UBE4B, a microRNA-9 target gene, promotes autophagy-mediated Tau degradation

Subramanian, Manivannan; Hyeon, Seung Jae; Das, Tanuza; Suh, Yoon Seok; Kim, Yun Kyung; Lee, Jeong-Soo; Song, Eun Joo; Ryu, Hoon; Yu, Kweon

doi:10.1038/s41467-021-23597-9

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Published: 02 June 2021

UBE4B, a microRNA-9 target gene, promotes autophagy-mediated Tau degradation

Nature Communications volume 12, Article number: 3291 (2021) Cite this article

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A Publisher Correction to this article was published on 07 July 2021

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Abstract

The formation of hyperphosphorylated intracellular Tau tangles in the brain is a hallmark of Alzheimer’s disease (AD). Tau hyperphosphorylation destabilizes microtubules, promoting neurodegeneration in AD patients. To identify suppressors of tau-mediated AD, we perform a screen using a microRNA (miR) library in Drosophila and identify the miR-9 family as suppressors of human tau overexpression phenotypes. CG11070, a miR-9a target gene, and its mammalian orthologue UBE4B, an E3/E4 ubiquitin ligase, alleviate eye neurodegeneration, synaptic bouton defects, and crawling phenotypes in Drosophila human tau overexpression models. Total and phosphorylated Tau levels also decrease upon CG11070 or UBE4B overexpression. In mammalian neuroblastoma cells, overexpression of UBE4B and STUB1, which encodes the E3 ligase CHIP, increases the ubiquitination and degradation of Tau. In the Tau-BiFC mouse model, UBE4B and STUB1 overexpression also increase oligomeric Tau degradation. Inhibitor assays of the autophagy and proteasome systems reveal that the autophagy-lysosome system is the major pathway for Tau degradation in this context. These results demonstrate that UBE4B, a miR-9 target gene, promotes autophagy-mediated Tau degradation together with STUB1, and is thus an innovative therapeutic approach for AD.

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Introduction

Alzheimer’s disease (AD) is one of the most common age-related neurodegenerative diseases, and includes the pathological hallmarks of extracellular amyloid plaques by abnormally folded amyloid-β 42 (Aβ-42) and intracellular neurofibrillary tangles (NFTs) by Tau hyperphosphorylation in the brain^1,2. Tau is an essential soluble intracellular protein that associates with and stabilizes axon microtubules³. Hyperphosphorylation disrupts the physiological function of Tau, resulting in microtubule destabilization and formation of intracellular NFTs, potentiating neurodegeneration and memory impairment in AD patients. Neurodegenerative disorders with Tau inclusions are referred to as tauopathies⁴.

Drosophila melanogaster has emerged as an important model system for investigating the pathology of tauopathies at the cellular and molecular levels. Drosophila tauopathy AD models exhibit visible phenotypes such as brain vacuole formation, neuromuscular junction defects, larval and adult locomotor defects, impairment of learning and memory, and reduced lifespan^5,6. Ectopic expression of human tau in Drosophila eyes induces a rough eye phenotype, which is widely used to screen for modifiers of Tau pathology⁷. Similarly, tissue-specific ectopic expression of human tau or mutant tau in the Drosophila brain or mushroom body induces neurodegeneration, manifesting in phenotypes such as locomotor and cognitive impairment⁸. The pathogenic mechanisms of Tau are similar in humans and Drosophila^9,10.

MicroRNAs (miRNAs) are non-coding RNAs comprised of 19 to 25 nucleotides, which suppress target mRNAs by pairing to complementary sequences in the 3’ UTRs of target genes, impairing translation and in some cases mRNA stability^11,12. miRNAs are implicated in diverse brain functions including development, cognition, and synaptic plasticity¹³. Dysregulation of miRNAs can be detrimental, and is associated with several human diseases ranging from metabolic and inflammatory disease to neoplasia^14,15,16. miRNA expression-profiling studies have identified that multiple miRNAs are dysregulated in the brains of AD patients, but the functional implications of these changes remain unclear¹⁷.

The ubiquitin-proteasome system (UPS) and autophagy-lysosome system (ALS) are the primary protein degradation pathways in eukaryotic cells¹⁸. Proteins that undergo proteasomal degradation are polyubiquitinated at Lys48 by ubiquitin ligases and targeted for 26S proteasome complex degradation and proteins poly-ubiquitinated at Lys63 are degraded by the ALS. Interestingly, proteostasis is disrupted in AD brains¹⁹. A prior study reported that Tau phosphorylation induces ubiquitination and subsequent degradation by the UPS²⁰. STUB1 (STIP1 homology and U-Box containing protein1) is an E3 ubiquitin ligase that ubiquitinates phosphorylated Tau for proteasomal degradation in vitro²¹ and has also been implicated in the clearance of truncated Tau by the ALS²².

In the present study, we report the results of a Drosophila miRNA library screening, which identified the evolutionarily conserved miR-9 and its target CG11070 as strong modifiers of the neurodegenerative Drosophila rough eye phenotype induced by human tau overexpression. Further, Drosophila CG11070 and its mammalian orthologue UBE4B (Ubiquitin conjugation E4 B) alleviated the neurodegenerative phenotypes induced by neuronal human tau overexpression in Drosophila. In addition, total and phosphorylated Tau in aged tau-overexpressing flies was decreased by CG11070 or mammalian UBE4B overexpression. In mammalian neuroblastoma cells and a mouse tau overexpression model, UBE4B, and STUB1 E3 ligases ubiquitinated Tau proteins and induced autophagy-mediated Tau degradation. These findings suggest that CG11070/UBE4B is a E4 ubiquitin ligase that regulates Tau degradation via the ALS, and is a putative therapeutic target for the treatment of tauopathies such as AD.

Results

Identification of miR-9 as a modifier of hTau in Drosophila by genome-wide miRNA screening

The overexpression of human tau (hTau) in Drosophila eyes using the eye-specific GMR-GAL4 promoter induces eye neurodegeneration, which is grossly characterized by decreased eye size in the rough eye phenotype⁷. This phenotype was used to screen 131 UAS-miRNA lines covering 144 Drosophila miRNAs²³. We analyzed the eye morphology of each line, quantitatively measured eye sizes, and arranged the library in the order of increasing eye size (Fig. 1a, Supplementary Fig. 1a, b). Eye-specific overexpression of many miRNAs in hTau flies (GMR > hTau) affected the eye size. Quantification of eye sizes was plotted in a volcano plot by the ratio of eye size to p-value of miRNA-overexpressing lines relative to the control GMR > hTau line (Fig. 1b). The most significant reductions in eye sizes were induced by the overexpression of miR-9a, miR-9b, and miR-9c (Fig. 1c, d), which are members of the evolutionarily conserved miR-9 family (Fig. 1e). The reduced eye size of miR-9a, miR-9b or miR-9c in the absence of hTau expression (Fig. 1c, d, and Supplementary Fig. 1) may be due to the involvement of these miRNAs during eye development where these miRNAs may regulate other target genes and affect morphology and development of eye sizes. However, when these miRNAs were expressed in the presence of hTau, the reduction of eye size was enhanced drastically. This indicated an important regulatory role for the miR-9 family in Drosophila Tau toxicity. We focused on miR-9a for subsequent studies, as miR-9a has 100% homology to mammalian miR-9 (Fig. 1e).

**Fig. 1: Genome-wide *Drosophila miRNA* library screening identified *miR-9* family miRNAs as modifiers of *hTau* in *Drosophila eyes*.**

Modulation of hTau by CG11070, a miR-9a target identified by secondary screening

Because miR-9a was identified as a modifier of Tau toxicity, we searched miR-9a target genes using three separate microRNA target prediction programs, TargetScan (www.targetscan.org), miRanda (www.microRNA.org), and miRbase (www.mirbase.org), detecting 34 miR-9a targets common to all three platforms (Supplementary Fig. 2a). We then screened the 34 putative target genes against the eye phenotype of hTau overexpression (GMR > hTau) using stably expressed RNAi knockdown in the GMR > hTau background, and arranged the RNAi lines in order of increasing eye size (Fig. 2a, Supplementary Fig. 2b, c). Volcano plot analysis was conducted by the ratio of eye size to p-value of the 34 RNAi lines relative to the control GMR > hTau flies. CG11070-RNAi flies exhibited the most prominent reduction in eye size (Fig. 2b, Supplementary Fig. 2c). The eye size of CG11070-RNAi flies in the GMR > hTau background (GMR > hTau ⁺ CG11070-RNAi) was significantly reduced relative to the eye size of control GMR > hTau flies (Fig. 2c, d).

**Fig. 2: *CG11070*, a *miR-9a* target identified from secondary screening, strongly modulated *hTau* in *Drosophila* eyes.**

Because microRNAs bind to 3′-UTR region of target mRNAs to suppress translation and/or mRNA stability, we assessed binding of miR-9a to CG11070 using a miRNA–mRNA pull-down assay²³. Similar to the known miR-9a/miR-9 targets senseless²⁴ and sNPFR1²³, we found that miR-9a bound to and enriched CG11070 mRNA in Drosophila S2 cells compared to its control scrambled miRNA (Fig. 2e). Consistently, eye-specific overexpression of miR-9a with GMR-GAL4 flies decreased CG11070 mRNA levels (Fig. 2f). Taken together, these findings suggested that CG11070 was a legitimate target of miR-9a.

The overexpression of Drosophila CG11070 and its mammalian orthologue UBE4B alleviated hTau phenotypes in Drosophila

CG11070 and UBE4B proteins had 26% amino acid identities and similar functional domains (Supplementary Fig. 3a). Interestingly, the 3′-UTR region of UBE4B mRNA also contained miR-9 binding sequences, similar to the 3′-UTR region of CG11070 mRNA (Supplementary Fig. 3b). In addition to miR-9a, UBE4B is also regulated by miR-26, miR-148/miR-152, and miR-15/16/195/424/497. However, CG11070 is regulated by miR-9a only in Drosophila. Therefore, CG11070/UBE4B was chosen for further analysis due to similarity in regulation by miR-9. Because the knockdown of CG11070 in GMR > hTau flies decreased eye size relative to control GMR > hTau flies, we determined if the overexpression of CG11070 and its mammalian orthologue UBE4B could alleviate hTau phenotypes in GMR > hTau flies.

In GMR > hTau flies, the overexpression of CG11070 (GMR > hTau + GC11070) and UBE4B (GMR > hTau + UBE4B) increased eye size, alleviating the rough eye neurodegenerative phenotype (Fig. 3a, b). Neuron-specific ectopic hTau expression (Elav > hTau) decreased Drosophila larva locomotion, which was alleviated by CG11070 and UBE4B overexpression (Fig. 3c, d). The bouton number of neuromuscular junctions (NMJ) in Drosophila larvae is directly correlated with locomotion, and is decreased in Drosophila AD models^25,26. Neuronal hTau-expressing larvae exhibited significantly reduced bouton numbers relative to the control, which was alleviated by the overexpression of CG11070 and UBE4B (Fig. 3e, f).

**Fig. 3: The overexpression of *Drosophila CG11070* and its mammalian orthologue *UBE4B* alleviated *hTau* phenotypes in *Drosophila*.**

The knockdown of miR-9a by miR-9a-sponge (SP) showed no change in the eye sizes when compared with GMR > hTau flies (Supplementary Fig. 4a, b). However, the knockdown by miR-9a-SP in neurons of Elav > hTau rescued the larval locomotion phenotype similar to the overexpression of CG11070 and hUBE4B (Supplementary Fig. 4c, d). Similarly, the knockdown by miR-9a-SP in neurons also rescued NMJ bouton numbers when compared with the overexpression of CG11070 and hUBE4B in neurons (Supplementary Fig. 4e, f). This data also confirmed that miR-9a and CG11070/UBE4B forms a common axis involved in regulating Tau toxicity in Drosophila.

Because the overexpression of CG11070 and UBE4B alleviated hTau phenotypes, we further examined whether the overexpression of these genes affected Tau degradation. We performed western blots on 30-day-old fly heads with eye-specific hTau expression (GMR > hTau), and identified that total hTau protein was significantly decreased by the overexpression of CG11070 (GMR > hTau + GC11070) and UBE4B (GMR > hTau + UBE4B) (Fig. 3g, h). In addition, phosphorylated Tau, detected by AT8 (p-S202/T205) antibody, was also significantly decreased by the overexpression of CG11070 and UBE4B (Fig. 3g, i). Similarly, other Tau phosphorylated forms, detected by AT180 (p-T231) and PHF-1 (p-S396/S404) antibodies, were also reduced in these genotypes (Supplementary Fig. 3c–e). Taken together, these findings suggested that the overexpression of either Drosophila CG11070 or human UBE4B rescued both larval locomotion/NMJ defects and adult eye phenotypes in hTau-overexpressing Drosophila by increasing Tau degradation.

Ubiquitination and degradation of Tau by UBE4B and STUB1 in mammalian neuroblastoma cells

To investigate the mechanism by which UBE4B degraded Tau, we first determined if UBE4B affected Tau ubiquitination in SH-SY5Y neuroblastoma cells (Fig. 4a). Previous studies have demonstrated that UBE4B has E4 ubiquitin ligase activity, and that Tau is ubiquitinated by STUB1 E3 ligase^21,27,28. STUB1 was chosen due to its role in ubiquitination of Tau proteins and is not regulated by miR-9. To confirm the effect of STUB1 on Tau ubiquitination, we evaluated ubiquitination of Tau by STUB1, and found that STUB1 overexpression alone did not affect Tau ubiquitination (Fig. 4a, lane 3). Similarly, UBE4B overexpression alone did not significantly affect Tau ubiquitination (Fig. 4a, lane 4). However, UBE4B and STUB1 co-expression significantly increased Tau ubiquitination (Fig. 4a, lane 5 compared with lane 3 and 4). Subsequently, we determined that STUB1 activity was important for UBE4B-mediated Tau ubiquitination, as co-expression with a dominant-negative mutant (STUB1^H260Q) failed to enhance Tau ubiquitination (Fig. 4a, lane 6). These results suggested that UBE4B and STUB1 co-regulated Tau ubiquitination.

**Fig. 4: Tau was ubiquitinated and degraded by UBE4B and STUB1 in mammalian neuroblastoma cells.**

Due to the essential regulatory roles of these ubiquitin ligases in protein degradation, we examined UBE4B and STUB1-mediated Tau degradation. By inhibiting protein synthesis with cycloheximide (CHX), we observed that Tau degradation was increased by STUB1 (Fig. 4b, lanes f–i, Fig. 4c) and UBE4B overexpression (Fig. 4b, lanes 1–4 and Fig. 4c), and was further increased by co-expression of STUB1 and UBE4B (Fig. 4b, lanes 6–9 relative to lanes 1–4, Fig. 4c). Because UBE4B overexpression alone degraded Tau (Fig. 4b, c), we knocked down endogenous STUB1 with siRNA (Supplementary Fig. 5a) and examined UBE4B-mediated Tau degradation (Supplementary Fig. 5b, c). Interestingly, UBE4B overexpression did not affect Tau degradation when STUB1 was knocked down (Supplementary Fig. 5b, lanes 6–9 compared with lanes 1–4). Similarly, the knockdown of STUB1 showed no change in Tau degradation when compared with the siControl (Supplementary Fig. 5c). Collectively, these results indicated that UBE4B was a critical factor that enhanced the ubiquitination activity of STUB1 to ubiquitinate and degrade Tau. To evaluate the biochemical interactions of UBE4B with STUB1 and Tau, we performed immunoprecipitation analyses. When Tau was co-expressed with UBE4B and STUB1, UBE4B co-precipitated with STUB1 (Fig. 4d). However, UBE4B did not directly interact with STUB1, as UBE4B did not co-precipitate with STUB1 in the absence of Tau (Fig. 4d). Previous studies demonstrated that STUB1 directly interacts with and ubiquitinates Tau, targeting it for degradation^21,27,28. UBE4B also directly interacted with Tau protein (Fig. 4e). These results suggested that UBE4B did not directly interact with STUB1, but rather that Tau mediated the interaction between STUB1 and UBE4B.

STUB1 knockdown reduces UBE4B-mediated alleviated hTau phenotypes in Drosophila

Since the knockdown of STUB1 in neuroblastoma cell lines showed no change in Tau levels in the presence of UBE4B (Supplementary Fig. 5b, c), we further examined the importance of STUB1 in the in vivo Drosophila model system. Studies from the in vivo model system showed that the knockdown of STUB1 gene in the eyes of flies expressing GMR > hTau + hUBE4B, significantly reduced eye phenotype when compared with GMR > hTau + hUBE4B flies (Fig. 5a, b). Similarly, the knockdown of STUB1 in neurons expressing Elav > hTau + hUBE4B also significantly reduced larval locomotion phenotype when compared with Elav > hTau + hUBE4B in neurons (Fig. 5c, d). However, NMJ phenotype showed no change in larvae expressing Elav > hTau + hUBE4B + STUB1-RNAi when compared with Elav > hTau + hUBE4B larvae (Fig. 5e, f). These studies indicate that alleviated hTau phenotypes by hUBE4B overexpressing are dependent on STUB1 function.

Degradation of Tau by UBE4B and STUB1 in the Tau-BiFC mouse model

To determine whether in vivo overexpression of UBE4B and STUB1 affects Tau degradation, we generated AAV-CMV-UBE4B and AAV-CMV-STUB1 constructs (Fig. 6a). AAVs were delivered to the dentate gyri of Tau-BiFC mice by stereotaxic injection (Fig. 6b, c) and pathological examinations were performed as shown in the work flow (Fig. 6d). In the Tau-BiFC mouse model system, Tau oligomerization can be visualized by BiFC fluorescence²⁹. Consistent with the in vitro data, the overexpression of either UBE4B or STUB1 induced degradation of Tau oligomer, as indicated by decreased Tau fluorescence relative to control (Fig. 6e). Similarly, co-overexpression of UBE4B and STUB1 in Tau-BiFC mice further decreased Tau fluorescence (Fig. 6e, f). Similar to reduction of oligomeric Tau levels, phosphorylated Tau levels in the dentate gyrus of Tau-BiFC mice, as detected by the AT8 (p-S202/T205) antibody, were also decreased by the overexpression of either UBE4B or STUB1, and were further decreased by co-expression of UBE4B and STUB1 (Fig. 6e and g). Additional phosphorylated forms of Tau were detected by AT180 (p-T231) and PHF-1 (p-S396/S404) antibodies. Both p-T231 and p-S396/S404 Tau levels were also decreased by the overexpression of either UBE4B or STUB1 alone, and were further decreased by co-expression of UBE4B and STUB1 (Supplementary Fig. 6a–c). These results demonstrated that UBE4B and STUB1 additively degraded Tau in vivo.

**Fig. 6: Tau oligomers were degraded by UBE4B and STUB1 in the Tau-BiFC mouse model.**

UBE4B and STUB1-mediated autophagy Tau degradation

The two reported major pathways of Tau degradation are the UPS and ALS^30,31. To determine the primary pathway of UBE4B and STUB1-mediated Tau degradation, we co-overexpressed UBE4B and STUB1 with Tau in neuroblastoma cells, and treated the cells with either the UPS inhibitor MG132 or the ALS inhibitors. Interestingly, MG132 treatment did not inhibit Tau degradation relative to the control cells (Fig. 7a). In contrast, Chloroquine significantly inhibited Tau degradation (Fig. 7b). In addition, pepstatin A (PEPA) alone and E64D plus PEPA (E64D + PEPA), autophagy inhibitors, significantly inhibited Tau degradation (Fig. 7c), suggesting that ALS was the major pathway of UBE4B and STUB1-mediated Tau degradation in neuroblastoma cells. Our in vitro study showed that the turnover of monomeric Tau molecule by UBE4B/STUB1 is preferentially mediated by autophagy-dependent manner rather than the proteasome pathway in SH-SY5Y cells (Fig. 7a–c).

**Fig. 7: Tau was degraded by UBE4B and STUB1 primarily via autophagy.**

Because autophagy inhibitor treatment blocked autophagic Tau degradation in vitro, we tested this in the in vivo system by injecting autophagy inhibitors into the dentate gyri of Tau-BiFC mice with co-expression of UBE4B and STUB1 (Fig. 7d, e). We measured oligomeric Tau and phosphorylated Tau levels in the dentate gyrus of Tau-BiFC mice detected with the AT8 antibody (S202/T205) (Fig. 7f–h). Inhibition of ALS by Chloroquine and E64D + PEPA significantly increased oligomeric and phosphorylated Tau (S202/T205) (Fig. 7f–h). Similarly, the phosphorylated forms of Tau p-S396/S404 (PHF-1) and p-T231 (AT180) were also significantly increased in Chloroquine and E64D + PEPA-treated Tau-BiFC mice (Supplementary Fig. 7a–d). Since ALS disruption changes LC3 and p62/SQSTM1 levels, which are inversely correlated with the efficiency of autophagy³², we measured LC3 and p62 levels in our model system. Co-expression of UBE4B and STUB1 significantly decreased LC3 and p62 levels relative to control in the dentate gyri of Tau-BiFC mice, whereas autophagy inhibitors elevated LC3 and p62 levels relative to the control in the dentate gyri of UBE4B + STUB1-expressed Tau-BiFC mice (Fig. 7i, j). Furthermore, autophagy inhibitors modulated BECN1 levels compared to the control in the dentate gyri of UBE4B + STUB1-expressed Tau-BiFC mice (Supplementary Fig. 8). Collectively, these results suggested that monomeric and oligomeric Tau degradations by STUB1 and UBE4B are mediated through autophagy pathway rather than the ubiquitin-proteasome system.

Discussion

In the present study, we demonstrated that Drosophila miR-9a regulated Tau toxicity and increased Tau phosphorylation in the eyes. MiR-9a-mediated Tau toxicity arose through disruption of the miR-9a target gene CG11070, the human orthologue of UBE4B, which when overexpressed mimicked the miR-9a knockdown phenotype. The overexpression of either Drosophila CG11070 or human UBE4B alleviated Tau-associated phenotypes, including increased eye size, improved larval crawling/NMJ phenotypes, and decreased total and phosphorylated Tau levels. Further, we demonstrated that clearance of Tau proteins by UBE4B occurred primarily through ubiquitin-dependent ALS rather than the proteasome system.

Previous studies have demonstrated that miRNAs mediate neurodegeneration, including that of AD^33,34. AD-related miRNAs regulate multiple stages of AD pathology and enhance Tau toxicity^35,36. Studies in AD cadaver samples demonstrated that miR-125b is increased in AD, and the overexpression of miR-125b in mice increases Tau hyperphosphorylation by regulating kinases and phosphatases³⁷. Also, downregulation of miR-132/212 promotes Tau phosphorylation, which in turn enhances AD phenotypes³⁸. In addition to these miRNAs, miR-138 regulates Tau phosphorylation through GSK3β³⁹ and miR-922 affects Tau phosphorylation through UCHL1⁴⁰. Functional analysis of miR-9 has revealed its involvement in the regulation of neuronal progenitor cells and differentiation of neuronal cells during development⁴¹. In AD patients, miR-9 expression is elevated⁴². In Drosophila, miR-9a plays an important role in the development of sensory organs by suppressing its target gene senseless²⁴ and also regulates body growth through sNPFR signaling²³. In the Drosophila Tau AD model, we observed increased Tau toxicity when miR-9a was overexpressed. The Drosophila miR-9a binding sequence was 100% identical to that of mammalian miR-9 (Fig.1e). Our studies clearly demonstrated that miR-9a regulated Tau toxicity, exacerbating the rough eye phenotype and significantly decreasing eye size.

miR-9a targets a multitude of genes that are involved in diverse cellular processes, and we used in silico analysis to identify 34 putative targets, which were predicted as miR-9a target genes using three separate in silico platforms. We then performed a secondary screening of the putative 34 miR-9a target genes to identify a distinct gene, CG11070, as a strong modifier of hTau (Fig. 2). Mammalian orthologues of Drosophila CG11070 include UBE4A and UBE4B. UBE4B, but not UBE4A, contained miR-9 binding sequences in its 3′ UTR like those of CG11070 (Supplementary Fig. 3b), and is extensively expressed in the brain⁴³. We therefore considered UBE4B as the mammalian orthologue of Drosophila CG11070 in the context of miR-9a regulation of Tau toxicity. The overexpression of UBE4B or CG11070 in Drosophila not only alleviated hTau eye neurodegenerative phenotypes, larval crawling defects, and synaptic dysfunction in NMJs (Fig. 3a, f), but also decreased total and phosphorylated Tau levels (Fig. 3g–i, Supplementary Fig. 3c–e). These results indicated that Tau degradation was strongly regulated by UBE4B/CG11070. Since Tau is involved in maintaining the stability of microtubules in neurons, the degradation of Tau proteins affects microtubule stability as seen in tauopathy and Alzheimer’s disease models. However, recent studies have shown that reduction in Tau proteins leads to increased accumulation of MAP6 proteins on the microtubules and enhances the stability of microtubules in neurons⁴⁴. The rescue phenotype seen in UBE4B/CG1170 may be a result of compromised function of Tau and MAP6 proteins in the neurons. Reduction in Tau proteins leads to reduced pTau levels in UBE4B/CG11070 rescue animals, due to increased ubiquitination of Tau proteins. Studies have shown that the acetylation of Tau inhibits degradation of phosphorylated Tau and contributes to increased tauopathy⁴⁵. Both ubiquitination and acetylation shares common Lys residue Lys280⁴⁶ which is involved in stability of Tau proteins. The UBE4B proteins are distinct from E3 ligases in their U-Box domains, which possess both E3 ligase and E4 ligase activities⁴⁷. UBE4B ligase ubiquitinates the tumor suppressor genes p53, p63 and p73, and Wallerian degeneration proteins in conjunction with various E3 ligases⁴⁸. In the Drosophila spinocerebellar ataxia type 3 model, UBE4B ubiquitinates ataxin, targeting it for proteasomal degradation. However, the dominant-negative UBE4B mutation enhances neurodegeneration⁴⁹. These previous studies suggest that UBE4B potentiates the degradation of various proteins through the UPS, unlike our current findings, which suggest synergistic regulation of Tau degradation through the ALS in conjunction with STUB1.

Prior reports have demonstrated that Tau is ubiquitinated by two E3 ubiquitin ligases, STUB1^21,27,28 and TRAF6⁵⁰, both of which are colocalized with NFTs in AD brains. In the present study, we found that Tau was ubiquitinated by the E3 ligase STUB1 in the presence of UBE4B, a further ubiquitin conjugation factor (Fig. 4a). In addition, Tau degradation was increased by UBE4B expression, and further increased by STUB1 and UBE4B co-expression (Fig. 4b, c). Studies on Tau degradation have shown that the proteasomal pathway plays a crucial role. Inhibition of proteasome in HEK cells increases the levels of full-length Tau proteins⁵⁰. Similar results were seen in SH-SY5Y cells with increased accumulation of both full-length Tau protein and mutant P301L Tau protein levels when proteasome degradation is inhibited^51,52. However, from our studies, STUB1- and UBE4B-mediated Tau degradation was inhibited by the autophagy inhibitors such as Chloroquine, PEPA, and E64D (Fig. 7b, c) but not by the proteasomal inhibitor MG132 (Fig. 7a), suggesting that STUB1/UBE4B-mediated Tau degradation was facilitated by autophagy rather than the UPS. In FTDP-17 mutant P301S mice, trehalose treatment promotes autophagy with reduced insoluble and pTau proteins⁵³. Similarly, the knockout of Atg7 (autophagy marker) in neurons give rise to increased pTau levels and neurodegeneration with aging⁵⁴. A number of autophagy inhibitors, including chloroquine, NH₄Cl, 3-methyl adenine (3-MA), and cathepsin inhibitors, delay Tau degradation and enhance the formation of high molecular weight Tau aggregates^55,56. Contrastingly, the autophagy inducer rapamycin facilitates insoluble Tau degradation, alleviating Tau toxicity in Drosophila³⁰. Trehalose, an mTOR-independent autophagy activator³¹, improves neuronal survival by decreasing Tau aggregation in tauopathy mouse model^57,58. This suggests that basal autophagy activity is essential for prevention of neuronal Tau aggregate accumulation, which is regulated in part by UBE4B activation. Considering autophagy inhibitors not only modulate neuronal autophagy pathway but also affect neuroinflammation pathway, it remains to be determined whether other neuroinflammatory pathways are involved in Tau pathology beyond the autophagy pathway in future studies.

Lys63-linked polyubiquitination of Tau facilitates the formation of disease-associated Tau inclusions, which are preferentially cleared by the ALS⁵⁹, while Lys48-linked polyubiquitinated Tau is likely degraded by the UPS⁶⁰. Lys63 ubiquitinated substrates are recognized by autophagy receptors present on autophagosomes, mediating ALS-dependent degradation^61,62,63. Recent studies have shown that the knockdown of UBE4B affects Lys48 and Lys63 polyubiquitination in Tax binding proteins and Tax mediated activation of NF-κB⁶⁴. Therefore, in the context of the present study, UBE4B acted synergistically with STUB1 to facilitate Tau degradation by ALS, and could polyubiquitinate Tau proteins by Lys63 ubiquitin linkage. The specific ubiquitin chain linkage for Tau ubiquitination by STUB1/UBE4B is a potential target for lysosomal targeting of Tau. However, the mechanism underlying ALS-mediated Tau degradation remains to be elucidated in future studies.

Tau oligomers play a central role in tauopathies. To understand the importance of Tau oligomers in vivo, Bimolecular Fluorescence Complementation (BiFC) technology was applied to visualize Tau oligomerization⁶⁵. In this system, full-length human Tau protein is fused to the non-fluorescent N- and C-terminal termini of Venus fluorescent protein. In the transgenic TauP301L-BiFC mouse model, Venus fluorescence is activated only when Tau is aggregated²⁹. In our studies using the TauP301L-BiFC mouse model, the overexpression of UBE4B, STUB1, or both significantly reduced the fluorescence generated by Tau oligomers (Fig. 6). Similarly, Tau phosphorylation was also decreased in the dentate gyri of TauP301L-BiFC mice with the overexpression of UBE4B, STUB1, or both. These results indicate that Tau oligomers, a primary cause of tauopathy, can be degraded by UBE4B and STUB1 in vivo. In TauP301L-BiFC mice, treatment with the autophagy inhibitors (Chloroquine, PEPA, and E64D) increased oligomeric and phosphorylated Tau levels (Fig. 7b, c, f–h, Supplementary Fig. 7). Notably, while the overexpression of UBE4B and STUB1 decreased protein levels of LC3, p62, and BECN1, autophagy markers, autophagy inhibitors significantly elevated LC3, p62, and BECN1 levels in the dentate gyri of AAV-STUB1/UBE4B injected TauP301L-BiFC mice (Fig. 7i–l and Supplementary Fig. 8). Collectively, these results further suggested that UBE4B promoted oligomeric Tau clearance via ALS through a STUB1-dependent mechanism.

In the present study, we identified that Drosophila CG11070 and its mammalian orthologue UBE4B, which are targets of miR-9a/miR-9, rescued human Tau phenotypes in flies by decreasing total and phosphorylated Tau levels. In mammalian neuroblastoma cells, UBE4B-mediated Tau clearance was accelerated by co-expression of STUB1, which encodes an ubiquitin E3 ligase for Tau. In the dentate gyri of Tau BiFC mice, the overexpression of UBE4B and STUB1 also decreased oligomeric Tau and phosphorylated Tau levels. These Tau degradations occurred primarily via ALS in mammalian in vitro and in vivo systems. These results demonstrated that UBE4B promoted autophagy-mediated Tau degradation synergistically with STUB1, providing an innovative therapeutic approach for AD.

Methods

Drosophila culture and stocks

Drosophila melanogaster were maintained at 25 ^oC on standard cornmeal, yeast, sugar, and agar medium. UAS-hTau, GMR-GAL4, and ElavGAL4 fly lines were obtained from Bloomington Stock Centre (Bloomington, USA). miR-9a target RNAi stocks were obtained from Bloomington Stock Centre (Bloomington, USA) and Vienna Drosophila Research Centre (Vienna, Austria). pUAS-CG11070 and pUAS-UBE4B flies were generated by the p-element-mediated germline transformation method with cDNA containing the coding regions of CG11070 or UBE4B.

Cell culture and transfections

SH-SY5y neuroblastoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (10 U/mL) and streptomycin (100 μg/mL). Cells were incubated at 37 °C in 5% CO₂ and transfected with desired plasmids using Effectene transfection reagent (Qiagen) following the manufacturer’s instructions. SiRNAs were transfected with lipofectamine 2000 transfection reagent.

Mouse model and virus injection

Male TauP301L-BiFC mice were a kind gift of Dr. Yunkyung Kim (KIST, KOR)²⁹. Brain specimens of TauP301L-BiFC mice were prepared as previously described⁶⁶. AAV-CMV-UBE4B and AAV-CMV-STUB1 viruses were injected using a stereotaxic micro-injector (Stoelting Co.). Control groups were injected with AAV-CMV. Neuropathological experiments were performed at 2 weeks after injection. Mice were housed on a 12:12 h light-dark cycle and maintained at 18–23 °C with humidity between 40 and 60% in pathogen-free facilities at Korea Institute of Science and Technology. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the Korea Institute of Science and Technology. All animal experiments were approved by the Korea Institute of Science and Technology Animal Care Committee.

Quantification of eye phenotypes in Drosophila screens

GMR > hTau flies were crossed with either UAS-miRNAs²³ or miR-9a target UAS-RNAi flies, and the progeny were scored for Tau toxicity in the eyes. Eye images were captured using a Digiretina 16 camera, and eye size was measured using Image J v1.44 software (National Institutes of Health, Bethesda, USA). Values obtained from these measurements were plotted using a volcano plot in Graphpad Prism 9.1.0.

Larval crawling assay

Wandering third instar larvae were briefly washed with PBS to remove residual food. Larvae were dried for a short time on clean filter paper and placed on a 2% agar grape juice-coated petri dish. Each genotype was allowed to crawl freely for 90 s. To quantify crawling distance, lines were drawn to track the crawling larvae, and total distance was measured using Image J v1.44 software. Approximately 10–20 animals were tested for each genotype^25,26.

Immunohistochemistry of neuromuscular junction

Third instar larvae were dissected in PBS, fixed in 4% formaldehyde in PBS for 15 min, and washed three times in 0.1% Triton X-100 in PBS. FITC-conjugated anti-HRP was used at 1:100 and incubated for 1.5 h at room temperature. Larvae were mounted in Slow Fade Antifade media. Confocal images were captured using Zeiss confocal microscopes. Quantification of the NMJ was performed by counting the number of boutons in each genotype using Image J v1.44 software with cell counter plugin^25,26.

Quantitative PCR

The heads of 20 adult Drosophila per group were collected and total RNA was isolated with Trizol reagent. After treating the RNA samples with RNase-free DNase I, cDNA was synthesized using the SuperScript III First-Strand Synthesis System (TAKARA, Japan). Quantitative reverse transcription–PCR (qRT–PCR) analysis was performed using a StepOnePlus Sequence Detection System (BioRAD, USA) with SYBR Green PCR Core reagents (BioRAD). Each experiment was performed at least in triplicate. The comparative cycle threshold was utilized to quantify the fold change of each specific mRNA after normalizing to rp49 levels.

miRNA–mRNA pull-down assay

The miRNA–mRNA pull-down assay was performed²³ with minor modifications. Briefly, cells were harvested 24 h after transfection and lysed in lysis buffer (Cell Signaling, USA) containing 20× protease inhibitor (Roche) and 60 U RNaseOUT (Invitrogen). Protein A Dynabeads (Invitrogen, USA) and 2 µg AGO-1-specific antibody were used for immunoprecipitation. The immunoprecipitate was treated with 20 µg/ml proteinase K for 10 min at 37 °C. RNA was extracted using the easy-BLUE kit (iNTRON, Korea), and cDNAs were synthesized with the SuperScript III First-Strand Synthesis System (Invitrogen). To determine if miR-9a directly bound CG11070, primers that amplified the fragments of its 3′-UTR that included the predicted miR-9a seed sequence matches were designed. Senseless and sNPFR1 were used as positive controls and tubulin was used as a negative control (Supplementary Table 1).

Western blot

Briefly, 20 fly heads for each genotype were homogenized in RIPA buffer, and lysates were loaded in each lane of 10% SDS gels and transferred to nitrocellulose membrane. Membranes were blocked in 5% BSA and incubated with primary antibodies at 4 °C overnight. After washing membranes with TBS-T, membranes were incubated with the appropriate secondary antibody. Using the ECL Western blotting detection reagent, membranes were developed and images were captured using FluorChem E image processor. Antibodies used were anti-Tau (1:1000, T46, Cat no. 13-6400, Invitrogen), anti-AT180 (1:1000, Cat no. MN1040, Invitrogen), anti-PHF-1 (1:1000, Cat no. MN1050, Invitrogen), anti-AT8 (1:1000, Cat no. MN1020, Invitrogen) and anti-β-actin (1:1000, Cat no. JLA20, DHSB). β-actin was used as a loading control. Signal intensity was quantified using ImageJ (NIH) software. Flies used were 30 days old after eclosion. For Western blot analysis of SH-SY5y cells, the following antibodies were used: anti-Tau (Cat no. ab64193, Abcam), anti-β-actin (Cat no. LF-PA0207, AB Frontier), anti-Myc (Cat no. C3956, Sigma), anti-HA (Cat no. H6908, Sigma), and anti-CHIP (Cat no. sc-133066, Santa Cruz Biotechnology).

Immunoprecipitation

Cells were transfected with the indicated plasmids. After 24 h of transfection, cells were harvested and lysed with IP buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 5 mM KCl, 0.1% Tween-20, 2 mM DTT, and protease inhibitor cocktail (Roche)). Lysates were centrifuged at 9700 × g for 30 min at 4 °C, and the collected supernatants were incubated with anti-HA agarose beads (Sigma) at 4 °C for 4 h. The beads were then washed with buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 5 mM KCl, 0.1% Tween-20, and 2 mM DTT, and the bound proteins were eluted with 2× SDS sample buffer. Samples were quantified by Western blot after heating at 95 °C for 10 min.

His-ubiquitin pull-down assay

PCS2-His-ubiquitin was co-transfected with the indicated plasmids and after 24 h of transfection, transfected cells were treated with 10 µM MG132 for an additional 6 h. Cells were lysed in urea lysis buffer (8 M Urea, 0.3 M NaCl, 0.5 M Na₂HPO₄, 0.05 M Tris, 0.001 M PMSF, 0.01 M imidazole, pH 8.0) and sonicated for 4 min. Cell lysates were transferred to equilibrated Ni-NTA agarose and incubated for 4 h at room temperature. Beads were then washed five times with urea wash buffer (8 M Urea, 0.3 M NaCl, 0.5 M Na₂HPO₄, 0.05 M Tris, 0.001 M PMSF, 0.02 M imidazole, pH 6.5) and conjugated proteins were eluted with 40 µL 2X Laemmli/Imidazole (200 mM imidazole). Eluted proteins were analyzed by Western blotting after heating the samples at 95 °C for 10 min.

Protein stability analysis

SH-SY5y cells were transfected with the indicated plasmids or siRNAs, and treated with 100 μg/mL cyclohexamide (CHX) or vehicle after 24 h of transfection. Cells were collected at the specified time points after CHX treatment and immunoblotted with antibodies against the specified proteins. To evaluate autophagic degradation, Tau was co-transfected with STUB1 and UBE4B for 24 h, and cells were treated with 50 µM Chloroquine or vehicle for 8 h. Cells were collected and lysed, and lysates were immunoblotted with antibodies against the specified proteins.

In vivo confocal microscopy and image analyses

Immunofluorescence staining for anti-Tau 5 (1:200, ab3931, Abcam), anti-pTau AT8 (1:200, Cat no. MN1020, Invitrogen), anti-pTau AT180 (1:200, Cat no. MN1040, Invitrogen), anti-pTau PHF-1 (1:200, Cat no. MN1050, Invitrogen), anti-LC3 (1:200, Cat no. M152-3, MBL), anti-P62 (1:200, Cat no. PM045, MBL) and anti-Beclin (1:200, Cat no. PD017, MBL) was performed in Tau-BiFC mice models²⁹. Fluorescence was observed by confocal microscopy (Nikon A1R, JAPAN). Pre-absorption with excess target protein or omission of primary antibody was used to demonstrate antibody specificity and remove background generated by the detection assay. Co-localization and quantitative assessment of images were conducted using NIH Image J v1.44 software.

Statistics and reproducibility

All experiments were performed more than three times. In case of animal study, ‘N’ represents the number of biologically independent animals. Boxplots were generated using the standard style except that the whiskers represent minimum to maximum. In bar charts, unless otherwise noted, data are presented as mean ± SEM, and comparisons between groups were conducted using the Student’s t-test considering p < 0.05 to be statistically significant. For multiple comparisons, we performed a one-way analysis of variance followed by pairwise t-tests using the Bonferroni method to adjust the p-value threshold for significance.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Change history

07 July 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-24572-0

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Acknowledgements

Drosophila stocks were obtained from Bloomington Sock Center (Bloomington, IN, USA) and Vienna Drosophila RNAi Center (VDRC, Vienna, Austria). This work was supported by grants from KRIBB Research Initiative Program, National Research Council of Science & Technology (CRC-15-04-KIST), KIST (2E30951, 2E30954 and 2E30962), and National Research Foundation of Korea (2015R1A5A1009024, 2017HID3A1A02054608, 2018M3C7A1056894, 2019R1A2C 2004052, 2019R1A2C2004149, 2020R1A4A4079494, and 2020M3E5D9079742).

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These authors contributed equally: Manivannan Subramanian, Seung Jae Hyeon.

Authors and Affiliations

Metabolism and Neurophysiology Research Group, KRIBB, Daejeon, Korea
Manivannan Subramanian, Yoon Seok Suh, Jeong-Soo Lee & Kweon Yu
Convergence Research Center of Dementia, KIST, Seoul, Korea
Manivannan Subramanian, Yun Kyung Kim, Jeong-Soo Lee & Kweon Yu
Center for Neuroscience, Brain Science Institute, KIST, Seoul, Korea
Seung Jae Hyeon & Hoon Ryu
Biomedical Research Institute, KIST, Seoul, Korea
Tanuza Das
Graduate School of Pharmaceutical Sciences and College of Pharmacy, Ewha Womans University, Seoul, Korea
Eun Joo Song
Department of Functional Genomics, UST, Daejeon, Korea
Kweon Yu

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Manivannan Subramanian
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Contributions

M.S., E.J.S., H.R., and K.Y designed the research, M.S., S.J.H., T.D., and Y.S.S. performed experiments, M.S., Y.K.K., J.L., E.J.S., H.R., and K.Y. analyzed the data, and M.S., E.J.S., H.R., and K.Y. wrote the manuscript.

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Subramanian, M., Hyeon, S.J., Das, T. et al. UBE4B, a microRNA-9 target gene, promotes autophagy-mediated Tau degradation. Nat Commun 12, 3291 (2021). https://doi.org/10.1038/s41467-021-23597-9

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Received: 03 August 2020
Accepted: 29 April 2021
Published: 02 June 2021
DOI: https://doi.org/10.1038/s41467-021-23597-9

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