loss-of-function studies reveal antagonistic interactions between ubqn and psn
A key question we sought to address is whether there is any genetic interaction between ubqn and psn in vivo and, if so, whether ubqn promotes or antagonizes psn function. Silencing of ubqn in all somatic cells using a ubiquitous Gal4 driver (tubulin-Gal4 or da-Gal4) resulted in lethality, suggesting that ubqn is essential for development. In contrast, silencing of ubqn in the developing wing using MS1096-Gal4 resulted in viable flies with wing phenotypes, including crumpled wings and defects in wing veins (Fig. 2B). These phenotypes were due to reduction of ubqn function since overexpression of ubqn suppressed these phenotypes (Fig. 2C). Overexpression of UBQLN1, which the ubqn RNAi does not recognize, also partially suppressed these phenotypes (data not shown), thus arguing against non-specific RNAi effects.


Figure 2
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  		Figure 2. ubqn antagonizes psn function in vivo. (A–F) Reduction of psn function suppresses, whereas psn overexpression enhances, wing phenotypes due to silencing of ubqn function. Silencing of ubqn function (B) causes multiple wing phenotypes. These include severely shortened longitudinal wing veins L3–L5, with L4 and L5 (red arrowheads) most consistently affected, missing crossveins, and a wrinkled wing blade. ubqn overexpression rescues the ubqn RNAi phenotypes (C). Though psnC1/+ or psnC2/+ flies have no wing phenotypes, they strongly suppress the phenotypes due to loss of ubqn function, as the veins L3–L5 extend further (D). psn overexpression does not show a phenotype (F), but it strongly enhances phenotypes due to loss of ubqn function (E). Genotypes: (A), w MS1096-Gal4/Y; UAS-GFP/+; (B) w MS1096-Gal4, UAS-RNAi-ubqn/Y; (C) w MS1096-Gal4, UAS-RNAi-ubqn/Y; UAS-ubqn/+; (D) w MS1096-Gal4, UAS-RNAi-ubqn/Y; psnC1/+; (E) w MS1096-Gal4, UAS-RNAi-ubqn/Y; UAS-psn/+; (F) w MS1096-Gal4/Y; UAS-psn/+. (G and H) Silencing of psn function results in pupal lethality (G) with almost no adult flies emerging. Simultaneous silencing of both ubqn and psn in the same tissue shows a partial rescue of the lethality, and allows the development of many pharate adults (H), i.e. pupae with visible adult structures such as red eyes. Genotypes for psn–: w MS1096-Gal4; UAS-RNAi-psn/+. Genotype for psn– ubqn–: w MS1096-Gal4, UAS-RNAi-ubqn; UAS-RNAi-psn/+. Error bars indicate standard deviation.

 
Consistent with a direct physical binding between Ubqn and Psn, we observed a strong dosage sensitive genetic interaction between ubqn knockdown and heterozygous loss-of-function of psn. Reduction of just 50% gene dosage of psn dominantly suppressed the ubqn loss-of-function phenotypes, though it did not cause any wing phenotypes on its own (Fig. 2D). Furthermore, although overexpression of psn in the wing had no phenotype in isolation (Fig. 2F), it strongly enhanced the wing phenotypes due to silencing of ubqn function (Fig. 2E). These results suggest ubqn antagonizes psn function in vivo.

To further test this hypothesis, we asked if loss of ubqn could rescue phenotypes associated with loss of psn. Silencing of psn function using MS1096-Gal4 resulted in early pupal lethality with almost no viable flies (Fig. 2G). Since the wing is not required for viability, this lethality is likely due to expression of the driver in tissues other than the developing wing. These pupae arrested early during development prior to the appearance of any visible adult structures. However, simultaneous silencing of ubqn and psn in the same tissue resulted in a number of viable flies (Fig. 2G). Even for arrested pupae, development proceeded farther, with adult structures such as eyes and abdomens now apparent (Fig. 2H). These observations suggest that the phenotypes caused by loss of psn function can be suppressed by a simultaneous loss of ubqn function, again indicating that ubqn antagonizes psn in vivo.

UBQLN1 has previously been reported to influence both the levels and the endoproteolytic processing of PS in mammalian cells (11). To explore the possibility that Ubqn antagonizes Psn function by regulating either the levels or the endoproteolysis of Psn, we manipulated Ubqn levels in both transgenic animals and S2 cells, but failed to observe any consistent differences in Psn levels or endoproteolysis (data not shown). Thus, at least in Drosophila, Ubqn might not antagonize Psn activity at the level of either Psn stability or endoproteolysis.

Overexpression of ubqn in the eye induces age-dependent neurodegeneration
UBQLN1 variants reportedly have a dominant inheritable association with an increased risk for AD, since the presence of one copy of the variant confers a predisposition to the disease (15). Thus, we explored the hypothesis that overexpression of ubqn can contribute to neurodegeneration. We focused our studies on the Drosophila eye since it is dispensable for viability and fertility, is highly enriched for neurons, and has been widely used to assess neurodegeneration in response to expression of human neurodegenerative disease genes (reviewed in 30,34). ubqn overexpression was accomplished by using GMR-Gal4 that drives transgene expression in all differentiated cells of the eye.

In young adult flies, ubqn overexpression in the eye, confirmed using anti-Ubqn antibodies (Fig. 3H), resulted in wild-type-appearing eyes with no developmental defects. To assay for adult onset neurodegeneration, flies were aged for 3 days, 18 days or 33 days post-eclosion, and their eyes were sectioned and stained with Toluidine Blue. Control flies (eye-specific LacZ expression) did not show significant alterations in tissue morphology over time (Fig. 3A–C). For ubqn overexpression, 3-day-old fly eyes showed normal tissue morphology, with organized arrays of photoreceptors and non-neuronal pigment cells (Fig. 3D). However, by day 18, ubqn overexpression resulted in readily visible neurodegeneration in photoreceptors with vacuoles and some cell loss, as well as degeneration in pigment cells (Fig. 3E). Eye degeneration progressively worsened in 33-day-old flies (Fig. 3F). Thus, overexpression of ubqn in the eye induces adult-onset, age-dependent retinal degeneration. Notably, there were no externally visible, morphological phenotypes in either control or ubqn overexpressing flies at any time.


Figure 3
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  		Figure 3. Eye-specific overexpression of ubqn causes adult onset, age-dependent retinal degeneration, which is apoptotic in nature. Toluidine blue stained horizontal sections of adult male eyes are shown, with red and green arrows pointing to a pigment cell and a photoreceptor, respectively. Control flies (expressing LacZ) do not show retinal degeneration with age (A–C). ubqn overexpression flies show normal histology at day 3 (D). However, they display a marked age-dependent increase in degeneration manifested as vacuolation (yellow arrowheads), cell loss and disorganization (E and F). (G) Overexpression of diap1 alone does not cause any retinal degeneration (data not shown) while simultaneous overexpression of diap1 with ubqn results in a marked rescue in the organization of the cells, as well as a reduction in the number of vacuoles. Regions of missing retinal tissue in some sections (blue arrowhead) suggest that this rescue is not complete. Genotypes: (A–C), w; UAS-LacZ/+; GMR-Gal4/+; (D–F), w; UAS-ubqn/ GMR-Gal4. (G), w; UAS-diap1/ GMR-Gal4, UAS-ubqn. Scale bar: 50 µm. (H) Western blotting of adult head lysates from control (w1118) flies and GMR-Gal4 driven transgenes using anti-Ubqn antibodies (arrowhead), which are specific for Drosophila Ubqn and do not recognize UBQLN1. A non-specific band (marked with *) serves as an internal loading control. While ubqn overexpression shows elevated Ubqn levels (lane 3), other genotypes display levels of Ubqn that are comparable with wildtype (lane 1) (also see Fig. 1B).

 
Eye degeneration induced by ubqn overexpression is apoptotic in nature
Many, but not all, neurodegenerative events involve activation of caspase-dependent cell death (35). To determine if ubqn overexpression-induced degeneration required caspase activity, we assayed flies that simultaneously overexpressed Drosophila inhibitor of apoptosis (diap1), the broad specificity caspase inhibitor (35), in the eye. As shown in Fig. 3G, ubqn-induced cell degeneration was largely rescued by coexpression of diap1, suggesting that the degeneration is, at least partly, apoptotic in nature.

ubqn overexpression-induced eye degeneration depends on psn
Since ubqn antagonizes psn function in the wing, we asked if psn overexpression could rescue ubqn-induced eye degeneration. Overexpression of one copy of wild-type psn (GMR-psn) had neither any developmental phenotypes on its own, nor any degeneration at day 18 post-eclosure (Fig. 4B). However, it strongly suppressed the eye degeneration caused by ubqn overexpression (Fig. 4E). These data suggest that decreased Psn activity mediates the degeneration induced by ubqn overexpression.


Figure 4
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  		Figure 4. Overexpression of psn suppresses, whereas expression of dominant negative Psn enhances the eye degeneration induced by ubqn overexpression. Toluidine Blue stained horizontal sections of eyes from adult males aged 18 days post-eclosion. Though overexpression of psn in the eye (B) does not result in any degeneration compared with control (A), it largely suppresses the retinal degeneration due to ubqn overexpression (compare E with D). Though expression of PsnD257A results in only minimal degeneration (C), it strongly enhances the eye degeneration induced by ubqn overexpression (compare F with D). Vacuolation indicative of cell degeneration is highlighted by yellow arrowheads. To control for transcriptional imbalance, an identical number of transgenes were used for (B–F). Genotypes: (A) w; UAS-LacZ/+; GMR-Gal4/+; (B) w; UAS-LacZ/GMR-psn; GMR-Gal4/+; (C) w; UAS-LacZ/GMR-psnD257A; GMR-Gal4/+; (D) w; UAS-LacZ/+; GMR-Gal4, UAS-ubqn/ +; (E) w; GMR- psn/+; GMR-Gal4, UAS-ubqn/+; (F) w; GMR-psnD257A/+; GMR-Gal4, UAS-ubqn/+. Scale bar: 50 µm.

 
Recent studies indicate that in addition to its well-known role as the catalytic core of the {gamma}-secretase complex, Psn also functions in multiple {gamma}-secretase independent contexts (36), some of which may be important for AD pathogenesis. Therefore, we asked if ubqn-induced eye degeneration was mediated by the reduction of {gamma}-secretase dependent functions of Psn. The aspartyl protease activity of {gamma}-secretase depends on two aspartate residues in PS. Mutation of either residue generates a dominant negative form of PS (36). The eye-specific expression of a dominant negative version of Drosophila Psn (PsnD257A) (GMR-psnDN) suppressed {gamma}-secretase-dependent functions without causing any developmental defects (26). Remarkably, though expression of PsnD257A was associated with only minimal neurodegeneration by 18 days (Fig. 4C), it strongly enhanced the phenotypes due to ubqn overexpression, resulting in increased vacuolation and cell loss by day 18 (Fig. 4F). These strong genetic interactions suggest that psn is a key mediator of ubqn overexpression-induced eye degeneration, and are consistent with a hypothesis in which ubqn antagonizes {gamma}-secretase-dependent function of psn, thereby leading to progressive neurodegeneration.

Expression of a human UBQ-8i variant results in more severe age-dependent eye degeneration than expression of human wild-type UBQLN1
An alternative splicing variant of UBQLN1, UBQ-8i, is reportedly associated with an increased risk of AD (15). These AD patients have increased expression of the UBQ-8i message (15). UBQ-8i encodes a variant lacking one Sti1-repeat due to the deletion of coding exon 8 (Fig. 1A). This truncated protein is likely to a exert dominant effects since the presence of one copy of the UBQ-8i allele confers a predisposition to AD. Since overexpression of fly ubqn results in eye degeneration, we asked if eye-specific expression of human UBQLN1 or UBQ-8i also show age-dependent degeneration.

As with overexpression of fly ubqn, eye-specific expression (using GMR-Gal4 driver) of either human form did not cause any developmental defects, and eye sections at day 3 showed normal histology (Fig. 5A and D). By day 18 post-eclosion, expression of wild-type UBQLN1 resulted in subtle, if any, neurodegeneration (Fig. 5B). However, by day 33, expression of UBQLN1 displayed visible degeneration (Fig. 5C). In contrast, expression of UBQ-8i led to an earlier age of onset and a more severe cellular degeneration with increased vacuolation and cell loss (Fig. 5E–F). By day 18, expression of UBQ-8i demonstrated pronounced cellular degeneration (Fig. 5E), which progressively worsened by day 33 (Fig. 5F). These phenotypic differences were not due to differences in expression levels of the two human isoforms, since western blotting of lysates with an anti-UBQLN1 antibody (Fig. 1B) indicates that the UBQ-8i variant is expressed at levels similar to wild-type UBQLN1. In addition, the expression of either human variant did not affect the levels of endogenous Ubqn (Fig. 1B and 3H). Together, these results suggest that UBQ-8i has an intrinsically greater capacity to induce eye degeneration.


Figure 5
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  		Figure 5. Expression of UBQ-8i displays enhanced eye degeneration and distinct developmental defects compared with expression of UBQLN1. UBQLN1 expression leads to adult onset, progressive retinal degeneration detectable at 33 days (C) but not at 18 days post-eclosion (B). Strikingly, UBQ-8i expression results in an earlier age of onset (day 18) as well as enhanced severity of this degeneration (compare B with E and C with F, respectively). The vacuolation (yellow arrowheads) and disorganization appear similar to that observed in animals overexpressing Drosophila ubqn (Compare C, F with Fig. 3F). (G and H) Scanning electron micrographs of eyes from adult flies expressing either UBQLN1 (G) or UBQ-8i (H) during development of the undifferentiated eye disc. UBQLN1 expression causes no phenotype while UBQ-8i expression results in outgrowths of cuticle through the eye (red arrowhead). Genotypes: (A–C), w; UAS-UBQLN1/+; GMR-Gal4/+; (D–F), w; UAS-UBQ-8i/+; GMR-Gal4/+; (G), w; ey-Gal4/UAS-UBQLN1; (H), w; ey-Gal4/UAS-UBQ-8i. Scale bar: 50 µm.

 
Furthermore, UBQ-8i also has the distinct ability to induce developmental defects when expressed in other tissues such as the undifferentiated eye imaginal disc [using eyeless (ey)-Gal4]. UBQLN1 expression showed a wild-type-appearing eye (Fig. 5G), whereas UBQ-8i expression resulted in a range of cuticle outgrowths through the eye tissue (Fig. 5H). These observations are consistent with the possibility that UBQ-8i is either hyperactive or possesses a different (neomorphic) activity when compared with the wild-type protein. Since we showed that Ubqn can bind to Psn, we asked if UBQ-8i differed from UBQLN1 in its ability to bind Psn. GST pull-down assays did not reveal any differences in the ability of UBQLN1 and UBQ-8i to bind Psn (Fig. 1C), suggesting that other aspects of UBQLN1 function are modified in the UBQ-8i variant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Previous studies have suggested links between UBQLN1 and AD. UBQLN1 is located at 9q22, a locus believed to contain a major AD risk gene (13,14), and UBQLN1 has been detected in neurofibrillary tangles in AD patient brains (11). UBQLN1 binds to PS (11) and modulates the trafficking of APP (21). Furthermore, a recent study has found that Drosophila ubqn can genetically interact with psn and influence the processing of human APP when overexpressed with human APP in the fly eye (32). Taken together, these observations emphasize the potential relevance of UBQLN1 in AD and underline the importance of studying the in vivo function of UBQLN1. Furthermore, some human genetic studies, but not others, have suggested that variants of UBQLN1 are risk factors conferring increased vulnerability to late-onset AD. Here, we have investigated the consequences of loss- and gain-of-function of ubqn, in the context of development and neurodegeneration. Our studies provide strong evidence that at least one aspect of the endogenous function of ubqn is to antagonize psn. This is likely accomplished, at least in part, by a direct physical interaction requiring the UBA domain of Ubqn. Many proteins have been found to bind Presenilins in various assays (37). However, with the exception of APP, Tau and components of the {gamma}-secretase complex, none has been shown to have strong in vivo genetic interactions with presenilins and to exhibit a genetic association with AD. We also found that ubqn overexpression can lead to eye degeneration and that a human AD-associated variant, UBQ-8i, has enhanced activity in inducing eye degeneration and causing developmental defects as compared to wild-type UBQLN1. Together, our studies provide in vivo functional support for a potential role of UBQLN1 in AD.

In a related study, Li et al. reported no phenotypes with eye-specific ubqn overexpression, based on observations of the external structure of the adult eye (32). Using the same GMR-Gal4 driver, we also observed no detectable defects in the external structure of the eye with ubqn overexpression. However, when we aged the flies and examined the inner structure of the eye, we observed adult-onset, progressive eye degeneration. The external eye largely comprises the lens, which is secreted by the underlying cone cells (38). Thus, when adult-onset eye degeneration (including photoreceptors and cone cells) occurs, it is not unusual to have a normal external structure along with a progressive degeneration of the underlying cells since the lens has already been secreted during development. Thus, while our data are in agreement with Li et al. (32), suggesting that there are no developmental defects upon eye-specific overexpression of ubqn, we have uncovered a post-developmental, adult onset degeneration, which we believe is more relevant to neurodegeneration.

The molecular mechanisms underlying the enhanced toxicity of UBQ-8i are unknown. In addition to being a catalytic core, PS also plays important roles in regulating the trafficking and assembly of {gamma}-secretase components, as well as functions independent of {gamma}-secretase activity. UBQ-8i might affect any or all of these processes. Moreover, UBQ-8i could also have altered interactions with normal binding partners of UBQLN1. In this context, the Sti1 repeats in UBQLN1, one of which is missing in UBQ-8i, have been shown to bind Protein Disulfide Isomerase (PDI) (39), an endoplasmic reticulum protein involved in stress responses (40). Furthermore, the Sti1 repeat-containing region of UBQLN1 mediates UBQLN1 homodimerization (41), thus the presence of UBQ-8i might dominantly alter the monomer/dimer ratio of UBQLN1. It remains to be explored if UBQ-8i has an altered ability to interact with PDI, and/or dimerize with itself.

Our data demonstrate that downregulation of Psn activity is important in mediating ubqn-induced neurodegeneration. This degeneration is likely to be amyloid-independent, since the Drosophila ortholog of APP, APP-like protein (Appl), does not contain a sequence homologous to Aβ (42), and there is no endogenous BACE activity reported in Drosophila (25). Familial AD (FAD) associated PS mutations have generally been considered to be ‘toxic gain-of-function’ leading to amyloid-dependent neurodegeneration. However, recent evidence suggests that both loss-of-function of PS and amyloid-independent degeneration might also be important in AD pathogenesis. Conditional knockout of PS in mice postnatally in the brain results in multiple defects including memory loss and synaptic plasticity impairments followed by neurodegeneration in the absence of amyloid production (43,44). Moreover, certain human mutations in PS cause neurodegeneration in the absence of amyloid pathology (45,46). The hypothesis that AD can be caused, at least in part, by loss of PS function is also supported by the following findings. In Drosophila (28,29), as in mammals (47–49), loss of psn function leads to inactivation of an endogenous {gamma}-secretase substrate Notch, which is a transmembrane receptor mediating cell–cell communication during development (50). This results in specific developmental phenotypes. Expression of fly psn mutations analogous to FAD mutations in PS cannot completely rescue these phenotypes, suggesting that these FAD equivalent psn mutations exhibit partial loss-of-function effects (27). Thus, FAD mutations in PS might have two consequences, one resulting in altered Aβ processing and amyloid-dependent neurodegeneration (Fig. 6A), and another leading to misregulation of other targets and ultimately amyloid-independent neurodegeneration (Fig. 6B). This could involve other substrates of {gamma}-secretase, or be due to {gamma}-secretase independent functions of PS.


Figure 6
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  		Figure 6. Model for the roles of PS and UBQLN1 in AD pathogenesis. Schematic depictions of PS (in red) and UBQLN1 (in green). Solid arrows indicate the existence of strong evidence supporting the connection, dashed lines with arrow suggest some evidence, while dashed lines with arrow and ‘?’ indicate speculative connections. (A) AD associated PS mutations lead to altered {gamma}-secretase cleavage of APP and subsequent neurodegeneration by amyloid-dependent mechanisms. Some AD-associated PS mutations might also be involved in amyloid-independent neurodegeneration (dashed arrow). (B) Loss of PS function in mice can lead to neurodegeneration via an amyloid-independent mechanism (43,44), indicating an alternate pathway. This could involve other substrates of {gamma}-secretase, or be due to {gamma}-secretase independent functions of PS. (C) UBQLN1 directly binds PS (11), and antagonizes PS activity (this work). Upregulated UBQLN1 activity, possibly associated with disease variants, might significantly inhibit PS function, resulting in neurodegeneration independent of amyloid production. Elevated UBQLN1 activity might also have downstream targets other than PS, which contributes to neurodegeneration directly.

 
It is important to note that ubiquilin has been shown to modulate APP processing and trafficking (21,32), and thus can potentially influence Aβ production and secretion. Our data suggesting that ubqn-induced degeneration can occur in an amyloid-independent fashion do not exclude the possibility that in humans, UBQLN1 may influence both amyloid-dependent and independent degeneration. While the role of amyloid-dependent processes is well established, the contribution of amyloid-independent degeneration to AD remains poorly studied. Thus, ubqn-induced amyloid-independent degeneration in flies may provide a good system to explore this process further.

The expression of PsnD257A was associated with only subtle eye degeneration. This could be because PsnD257A expression only partially compromised {gamma}-secretase activity (26). This was indicated by the lack of developmental defects that are normally associated with complete loss of psn function (26,51). Thus, while PsnD257A expression failed to reach the threshold for causing degeneration, ubqn overexpression led to a more severe inhibition of Psn activity. We were unable to determine if complete loss of psn function led to more robust adult-onset degeneration due to severe defects in eye development. Alternatively, it is possible that psn represents only one of multiple targets associated with ubqn overexpression (Fig. 6C). Since psn expression can rescue the ubqn overexpression-induced eye degeneration, Psn is likely the primary target of Ubqn.

UBQLN1 has also been found in the brains of patients with other neurodegenerative diseases. UBQLN1 is present in Lewy bodies in Parkinson's disease patients (11), and colocalizes with polyglutamine aggregates (12). UBQLN1 is also associated with neuronal intranuclear inclusions in a mouse model of Huntington's disease (52), and ubiquilin overexpression in C. elegans suppresses polyglutamine-induced toxicity (53). Thus, UBQLN1 may play a more general role in neurodegenerative diseases. Alternatively, while UBQLN1 may be involved in multiple processes under normal circumstances, its variants may be risk factors specifically for AD. It will be interesting to determine if UBQLN1 has a direct role in the pathogenesis of other neurodegenerative disorders, and our fly model may also be useful for elucidating interactions of ubqn with processes related to these diseases.


   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
DNA construct and antibody generation
A microRNA-based technology (54) was used for RNAi silencing. To silence ubqn, the coding region and the 3'-UTR were independently targeted. psn was targeted within its coding region. PCR products of these microRNA precursors were cloned into pUASt.

To generate UAS-ubqn, an EcoRI–XhoI fragment of the ubqn cDNA (EST LD38919, the Drosophila Genomic Resource Center) was excised and cloned into pUASt. To generate UAS-UBQLN1 or UAS-UBQ-8i, a NotI–Acc65I fragment (IMAGE clone 5263680) or a NaeI–XhoI fragment (IMAGE clone 4110398) was cloned into pUASt, respectively. GMR-psn was generated by inserting psn cDNA into pGMR. To inducibly express Psn in S2 cells, the N-terminally myc-tagged psn (a gift from M. Fortini) was excised and placed into pMT (Invitrogen). For generating GST-Ubqn, GST-Ubqn{Delta}UBA, GST-UBQLN1 and GST-UBQ-8i, the entire ubqn open reading frame (ORF), the sequence encoding amino acids 1–490 of Ubqn, and the ORFs of UBQLN1 and UBQ-8i were PCR amplified and cloned into pGEX-4T1 (Amersham), respectively. All cloned PCR products were confirmed by DNA sequencing.

A fusion of GST to Ubqn residues 85–490 (with an intervening TEV protease recognition site), corresponding to the region between the UBL and UBA domains, was purified, cleaved by TEV protease to remove GST and used to immunize rabbits (Covance).

Drosophila genetics
Multiple fly lines were generated (Rainbow Transgenic Flies) and tested for each transgene. Null alleles of psn (psnC1 and psnC2) were generously provided by Gary Struhl (28), UAS-psn flies, by Mark Fortini (55) and UAS-diap1, by Bruce Hay (56). GMR-PsnD257A was as described previously (26).

For pupal rescue experiments, homozygous UAS-RNAi-psn (insertion on an autosome) males were crossed to females homozygous either for MS1096-Gal4 or MS1096-Gal4, UAS-RNAi-ubqn (on the X chromosome). Four-day collections of progeny were aged and scored in two ways: the number of pupae in which red eye pigments were visible, and the number of viable adult escapers were each scored as a percent of total pupae present. Over 200 pupae were counted for each experiment. Three independent experiments were performed at different times and temperatures.

Histology
For examining eyes, adult males were aged at 25°C, their heads fixed in fixative (2% paraformaldehyde, 1% glutaraldehyde (Electron Microscopy Sciences), 0.1M Phosphate buffer pH 7.2), postfixed in 2% osmium tetroxide, dehydrated and embedded in Epon as described (57). Two-micron sections were prepared on a Leica microtome and stained with Toluidine Blue. Each set of experiments with controls were set up at the same time and prepared in identical fashion. A scanning electron microscope (Hitachi 2460 N) was used to take images of freshly sacrificed flies. For examining wings, wings from adult males were dissected in ethanol, mounted in Fluormount (Southern Biotech), and imaged on an Olympus SZX7 under identical magnification.

Lysate preparation and western blotting
S2 cells were harvested (24 h post-induction), and the cell pellets lysed in modified RIPA buffer [50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 5 mM EDTA, 5 mM EGTA, 2 mM DTT, Complete protease inhibitor (Roche)]. Total soluble protein was measured by Bradford Assay (BioRad). Lysates were boiled in 2x Laemmli sample buffer and resolved by SDS–PAGE followed by Western blotting.

Third instar larvae (22°C) and heads from age and sex-matched adults were lysed in lysis buffer [1X RIPA, 10 mM EDTA], and soluble protein content determined as earlier. For larvae, 20 µg of total protein per genotype and, for adults, total protein from four heads per genotype was analyzed by western blotting, respectively. Antibodies used were anti-Myc (Upstate), anti-Ubqn, anti-Tubulin and anti-UBQLN1 (Affinity Bioreagents).

S2 cell culture and transfection
S2 cells were grown in Schneider's Drosophila medium (Invitrogen) supplemented with 10% FBS, 50 units/ml penicillin and 50 µg/ml streptomycin at room temperature. Transfections were carried out using MaxFect transfection reagent (Molecula). Typically, 1.5x106 cells plated in a 12-well dish were transfected with 0.7–1 µg total plasmid DNA plus 5 µl MaxFect reagent. Metallothionein promoter expression was induced with 0.5 mM copper sulfate 24 h after transfection.

GST pulldown assay
Ten micrograms GST fusion proteins were retained on 15 µl glutathione beads, and mixed with 400 µg S2 cell lysate in 1 mL total volume. Retained proteins were eluted by boiling in Laemmli sample buffer, and detected by western blotting.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This work was supported by a UCLA Neurobehavioral Genetics training grant to R.F. as well as grants and funds from the National Institute of Health (RO1 and KO8), the Alfred P. Sloan Foundation, the Alzheimer's Association, the American Federation of Aging Foundation, the Larry Hillblom Foundation, the Glenn Family Foundation and the Ellison Medical Foundation to M.G.


   ACKNOWLEDGEMENTS
 
We thank Hao Zhao, Hong Yu and Jinhui Wang for generating several transgenic flies, Rosalind Young from Benzer lab for teaching eye sectioning, Frank Laski for use of his microtome, Mark Fortini, Gary Struhl and Bruce Hay for fly stocks and DNA, C. Lee, B. Hay, M. Dodson and I. Clark for comments on the manuscript, and the Guo lab members for discussions.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} Present address: Gevo, Inc., Pasadena, CA 91106, USA. Back


   REFERENCES
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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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