Differential ubiquitination and degradation of huntingtin fragments modulated by ubiquitin-protein ligase E3A Kavita P. Bhata, Sen Yana,b, Chuan-En Wanga, Shihua Lia, and Xiao-Jiang Lia,b,1 a Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322; and bState Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin and alters the levels of toxic proteins. In Huntington disease, polyglutamine expansion causes N-terminal huntingtin (Htt) to misfold, inducing neurodegeneration. Here we report that shorter N-terminal Htt fragments are more stable than longer fragments and find differential ubiquitination via K63 of ubiquitin. Aging decreases proteasome-mediated Htt degradation, at the same time increasing K63-mediated ubiquitination and subsequent Htt aggregation in HD knock-in mice. The association of Htt with the K48-specific E3 ligase, Ube3a, is decreased in aged mouse brain. Overexpression of Ube3a in HD mouse brain reduces K63-mediated ubiquitination and Htt aggregation, enhancing its degradation via the K48 ubiquitin–proteasome system. Our findings suggest that aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt. misfolding

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untington disease (HD) is caused by an autosomal-dominant mutation in the HD gene that results in an expansion of CAG triplets (n > 35), leading to an expansion of polyglutamine (polyQ) repeats in the N-terminal region of huntingtin (Htt), as well as protein misfolding and aggregation (1, 2). Although Htt is ubiquitously expressed in the brain and body, mutant Htt (mHtt) causes selective neurodegeneration that preferentially occurs in the striatum and extends to other brain regions as disease progresses (1, 3, 4). The selective neurodegeneration in HD is apparently associated with the age-dependent accumulation of misfolded Htt in neuronal cells, which results in the formation of aggregates in the brain. Brains from patients with HD show Htt aggregates in neuronal nuclei and processes, which are formed by N-terminal fragments of mHtt (5, 6). The N-terminal fragments of mHtt in the nucleus can dysregulate gene transcription, and mHtt in neuronal processes can affect intracellular trafficking and neuron transmitter release (7–10). Similarly, most age-dependent neurodegenerative diseases, including Alzheimer’s disease and Parkinson disease, show increased accumulation of misfolded proteins in neuronal cells with age (11, 12). Thus, understanding how mHtt accumulates in the brain will shed light on the mechanisms for several age-dependent neurodegenerative diseases. The accumulation of misfolded proteins is caused largely by impaired clearance of these proteins in aged neurons (13). Previous studies showed that the ubiquitin–proteasome system (UPS) and the autophagic pathway can degrade mHtt (14–20). Indeed, the neuronal aggregates seen in HD and other neurodegenerative diseases are found to contain ubiquitinated proteins (5, 6). Although these ubiquitin-enriched aggregates are a hallmark of HD and other neurodegenerative diseases, how they are linked to the development of neurodegeneration remains unclear. Previous studies revealed that Htt aggregates are more likely to be ubiquitinated in the nucleus than in the cytoplasm (6, 21, www.pnas.org/cgi/doi/10.1073/pnas.1402215111

22), although the mechanism behind this difference is unclear. Ubiquitination is important not only for diverse cellular functions, such as trafficking and localization of proteins, but also for degradation of misfolded proteins via the 26S proteasome (23). Ubiquitination involves the concerted action of three enzymes: ubiquitin-activating (i.e., E1), ubiquitin-conjugating (i.e., E2), and a number of ubiquitin-ligating (i.e., E3) enzymes (24), which confer substrate specificity by facilitating the interaction between a particular substrate and an E2 (25). The E3 ubiquitin ligase Ube3a (also named E6-AP) is involved in catalyzing the last but crucial step in ubiquitination (26) and is also implicated in several neurodegenerative diseases, including Alzheimer’s disease, Parkinson disease, ALS, and spinocerebellar ataxia type 1 (SCA1) (27–29). Although these observations underscore the importance of Ube3a and other E3 ligases, we still do not know how ubiquitination of mHtt regulates its degradation. Here we demonstrate that toxic mHtt fragments are stable and exhibit differential polyubiquitination patterns. Furthermore, Ube3a plays a critical role in mHtt ubiquitination, and its decline with age contributes to differential Htt ubiquitination and degradation. Overexpression of Ube3a increases mHtt clearance by enhancing ubiquitin-mediated proteasomal degradation, thereby reducing the accumulation of mHtt in HD knock-in (KI) mouse striatum. Our findings offer valuable insight into the age-dependent accumulation and aggregation of mutated Htt, as well as an additional therapeutic target for HD. Significance Ubiquitination of misfolded proteins is an important process in a variety of age-dependent neurodegenerative diseases including Alzheimer’s disease, Parkinson disease, and Huntington disease (HD). However, we know little about how ubiquitination regulates the degradation and accumulation of misfolded proteins. By using HD knock-in mice and cultured cells, we found that the pathogenic form of the HD protein is differentially ubiquitinated via K48 and K63 in ubiquitin. K48-mediated ubiquitination promotes huntingtin (Htt) degradation while K63mediated ubiquitination accelerates its aggregation. The K-48– mediated ubiquitination depends on Ube3a, whose expression declines in aged brain. Overexpression of Ube3a in old HD KI mouse brain can reduce mutant Htt accumulation and aggregation. The age-dependent decline in Ube3a may contribute to the late-onset neuropathology in HD. Author contributions: K.P.B., S.L., and X.-J.L. designed research; K.P.B., S.Y., and C.-E.W. performed research; S.Y. and C.-E.W. contributed new reagents/analytic tools; K.P.B., S.L., and X.-J.L. analyzed data; and K.P.B. and X.-J.L. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1402215111/-/DCSupplemental.

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Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved March 7, 2014 (received for review February 4, 2014)

Results Differential Stability and Ubiquitination of N-terminal mHtt Fragments.

Studies have shown that full-length mHtt is cleaved to form various toxic N-terminal fragments that, in turn, form intranuclear and neuropil aggregates (20, 30–32). Mouse models expressing N-terminal fragments display a rapidly progressive phenotype (33, 34) whereas mouse models that express full-length mHtt, including KI models, display a slowly progressive phenotype (35–37). We thought it would be interesting to examine how different N-terminal Htt fragments persist in cells. To explore this, we performed cycloheximide chase assays to determine the t1/2 of three N-terminal mHtt fragments: 1–67 aa of exon1 Htt with an additional 150Q repeat (N67-150Q), 1–212 aa of Htt with a 120Q repeat (N212-120Q), and 1–508 aa of mHtt with a 120Q repeat (N508-120Q; Fig. 1A). We found that the shortest fragment of N67-150Q has the longest t1/2 of approximately 16 h, whereas N212-120Q has a t1/2 of 5 h, and N508-120Q has a t1/2 of only 1.5 h (Fig. 1B). Inhibiting proteasomal activity with MG132 stabilized the level of N508-120Q, making it insignificantly different from the shorter Htt fragments. Although the autophagic inhibitor bafilomycin A could also reduce the degradation of N508120Q, N508-120Q appeared to degrade faster than N67-150Q and N212-120Q (Fig. 1B). These results suggest that shorter N-terminal fragments of Htt are more stable than longer fragments and are preferentially degraded by the UPS. There are seven lysine (K) residues in the ubiquitin molecule (K6, K11, K27, K29, K33, K48, and K63), which account for the diversity of the ubiquitin signal on the target proteins (38). Mutating these lysine residues to arginine (R) blocks any polyubiquitination at those sites (39). By using antibodies specific to K48 and K63 of ubiquitin, we found that Htt aggregates in HD KI mouse brains are ubiquitinated by K48 and K63 of ubiquitin (Fig. S1). These differentially ubiquitinated Htt proteins may have different functions, as a previous study has demonstrated

Fig. 1. Shorter N-terminal Htt fragments have longer half-lives in aging HD KI mice. (A) Schematic representation of N-terminal Htt fragments used in this study. (B) Cycloheximide chase (100 μM) assay of HEK293 cells to determine the t1/2 of transfected mHtt fragments (N-67-150Q, 212-120Q, and N508-150Q). The transfected cells were untreated or treated with MG132 (10 mM) or bafilomycin A (100 nM). The relative protein levels were assessed by densitometric analysis of the protein bands (Right), and the value at 0 h was considered 100%. All Western blots are representative of results from at least three independent experiments, and data are presented as mean ± SEM. NT, nontransfected cells.

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that differential polyubiquitination via K48 and K63 regulates apoptosis differently (40). Because how mHtt is ubiquitinated remains unknown, we next examined the ubiquitination topologies of Htt by expressing various ubiquitin mutants that have replaced specific lysine residues with arginine (Fig. 2A). We focused on ubiquitin K48- and K63-mediated ubiquitination, as K48-mediated ubiquitination normally results in protein degradation by the UPS, whereas K63-mediated ubiquitination leads to protein aggregation (39, 41–43). We previously established HEK293 cell lines that stably express full-length normal Htt (fHtt-23Q) and mHtt (fHtt120Q) (20). In these cells, we transfected WT ubiquitin, K48, which carries only one lysine at residue 48, and K48R, which replaces lysine with arginine at amino acid position 48. We also transfected mutant ubiquitin, K63 or K63R, which contains only one lysine at the residue 63 or replaces lysine with arginine at amino acid position 63. Htt in transfected cells was then immunoprecipitated, and the Htt immunoprecipitates were probed with an antibody to ubiquitin to detect Htt ubiquitination. Although transfection of K48 led to reduced ubiquitination of normal and mutant Htt compared with transfection of WT ubiquitin, K48R transfection appeared to selectively result in a greater extent of ubiquitination of fHtt-120Q than WT ubiquitin transfection (Fig. 2B). These results suggest that ubiquitination of Htt is carried out mainly by lysine residues other than K48. In contrast, transfection of K63 and WT ubiquitin led to a similar extent of ubiquitination in normal Htt and mHtt. Importantly, mHtt ubiquitination levels were dramatically reduced when K63 was mutated to arginine, whereas normal Htt maintains a similar level of ubiquitination (Fig. 2B), indicating that polyQ expansion may alter mHtt topology, leading mHtt to be ubiquitinated preferentially via K63, whereas its WT counterpart maintains a propensity for other lysine residues. Because degraded Htt fragments are likely ubiquitinated and then removed by the UPS, it was important to verify that N-terminal mHtt fragments are also preferentially ubiquitinated via K63 in ubiquitin. We therefore transfected N67-150Q, N212-120Q, or N503-120Q with ubiquitin or its mutants at K63. Immunoprecipitation of these mHtt fragments also revealed that replacing lysine with arginine (K63R) markedly reduced mHtt ubiquitination (Fig. 2C). However, replacing K48 with arginine (K48R) did not alter the ubiquitination of N-terminal mHtt (Fig. 2D). These results also suggest that N-terminal Htt fragments are ubiquitinated mainly via K63 in ubiquitin. Because protein degradation in the nucleus is mediated primarily via the UPS, we wanted to investigate whether the ubiquitination of nuclear mHtt also depends on K63. To this end, we generated nuclear localization sequence (NLS)-tagged N212120Q and confirmed its nuclear localization in HEK293 cells (Fig. S2A). In vitro ubiquitination assays indicated that NLS-tagged N212-120Q is also preferentially ubiquitinated via lysine 63 of ubiquitin (Fig. S2 B and C). Taken together, the preferential ubiquitination of N-terminal mHtt fragments via K63 of ubiquitin indicates a specific conformation mediated by polyQ expansion, which may confer the propensity for aggregation. The E3 Ligase Ube3a Binds to mHtt N-terminal Fragments. Although protein ubiquitination via K63 in ubiquitin often leads to protein aggregation, K48-mediated ubiquitination normally leads to protein degradation via the UPS. However, this process requires E3 ubiquitin ligases, which catalyze the last rate-limiting step in the process of ubiquitination (44). A number of E3 ligases are implicated in neurodegenerative diseases, including Alzheimer’s disease, Parkinson disease, ALS, and SCA1 (27–29). By examining several E3 ligases in Htt immunoprecipitates from the striatum tissues of HD KI mice, we found that Ube3a, a HECT domain E3 ligase, associates with mHtt, and this association is decreased in old KI mouse brain striatum (Fig. 3A). Results in the cortex and cerebellum of HD KI mice were similar (Fig. S3), indicating that the decreased binding of Ube3a to mHtt is a phenomenon in aged HD KI mice brain. To assess the association of Ube3a with N-terminal mHtt fragments, we performed a coimmunoprecipitation assay in HEK293 cells transfected with N-terminal Htt Bhat et al.

Fig. 2. Polyubiquitination of full-length and N-terminal fragments of mHtt preferentially uses lysine 63 (K63) of ubiquitin. (A) Schematic representation of ubiquitin lysine mutants used for in vitro ubiquitination assay. (B) In vitro ubiquitination assay using ubiquitin lysine mutants to determine ubiquitination topology of mHtt in full-length Htt (i.e., fHtt-23Q and fHtt-120Q) expressed in stably transfected cell lines. Various lysine mutants of HAubiquitin were expressed in the fHtt-23Q and fHtt-120Q cell lines, and mHtt was immunoprecipitated and detected by anti-HA for ubiquitin (Ubi.; Upper) and anti-Htt (Lower). (C and D) In vitro ubiquitination assay of HEK293 cells to determine ubiquitination topology of N-terminal mHtt fragments (N67150Q, N212-120Q, and N508-1200Q) by using ubiquitin lysine mutant K63R (C) or K48R (D). All Western blots are representative of results from at least three independent experiments. C, Htt transfected cells without ubiquitin contransfection; NT, nontransfected cells.

Overexpressing Ube3a Promotes mHtt Degradation. Given that Ube3a interacts with mHtt and is involved in UPS-mediated degradation, we wanted to determine whether overexpression of Ube3a could promote Htt degradation and reduce its aggregation. To this end, we transfected HEK293 cells with one of the N-terminal mHtt fragments (N67-150Q, N212-120Q, and N508120Q) and Ube3a. With an increase in transfected Ube3a, shorter N-terminal mHtt appeared to degrade more quickly (Fig. 5 A and C). After inhibiting proteasomal activity with MG132, the mHtt protein levels were stabilized (Fig. 5 B and D). These results confirmed that Ube3a is involved in the degradation of mHtt via the UPS. We also transfected fHtt-23Q and fHtt-120Q

fragments. In this experiment, we immunoprecipitated transfected Htt and then examined the association of the endogenous Ube3a with transfected Htt. N508-23Q and N508-120Q bind to Ube3a to a similar extent; however, N67-150Q and N212-120Q bind more Ube3a than their WT Htt counterparts (Fig. 3 B and C). These differences suggest that polyQ expansion results in more binding of shorter N-terminal mHtt to Ube3a than larger Htt fragments, which may be the initial step for the UPS to degrade the shorter Htt fragments that are more prone to misfolding and aggregation than larger Htt fragments. Double immunofluorescent staining of transfected HEK293 cells also confirmed the colocalization of Ube3a with aggregated Htt (Fig. 3D). Age-Dependent Decrease in Ube3a in the Mouse Brain. Next we wanted to examine whether levels of Ube3a are altered in the presence of mHtt. By using Western blotting to analyze HD KI mice at different ages (5, 11, and 22 mo), we saw an age-related decrease in levels of Ube3a in HD KI mice (Fig. 4A). Brain tissues from HD KI mice at 22 mo of age appear to have a lower level of Ube3a than age-matched WT mice (Fig. 4B). Because Ube3a is known to be decreased in the primate brain cortex (45), we wondered whether the decreased Ube3a in old KI mouse brain was caused by aging. By examining WT mouse brain tissues, we also found that aging reduced the levels of Ube3a in the mouse striatum and cortex, and quantitative analysis of the ratio of Ube3a to tubulin on Western blots also verified the agedependent decrease in Ube3a (Fig. 4C). However, the expression of mHtt may further down-regulate the expression of Ube3a, as reported in R6/2 mice that overexpress N-terminal mHtt (46). By Bhat et al.

Fig. 3. Ube3a interacts with mHtt fragments in vivo and in vitro. (A) Coimmunoprecipitation assay by using Htt-specific antibody to immunoprecipitate Htt (Upper) from the striatum of 5-, 11-, and 22-mo-old HD KI mice and 22-mo-old WT mice to determine Ube3a (Lower) binding to Htt in vivo. Bracket indicates aggregated mHtt, and arrow indicates full-length mHtt. (B) Coimmunoprecipitation assay in HEK293 cells using anti-Htt to immunoprecipitate Ube3a and N-terminal Htt fragments (N67-23Q/150Q, N212-23Q/120Q, and N508-23Q/120Q; two samples for each Htt fragment). Inputs are shown (Top) along with the precipitated Ube3a (Middle) and Htt (Bottom). (C) Densitometric analysis of the amount of immunoprecipitated Ube3a (Ube3a/input) in each lane, which is normalized to the total amount of Ube3a in input. All Western blots are representative of results from at least three independent experiments, and data are mean ± SEM (D) Fluorescent immunocytochemistry showing the colocalization of Ube3a with mHtt aggregate (arrow) in fHtt-120Q cell lines. NT, nontransfected cells. (Scale bar: 10 μm.)

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using fractionation of mouse brain striatum, we did see a moderate decrease in the nuclear Ube3a in a 12-mo-old KI mouse compared with an age-matched WT mouse (Fig. S4). The age-dependent decrease in Ube3a could lead to an increase in K63-specific ubiquitination of mHtt and its aggregation, as mHtt fragments have a propensity to ubiquitinate via K63 of ubiquitin (Fig. 2). To test this idea, we immunoprecipitated Htt from the striatum tissues in KI mice at different ages. As expected, we saw more aggregated Htt in old KI mice (Fig. 4D). The Htt immunoprecipitates were then probed with the antibody to K63 of ubiquitin. The Htt immunoprecipitates from KI mice at 11 and 22 mo showed more abundant K63 immunoreactive signals than those from WT or KI mouse at 5 mo of age (Fig. 4D). This result indeed supports the idea that more K63 ubiquitinmediated Htt aggregates occur in older HD KI mouse brain.

gel, compared with the AAV-GFP control. At 22 mo, however, we observed a marked reduction of Htt aggregates after injection of AAV-Ube3a (Fig. 6B). Such a marked reduction was also evident in the input before Htt immunoprecipitation (Fig. 6B, input). To further verify the long-term effect of Ube3a overexpression, we also performed biochemical studies after stereotaxic injection of AAV-Ube3a for 1 mo and 3 mo. We found that the expression levels of AAV-Ube3a were similar at the 1-mo and 3-mo time points (Fig. 6C). However, with an increase in time after AAVUbe3a injection (from 1 mo to 3 mo), we saw a further reduction of Htt aggregates in the total tissue lysates (Fig. 6C). More importantly, we saw a concomitant reduction in K63 ubiquitination

Fig. 4. Age-dependent decrease in Ube3a in HD KI mice. (A) Western blot analysis of mouse brain cortex and striatum from 5-, 11-, and 22 mo-old HD KI mice and 22-mo-old WT mouse. Arrow indicates full-length mHtt. (B) The relative levels of Ube3a in HD KI mouse brains at different ages. (C) Western blotting of the brain cortex and striatum in normal mice at 6, 12, and 22 mo of age. Densitometric analysis of the relative levels of Ube3a (the ratio of Ube3a to tubulin) is shown beneath the blots (*P < 0.05). (D) Coimmunoprecipitation assay using anti-Htt to immunoprecipitate Htt (Left) from HD KI mouse brain striatum at 5, 11, and 22 mo of age to determine in vivo Htt-specific K63 ubiquitination (Right). All Western blots are representative of results from at least three independent experiments, and data are mean ± SEM. Bracket indicates aggregated mHtt (Left) and K63 ubiquitinated mHtt (Right).

stable cell lines with Ube3a siRNA and saw a robust stabilization of full-length mHtt and a high molecular weight form of mHtt compared with cells transfected with scrambled siRNA or untransfected cells (Fig. 5 E and F). Considering that Ube3a was reduced to approximately 50% of the endogenous level (Fig. 5E), the normal level of Ube3a appears to be critical for degrading mHtt, explaining how an age-dependent decrease in Ube3a could be involved in the age-related increase of misfolded and aggregated mHtt in HD mouse brains. To determine whether Ube3a overexpression can promote the degradation of mHtt in vivo, we generated an AAV9-myc-Ube3a viral vector and injected it into the striatum of young and old HD KI mice with AAV9-GFP as a control. Four to 12 wk after injection, the mice were killed, and their brain sections were stained to reveal mHtt, ubiquitin, and Ube3a. We saw robust expression of Ube3a and GFP via stereotaxic injection of the AAV viral vectors (Fig. S5A). Double immunofluorescent staining showed that Ube3a was colocalized with mHtt aggregates throughout the striatum of HD KI mouse brain (Fig. 6A). Furthermore, mouse brain sections from 24-mo-old mice injected with AAV-Ube3a showed a substantial decrease in mHtt aggregates compared with AAV-GFP–injected mouse brain sections (Fig. 6A, Right). To better quantify aggregated Htt and to test whether overexpressing Ube3a could inhibit Htt aggregation in KI mouse brains, we immunoprecipitated mHtt from the striatum in KI mice that were injected with AAV-GFP or AAV-Ube3a at different ages. At 10 mo, we saw no obvious effect of overexpressed Ube3a on Htt aggregates, which remained in the stacking 4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1402215111

Fig. 5. Overexpressing Ube3a reduces mHtt stability and t1/2, whereas knocking down Ube3a has the opposite effect. (A) Western blot analysis of the effect of increasing transfected Ube3a (0, 1, and 2 μg DNA) on the protein stability and ubiquitination levels of N-terminal mHtt fragments (N63-150Q, N212-120Q, and N508-150Q) in HEK293 cells. Because anti-Htt (mEM48) reacts more intensively with shorter mHtt fragments with longer polyQ domain, the individual panels containing N63-150Q, N212-120Q, or N508-150Q are different exposures of Western blots from the same experiment to clearly show Ube3a-mediated differences at their levels. (B) Western blot analysis of the effects of MG132 (10 μM) and Ube3a (2 μg) on the protein stability of N-terminal mHtt fragments (N63-150Q, N212-120Q, and N508-120Q) in HEK293 cells. In A and B, ratios of mHtt to tubulin are normalized by the control without Ube3a and are shown beneath the blot probed with anti-tubulin. (C and D) Densitometric analysis of the relative levels of mHtt (ratio of mHtt to tubulin) in A (C) and B (D). *P < 0.05 and **P < 0.01. (E) Knockdown assay to determine the effects of Ube3a siRNA on the protein stability of mHtt fragments in fHtt-23Q and fHtt-120Q cell lines. The blots were probed with 1C2 antibody that reacts with expanded polyQ repeats and mEM48 antibody for Htt. Arrow indicates a high molecular weight form of mHtt recognized by 1C2. (F) Densitometric analysis of the results in C quantifying the levels of full-length mHtt that are normalized to the corresponding tubulin levels. All Western blots are representative of results from at least three independent experiments, and data are presented as mean ± SEM (*P < 0.05). C, control siRNA transfected cells; NT, nontransfected cells; siRNA, Ube3a siRNA transfected cells.

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after a prolonged period of AAV-Ube3a overexpression (Fig. 6D). These results support our in vitro observations that Ube3a promotes mHtt degradation via the 26S proteasome by modulating mHtt ubiquitination topology and thus reducing its aggregation. Indeed, other studies have shown that preventing the aggregation of detergent-insoluble K63-ubiquitinated mHtt by HSJ1a chaperone is neuroprotective (47). Based on these results, we propose a model that explains the age-dependent accumulation and aggregation of mHtt in the brain. In this model, aging can reduce the expression level of Ube3a, thus causing less Htt to be degraded by the UPS and more Htt to be ubiquitinated via K63 of ubiquitin, subsequently increasing Htt aggregation (Fig. 7). Discussion Many neurodegenerative diseases, including Alzheimer’s disease and Parkinson disease, show the age-dependent accumulation of misfolded proteins, resulting in the formation of aggregates or inclusions in the brain. HD provides us with an ideal model to investigate how aging can cause the accumulation of misfolded proteins, as polyQ expansion in Htt alters Htt conformation and also leads to the age-dependent aggregation of Htt and neurodegeneration. In the present study, we have shown that the shorter N-terminal mHtt fragments have longer t1/2 values and a greater propensity to form aggregates. Consistently, the shorter Htt fragments have more of a propensity for polyubiquitination via K63 of ubiquitin. In addition, Ube3a tends to bind shorter mutant N-terminal Htt for degradation via the UPS. However, an age-dependent decrease in Ube3a appears to cause more mHtt to be ubiquitinated via K63 of ubiquitin for its aggregation. Our findings thus provide mechanistic insight into the age-related accumulation and aggregation of mHtt. Proteolysis of mHtt in the brain is known to be the initial step toward neuropathological cascades in HD (9). N-terminal Htt fragments generated by proteolysis are believed to become misfolded and targeted by the UPS (17). The UPS-mediated protein degradation process involves the ubiquitination of proteins Bhat et al.

Fig. 7. A proposed model for age-dependent mHtt toxicity. In young HD KI mice, Ube3a is abundant and ubiquitinates mHtt via lysine 48 of ubiquitin and targets it for proteasomal degradation. With aging, the level of Ube3a decreases, reducing K48-mediated mHtt ubiquitination as well as its targeting to the proteasome for degradation and promoting mHtt ubiquitination via K63. As a result, more K63 polyubiquitinated mHtt fragments accumulate and form aggregates.

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Fig. 6. Ube3a overexpression reduces mHtt aggregation and K63 ubiquitination in the striatum of HD KI mice. (A) Fluorescent immunohistochemistry staining of 24-mo-old HD KI mouse brain injected with AAV9-Myc-Ube3a. Three months after injection, mouse brain sections were stained with antiHtt (mEM48, red) and anti-myc for the injected viral Ube3a (green). Relative amounts of Htt aggregates quantified as fluorescent signals per image are shown (Right; n = 15 images). (B) Coimmunoprecipitation assay using antiHtt to immunoprecipitate Htt (Left) from the striatum from 10- and 22-mo-old HD KI mice that had been injected with AAV-GFP or AAV-Ube3a. Bracket indicates aggregated mHtt. (C and D) Western blot analysis of 10-mo-old HD KI mouse striatum injected with AAV-GFP or AAV-Ube3a for 1 or 3 mo to detect levels of mHtt aggregation (C) and K63 ubiquitination (D). Bracket indicates aggregated mHtt (B and C) and K63 specific ubiquitination (D). Arrow indicates full-length mHtt. (Scale bar: 20 μm.)

via various lysine residues in ubiquitin, a 76-aa protein that is highly conserved in various species. It is generally thought that K48-linked polyubiquitination targets its substrates to the proteasome for degradation, whereas K63-linked polyubiquitination is important for targeting substrates to different cellular locations (48, 49). Different functions of ubiquitinated proteins are also supported by the finding that ubiquitinated proteins via K-48– and K-63–mediated ubiquitination accumulate in the mitochondria but differentially regulate apoptosis (40). Most other lysine-mediated linkage types, apart from K63 linkages, are also thought to target proteins for proteasomal degradation (48). One of the interesting findings in the present study is that mutant N-terminal Htt is preferentially ubiquitinated via K63 of ubiquitin. Also, there is a correlation between the K63-linked polyubiquitination of mHtt and its aggregation. Our studies suggest that polyQ expansion may alter Nterminal Htt conformation, leading to preferential ubiquitination by K63 of ubiquitin, thereby promoting Htt aggregation. We also show that N-terminal mHtt is ubiquitinated via K48 of ubiquitin, leading to UPS-dependent degradation. The targeting of K48-ubiquitinated proteins to the UPS for degradation requires Ube3a, one of the important E3 ligases. The critical function of Ube3a is evidenced by the fact that loss of Ube3a causes Angelman syndrome, a neurodevelopmental disorder with severe learning and memory impairment (50, 51). Also, lack of Ube3a was found to accelerate the polyQ-induced Purkinje cell pathology in the SCA1-transgenic mice (52). Our finding that shorter N-terminal mHtt binds more Ube3a supports the idea that shorter N-terminal mHtt fragments are prone to misfolding and are therefore more likely to be targeted by the UPS. Given that the E3 ubiquitin ligase plays a critical role in substrate selectivity and exists with great diversity (53), the association we found between mHtt and Ube3a points to Ube3a as a potential therapeutic target for Htt degradation. Although Ube3a overexpression is known to promote UPSmediated degradation of transfected mHtt in cultured cells (54, 55), how Ube3a expression levels impact HD pathology remains unclear. In our studies, we provide in vivo evidence that Ube3a levels can reduce mHtt aggregation in the HD KI mouse brain. More importantly, we saw that there is a concomitant decrease in K63 ubiquitination in mHtt in the presence of overexpressed Ube3a. Our findings suggest that promoting the K48-Ube3a– mediated degradation of mHtt could reduce the accumulation and aggregation of mHtt via K63 of ubiquitin. Thus, the dynamic balance between differential ubiquitination of mHtt appears to be important for regulating Htt levels and influencing the related neuropathology in HD. The age-dependent accumulation of mHtt and its aggregation in the brain are well-documented phenomena; however, the mechanism behind this remains elusive. In our studies, we

discovered that levels of Ube3a decline with age in the mouse brain. This finding is consistent with an earlier report that Ube3a expression decreases in various cortex regions in human, monkey, and cats (45). Because aging decreases proteasome activity in the brain (17), it will be interesting to explore whether the general decline of the UPS with age results from the decreased levels of Ube3a in older neurons. A key implication of this agedependent change in Ube3a is that this decrease may also underlie the age-dependent accumulation of misfolded proteins in other neurodegenerative diseases. Thus, by investigating the agerelated accumulation of aggregated Htt, our results suggest that age-related Ube3a activity may underlie the late-onset accumulation of misfolded proteins in other neurodegenerative diseases. 1. Gusella JF, MacDonald ME, Ambrose CM, Duyao MP (1993) Molecular genetics of Huntington’s disease. Arch Neurol 50(11):1157–1163. 2. Snell RG, et al. 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Materials and Methods Animal procedures were approved by the institutional animal care and use committee of Emory University. Full-length mHtt CAG140Q (i.e., HD KI) mice were provided by Michael Levine (University of California, Los Angeles, CA) and were maintained at the Emory University Animal facility in accordance with the institutional animal care and use committee guidelines. Additional information is provided in SI Materials and Methods. ACKNOWLEDGMENTS. We thank Dr. Kah-Leong Lim (Department of Biological Sciences, National University of Singapore) for providing myc-tagged WT-ubiquitin, K48, K48R, K63, and K63R ubiquitin lysine mutant constructs; and Cheryl Strauss for critical reading of this manuscript. This work was supported by National Institutes of Health Grants AG19206(to X.J.L.), NS041449 (to X.J.L.), AG031153 (to S.H.L.), and NS0405016 (to S.H.L.).

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Bhat et al.

Differential ubiquitination and degradation of huntingtin fragments modulated by ubiquitin-protein ligase E3A.

Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin a...
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