Mol. Cells 2022; 45(11): 855-867
Published online September 28, 2022
https://doi.org/10.14348/molcells.2022.0104
© The Korean Society for Molecular and Cellular Biology
Correspondence to : cchung42@jhmi.edu(CGC); jeonghyang.park@mitoimmune.com(JHP); sblee@dgist.ac.kr(SBL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.
Keywords acetylation, ATXN3, calcium, CBP, Importin α, nucleocytoplasmic transport
Proper subcellular localization of proteins is essential for their functioning and/or interactions with binding partners, of which dysregulation often leads to diverse cellular defects (Bauer et al., 2015). In line with this, disruption of nucleocytoplasmic transport (NCT) of proteins is known to cause diverse neurodegenerative diseases (NDs), such as amyotrophic lateral sclerosis (ALS) and polyglutamine (polyQ) diseases, through aberrant accumulation of toxic disease-related proteins (e.g., TDP-43 in ALS and polyQ proteins in polyQ diseases) in afflicted neurons (Chung et al., 2018). However, our understanding on the molecular and cellular basis underlying NCT dysregulation in these NDs remains incomplete. Recently, we showed that NCT of TDP-43 is controlled by the intracellular calcium-Calpain-A (CalpA)-Importin α3 (Imp α3) signaling pathway in
As one of ND-related proteins known to mislocalize in the disease context, ataxin-3 (ATXN3), also known as SCA3, is a deubiquitinating enzyme that normally localizes in the cytosol (Nijman et al., 2005). Upon genetic mutation of CAG repeats encoding polyQ repeats, mutated ATXN3 proteins aberrantly translocate into the nucleus, thereby inducing nuclear proteotoxicity in neurons (Bichelmeier et al., 2007; Evert et al., 1999; Lee et al., 2020; 2011). Mutated ATXN3 proteins are known to undergo proteolytic cleavage into smaller toxic fragments having higher toxicity (Breuer et al., 2010; Haacke et al., 2006; Simoes et al., 2022). Generally, previous studies have focused only on the proteotoxicity of mutated ATXN3 proteins in neurons with using ATXN3 proteins containing normal length of polyQ repeats as a control (Peng et al., 2022; Sowa et al., 2022). Given that the unmutated ATXN3 proteins are known to occasionally translocated into the nucleus (Fujigasaki et al., 2000; Perez et al., 1999; Reina et al., 2010; Tait et al., 1998), the proper localization of unmutated ATXN3 can be dynamically regulated in a cellular context-dependent manner, as was shown for TDP-43 proteins (Park et al., 2020a). It is reported that unmutated ATXN3 proteins can also undergo proteolytic cleavage (Simoes et al., 2022). Notably, we observed that unmutated ATXN3 proteins can confer aberrant protein toxicity, when truncated, leading to defective neuronal remodeling during metamorphosis of
All flies were maintained at 25°C and 60% humidity. The following lines were obtained from Bloomington
Immunohistochemistry was performed as previously described (Chung et al., 2017; Kim et al., 2021; Kwon et al., 2018; Park et al., 2020b). larvae (120 h after egg laying [120 h AEL]), pupae (18 h after puparium formation [18 h APF]), and adult flies were dissected in Schneider’s Insect Medium (Cat. No. S0146; Sigma, USA) to obtain fillet or brain samples for immunohistochemical analyses. Samples were fixed in 4% Paraformaldehyde for 20 min, washed in 0.3% PBST (Triton-X100 0.3% in phosphate-buffered saline [PBS]) for 10 min (repeated three times), and blocked in blocking buffer (5% Normal donkey serum or normal goat serum in 0.3% PBST) for 45 min at room temperature. The samples were incubated at 4°C with primary antibody. The following primary antibodies were used in this study: mouse anti-Flag (1E6, 1:400 dilution; Wako, Japan) for detecting the Flag epitope; rat anti-HA (3F10, 1:200 dilution; Roche, Switzerland) for detecting the HA epitope; and goat anti-HRP Alexa Fluor 488 (1:400 dilution; Jackson Immunoresearch Laboratories, USA) and anti-HRP Alexa Fluor 647 (1:400 dilution; Jackson Immunoresearch Laboratories) for detecting neuronal plasma membrane. To detect primary antibodies, the following secondary antibodies were used: goat anti-mouse Alexa Fluor 594 (1:200 dilution; Invitrogen, USA) and goat anti-mouse Alexa Fluor 488 (1:400 dilution; Invitrogen).
All images were acquired using LSM 700, 800 confocal microscope (Zeiss, Germany) and Zen software (Zeiss). All images of samples used after immunohistochemistry experiments or immediately were taken at 200× and 400× magnifications using 20× and 40× objective lens, respectively. Images of the
To perform quantification of dendrite remodeling defects in C4da neurons, we used a method as previously described (Wolterhoff et al., 2020), we determines the number of neurons that still have dendrites attached to soma to reflect the phenotypic penetrance, and these data were analyzed using a two-tailed Fisher’s exact test.
The lifespan assay was performed as previously described (Han et al., 2020). Adult flies were collected under CO2 anesthesia and housed at a density of 20 flies per vial. Flies were passed every other day, and the number of dead flies was recorded. At least 80 flies were used for each experimental group tested.
The cytoplasm-to-nucleus (Cyt/Nuc) ratios of immunostained proteins (HA-ATXN3tr-27Q, HA-ATXN3tr-78Q, 2xFlag-Imp α3, 2xFlag-Imp α3.K17R) was analyzed using a method previously described (Park et al., 2020a), the mean pixel intensities of them in the nucleus and the cytosol were measured using ImageJ (National Institutes of Health, USA). Statistical analysis was performed using Prism 8 (GraphPad Software, USA), with Student’s unpaired
We first explored whether additional disease-related proteins other than TDP-43 undergo context-dependent nucleocytoplasmic translocation similar to that shown in TDP-43 (Park et al., 2020a) in
Given that ATXN3tr-78Q conferring strong neuronal toxicity predominantly accumulates in the nucleus of neurons (Kwon et al., 2018; Lee et al., 2011; Park et al., 2020a), we suspected that nuclear accumulated ATXN3tr-27Q may also induce ectopic neuronal toxicity. Supporting this possibility, a previous study reported that overexpression of ATXN3tr-27Q in C4da neurons induced defects in neuronal remodeling during pupal stage when ATXN3tr-27Q translocates into the nucleus (Chung et al., 2017). To confirm this, we examined dendrite phenotypes of C4da neurons expressing ATXN3tr-27Q during larval and pupal stages. We confirmed that overexpression of ATXN3tr-27Q induced obvious defects in dendrite pruning at 18 h after pupal formation (18 h APF) (Figs. 1C and 1D), while overexpression of ATXN3tr-27Q did not induced any noticeable changes in dendrite arborization at the larval stage (Figs. 1C and 1D) as previously reported (Chung et al., 2017). Then, we also examined dendrite phenotypes of C4da neurons expressing ATXN3tr-27Q at adult stage. Because overexpression of ATXN3tr-27Q during pupal stage induced obvious defects in neuronal remodeling, we used inducible expression system (Supplementary Fig. S2A) to check the effect of nuclear translocation of ATXN3tr-27Q only during adult stage on dendrites with excluding the preceding effects during pupal stage. We confirmed that inducibly expressed ATXN3tr-27Q also translocated into the nucleus of C4da neurons in adult flies along aging (Supplementary Fig. S2B), similar to constantly expressed ATXN3tr-27Q through development (Fig. 1A). We found that ATXN3tr-27Q can induce obvious changes in dendrite morphology of C4da neurons in aged adult (45 days after hatching from pupa) upon nuclear accumulation, compared to the control (Supplementary Fig. S2C). These results indicate that ATXN3tr-27Q can induce ectopic neuronal toxicity in C4da neurons upon nuclear accumulation.
Next, we asked whether the observed dynamic NCT of ATXN3tr-27Q in C4da neurons also occurs in other neuronal cell types in
In a previous study, we showed that intracellular calcium level is a critical regulator of NCT of TDP-43 in C4da neurons (Park et al., 2020a). Based on this, we explored whether the level of intracellular calcium is also crucial for NCT of ATXN3tr-27Q in C4da neurons. To this end, we genetically manipulated intracellular calcium level in C4da neurons expressing ATXN3tr-27Q and then examined subcellular localization of ATXN3tr-27Q as well as neuronal phenotypes. We used RNAi of ryanodine receptor (RyR), one of major calcium-releasing channel in the endoplasmic reticulum (ER), to lower intracellular calcium level of C4da neurons. Knockdown (KD) of
Next, we examined whether NCT of ATXN3tr-27Q involves Imp α3 that has been identified as a key karyopherin mediating nuclear translocation of TDP-43 (Park et al., 2020a). To this end, we knocked down Imp α3 in C4da neurons expressing ATXN3tr-27Q and examined subcellular localization of ATXN3tr-27Q during pupal stage. KD of
Given that ATXN3tr-27Q induces ectopic neuronal toxicity upon nuclear accumulation (Fig. 1), we then investigated whether inhibition of ATXN3tr-27Q nuclear translocation during pupal stage can protect neurons from pruning defects. Inhibition of ATXN3tr-27Q nuclear translocation by KD of either
Given the involvement of Imp α3 in NCT of ATXN3tr-27Q, we speculated that overexpression of Imp α3 may facilitate nuclear translocation of ATXN3tr-27Q during larval stage, as shown for TDP-43 in a previous study (Park et al., 2020a). Interestingly, we found that overexpression of Imp α3 did not induce nuclear translocation of ATXN3tr-27Q during larval stage different from TDP-43 (Figs. 3A and 3B), implicating that NCT of ATXN3tr-27Q may involve certain mediators between intracellular calcium and Imp α3 that are different from those involved in NCT of TDP-43. Thus, we screened available calcium-dependent regulators using RNAi lines, as previously described (Park et al., 2020a), to identify specific mediators of NCT of ATXN3tr-27Q (Fig. 3C). We expressed RNAi line of calcium-dependent regulators in C4da neurons expressing ATXN3tr-27Q and examined whether nuclear accumulation of ATXN3tr-27Q is affected by KD of these regulators during pupal stage. For NCT of TDP-43, a previous study identified CalpA as one of critical mediators between intracellular calcium and Imp α3 (Park et al., 2020a). Notably, our genetic screening identified that CBP is primarily involved in NCT of ATXN3tr-27Q (Fig. 3D). Then, we examined whether genetic manipulation of CBP can alter neuronal phenotypes caused by nuclear accumulation of ATXN3tr-27Q. KD of
CBP is known as a HAT (histone acetyltransferase) that regulates expression profiles of genome through post-translational modification of histones (Bannister and Kouzarides, 1996). Notably, it is also known that CBP can acetylate target proteins other than histones, such as a subset of Importins (Importin α7 and Rch1) (Bannister et al., 2000) and HNF4 (hepatocyte nuclear factor 4) (Soutoglou et al., 2000). To understand how CBP mediates calcium-dependent NCT of ATXN3tr-27Q in C4da neurons, we first asked whether CBP-dependent acetylation of histones is involved in this process. To this end, we knocked down several histone deacetylases (HDACs) that counteract function of CBP on histone modification (Gaub et al., 2010; Saha and Pahan, 2006) and examined subcellular localization of ATXN3tr-27Q at the larval stage. We found that KD of
Then, to determine whether CBP has a regulatory function on Imp α3, we examined the effect of
Notably, a previous study reported that CBP acetylates a subset of Imp α in cultured mammalian cells (Bannister et al., 2000). Although a previous study reported that CBP does not acetylate the mammalian homolog of
Next, we decided to verify that CBP-mediated acetylation of Imp α3 described above (Fig. 4) is involved in the calcium-dependent NCT of ATXN3tr-27Q in C4da neurons. To this end, we first overexpressed either CBP or CBP.F2161A in C4da neurons expressing ATXN3tr-27Q and examined whether enzymatic activity of CBP is important for the subcellular localization of ATXN3tr-27Q. Overexpression of CBP.F2161A acting as a dominant-negative form indeed prevented nuclear translocation of ATXN3tr-27Q during pupal stage, while overexpression of CBP induced nuclear translocation of ATXN3tr-27Q (Figs. 5A and 5B). Then, we explored whether the status of CBP-mediated acetylation in Imp α3 is associated with NCT of ATXN3tr-27Q by comparing the effects of overexpressed Imp α3 with overexpressed Imp α3.K17R on subcellular localization of ATXN3tr-27Q in C4da neurons. We found that overexpression of Imp α3.K17R prevented nuclear translocation of ATXN3tr-27Q during pupal stage (Figs. 5C and 5D), consistent with the above results using overexpression of CBP.F2161A (Figs. 5A and 5B). Taken together, these results demonstrate that CBP-mediated acetylation of
In this study, we demonstrate that CBP-mediated acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3tr-27Q in
To figure out the target-selective nature of calcium-dependent regulatory mechanisms underlying NCT of these disease-related proteins, we need to also understand how CBP-dependent acetylation affects molecular features of Imp α. IBB domain containing the known acetylation site of Imp α is required for the interaction with Imp β (Bannister et al., 2000). According to a previous study, the acetylation of Imp α is known to increase the binding affinity to Imp β. From these previous results, we speculate that the enhanced formation of heterodimer between Imp α and Imp β may be responsible for target selectivity distinguishing between TDP-43 and ATXN3. Future studies on the molecular details underlying the target selectivity of CBP-dependent acetylation of Imp α are highly demanded.
In this study, we demonstrate that nuclear accumulation of ATXN3tr-27Q can induce ectopic neuronal toxicity (e.g., aberrant neuronal remodeling during metamorphosis and morphological changes of dendrites at the adult stage) in
We showed here that reducing intracellular calcium concentration can prevent ectopic neuronal toxicity induced by nuclear accumulation of ATXN3tr-27Q. In our study, we primarily used the genetic manipulation of intracellular calcium level (Figs. 2A and 2B), but we also showed that the use of BAPTA can lead to the effects in the regulation of NCT of ATXN3tr-27Q (Supplementary Fig. S2C) comparable to the effects of genetic manipulation. This implies that pharmacochemical approach to prevent ectopic neuronal toxicity induced by mislocalized proteins under the control of intracellular calcium level may be feasible. For the functional homeostasis of neurons, there are distinct mechanisms regulating intracellular calcium levels that involve concerted actions of plasma membrane calcium channels and intracellular calcium reservoirs such as ER and mitochondria (Bagur and Hajnoczky, 2017; Bootman and Bultynck, 2020). Given that ectopic neuronal toxicity associated with calcium-dependent protein mislocalization can be a contributing factor for either the neuronal disorders or aging-dependent neuronal impairments in humans, diverse chemicals regulating intracellular calcium level through different mode-of-actions need to be tested for their effectiveness and side effects. For example, there exist mitochondria-specific chemicals (MIT-001/NecroX-7 and NecroX-5) that show a strong regulatory effect on the intracellular calcium level without inducing serious side effects (Hwang et al., 2018; Park et al., 2017; Thu et al., 2012), which can be good candidates for the potential future application to humans. We believe that our study will pave a new avenue toward understanding the exact nature of calcium-dependent NCT of proteins in neurons and be useful to develop an effective therapeutic intervention targeting neuronal toxicity associated with dysregulated NCT of proteins.
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (2022R1A4A2000703 and 2021R1A2C1003817) and the Korea Brain Research Institute (KBRI) Research Program (22-BR-03-02), funded by the Ministry of Science and ICT, Republic of Korea, and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) and Korea Dementia Research Center (KDRC), funded by the Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea (HU21C0027).
J.H.C., E.S.K., and S.B.L. wrote the manuscript. J.H.C., M.G.J., and N.Y.L. performed experiments. J.H.C., M.G.J., and N.Y.L. analyzed the data. E.S.K., J.H.P., C.G.C., S.H.K., and S.B.L. provided expertise and feedback. S.B.L. supervised the research.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2022; 45(11): 855-867
Published online November 30, 2022 https://doi.org/10.14348/molcells.2022.0104
Copyright © The Korean Society for Molecular and Cellular Biology.
Jae Ho Cho1,4 , Min Gu Jo1,4
, Eun Seon Kim1
, Na Yoon Lee1
, Soon Ha Kim2
, Chang Geon Chung3,*
, Jeong Hyang Park2,*
, and Sung Bae Lee1,*
1Department of Brain Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea, 2MitoImmune Therapeutics Inc., Seoul 06123, Korea, 3Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, 4These authors contributed equally to this work.
Correspondence to:cchung42@jhmi.edu(CGC); jeonghyang.park@mitoimmune.com(JHP); sblee@dgist.ac.kr(SBL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, in the current study, we investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, we observed dynamic changes in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. We further showed that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons.
Keywords: acetylation, ATXN3, calcium, CBP, Importin &alpha,, nucleocytoplasmic transport
Proper subcellular localization of proteins is essential for their functioning and/or interactions with binding partners, of which dysregulation often leads to diverse cellular defects (Bauer et al., 2015). In line with this, disruption of nucleocytoplasmic transport (NCT) of proteins is known to cause diverse neurodegenerative diseases (NDs), such as amyotrophic lateral sclerosis (ALS) and polyglutamine (polyQ) diseases, through aberrant accumulation of toxic disease-related proteins (e.g., TDP-43 in ALS and polyQ proteins in polyQ diseases) in afflicted neurons (Chung et al., 2018). However, our understanding on the molecular and cellular basis underlying NCT dysregulation in these NDs remains incomplete. Recently, we showed that NCT of TDP-43 is controlled by the intracellular calcium-Calpain-A (CalpA)-Importin α3 (Imp α3) signaling pathway in
As one of ND-related proteins known to mislocalize in the disease context, ataxin-3 (ATXN3), also known as SCA3, is a deubiquitinating enzyme that normally localizes in the cytosol (Nijman et al., 2005). Upon genetic mutation of CAG repeats encoding polyQ repeats, mutated ATXN3 proteins aberrantly translocate into the nucleus, thereby inducing nuclear proteotoxicity in neurons (Bichelmeier et al., 2007; Evert et al., 1999; Lee et al., 2020; 2011). Mutated ATXN3 proteins are known to undergo proteolytic cleavage into smaller toxic fragments having higher toxicity (Breuer et al., 2010; Haacke et al., 2006; Simoes et al., 2022). Generally, previous studies have focused only on the proteotoxicity of mutated ATXN3 proteins in neurons with using ATXN3 proteins containing normal length of polyQ repeats as a control (Peng et al., 2022; Sowa et al., 2022). Given that the unmutated ATXN3 proteins are known to occasionally translocated into the nucleus (Fujigasaki et al., 2000; Perez et al., 1999; Reina et al., 2010; Tait et al., 1998), the proper localization of unmutated ATXN3 can be dynamically regulated in a cellular context-dependent manner, as was shown for TDP-43 proteins (Park et al., 2020a). It is reported that unmutated ATXN3 proteins can also undergo proteolytic cleavage (Simoes et al., 2022). Notably, we observed that unmutated ATXN3 proteins can confer aberrant protein toxicity, when truncated, leading to defective neuronal remodeling during metamorphosis of
All flies were maintained at 25°C and 60% humidity. The following lines were obtained from Bloomington
Immunohistochemistry was performed as previously described (Chung et al., 2017; Kim et al., 2021; Kwon et al., 2018; Park et al., 2020b). larvae (120 h after egg laying [120 h AEL]), pupae (18 h after puparium formation [18 h APF]), and adult flies were dissected in Schneider’s Insect Medium (Cat. No. S0146; Sigma, USA) to obtain fillet or brain samples for immunohistochemical analyses. Samples were fixed in 4% Paraformaldehyde for 20 min, washed in 0.3% PBST (Triton-X100 0.3% in phosphate-buffered saline [PBS]) for 10 min (repeated three times), and blocked in blocking buffer (5% Normal donkey serum or normal goat serum in 0.3% PBST) for 45 min at room temperature. The samples were incubated at 4°C with primary antibody. The following primary antibodies were used in this study: mouse anti-Flag (1E6, 1:400 dilution; Wako, Japan) for detecting the Flag epitope; rat anti-HA (3F10, 1:200 dilution; Roche, Switzerland) for detecting the HA epitope; and goat anti-HRP Alexa Fluor 488 (1:400 dilution; Jackson Immunoresearch Laboratories, USA) and anti-HRP Alexa Fluor 647 (1:400 dilution; Jackson Immunoresearch Laboratories) for detecting neuronal plasma membrane. To detect primary antibodies, the following secondary antibodies were used: goat anti-mouse Alexa Fluor 594 (1:200 dilution; Invitrogen, USA) and goat anti-mouse Alexa Fluor 488 (1:400 dilution; Invitrogen).
All images were acquired using LSM 700, 800 confocal microscope (Zeiss, Germany) and Zen software (Zeiss). All images of samples used after immunohistochemistry experiments or immediately were taken at 200× and 400× magnifications using 20× and 40× objective lens, respectively. Images of the
To perform quantification of dendrite remodeling defects in C4da neurons, we used a method as previously described (Wolterhoff et al., 2020), we determines the number of neurons that still have dendrites attached to soma to reflect the phenotypic penetrance, and these data were analyzed using a two-tailed Fisher’s exact test.
The lifespan assay was performed as previously described (Han et al., 2020). Adult flies were collected under CO2 anesthesia and housed at a density of 20 flies per vial. Flies were passed every other day, and the number of dead flies was recorded. At least 80 flies were used for each experimental group tested.
The cytoplasm-to-nucleus (Cyt/Nuc) ratios of immunostained proteins (HA-ATXN3tr-27Q, HA-ATXN3tr-78Q, 2xFlag-Imp α3, 2xFlag-Imp α3.K17R) was analyzed using a method previously described (Park et al., 2020a), the mean pixel intensities of them in the nucleus and the cytosol were measured using ImageJ (National Institutes of Health, USA). Statistical analysis was performed using Prism 8 (GraphPad Software, USA), with Student’s unpaired
We first explored whether additional disease-related proteins other than TDP-43 undergo context-dependent nucleocytoplasmic translocation similar to that shown in TDP-43 (Park et al., 2020a) in
Given that ATXN3tr-78Q conferring strong neuronal toxicity predominantly accumulates in the nucleus of neurons (Kwon et al., 2018; Lee et al., 2011; Park et al., 2020a), we suspected that nuclear accumulated ATXN3tr-27Q may also induce ectopic neuronal toxicity. Supporting this possibility, a previous study reported that overexpression of ATXN3tr-27Q in C4da neurons induced defects in neuronal remodeling during pupal stage when ATXN3tr-27Q translocates into the nucleus (Chung et al., 2017). To confirm this, we examined dendrite phenotypes of C4da neurons expressing ATXN3tr-27Q during larval and pupal stages. We confirmed that overexpression of ATXN3tr-27Q induced obvious defects in dendrite pruning at 18 h after pupal formation (18 h APF) (Figs. 1C and 1D), while overexpression of ATXN3tr-27Q did not induced any noticeable changes in dendrite arborization at the larval stage (Figs. 1C and 1D) as previously reported (Chung et al., 2017). Then, we also examined dendrite phenotypes of C4da neurons expressing ATXN3tr-27Q at adult stage. Because overexpression of ATXN3tr-27Q during pupal stage induced obvious defects in neuronal remodeling, we used inducible expression system (Supplementary Fig. S2A) to check the effect of nuclear translocation of ATXN3tr-27Q only during adult stage on dendrites with excluding the preceding effects during pupal stage. We confirmed that inducibly expressed ATXN3tr-27Q also translocated into the nucleus of C4da neurons in adult flies along aging (Supplementary Fig. S2B), similar to constantly expressed ATXN3tr-27Q through development (Fig. 1A). We found that ATXN3tr-27Q can induce obvious changes in dendrite morphology of C4da neurons in aged adult (45 days after hatching from pupa) upon nuclear accumulation, compared to the control (Supplementary Fig. S2C). These results indicate that ATXN3tr-27Q can induce ectopic neuronal toxicity in C4da neurons upon nuclear accumulation.
Next, we asked whether the observed dynamic NCT of ATXN3tr-27Q in C4da neurons also occurs in other neuronal cell types in
In a previous study, we showed that intracellular calcium level is a critical regulator of NCT of TDP-43 in C4da neurons (Park et al., 2020a). Based on this, we explored whether the level of intracellular calcium is also crucial for NCT of ATXN3tr-27Q in C4da neurons. To this end, we genetically manipulated intracellular calcium level in C4da neurons expressing ATXN3tr-27Q and then examined subcellular localization of ATXN3tr-27Q as well as neuronal phenotypes. We used RNAi of ryanodine receptor (RyR), one of major calcium-releasing channel in the endoplasmic reticulum (ER), to lower intracellular calcium level of C4da neurons. Knockdown (KD) of
Next, we examined whether NCT of ATXN3tr-27Q involves Imp α3 that has been identified as a key karyopherin mediating nuclear translocation of TDP-43 (Park et al., 2020a). To this end, we knocked down Imp α3 in C4da neurons expressing ATXN3tr-27Q and examined subcellular localization of ATXN3tr-27Q during pupal stage. KD of
Given that ATXN3tr-27Q induces ectopic neuronal toxicity upon nuclear accumulation (Fig. 1), we then investigated whether inhibition of ATXN3tr-27Q nuclear translocation during pupal stage can protect neurons from pruning defects. Inhibition of ATXN3tr-27Q nuclear translocation by KD of either
Given the involvement of Imp α3 in NCT of ATXN3tr-27Q, we speculated that overexpression of Imp α3 may facilitate nuclear translocation of ATXN3tr-27Q during larval stage, as shown for TDP-43 in a previous study (Park et al., 2020a). Interestingly, we found that overexpression of Imp α3 did not induce nuclear translocation of ATXN3tr-27Q during larval stage different from TDP-43 (Figs. 3A and 3B), implicating that NCT of ATXN3tr-27Q may involve certain mediators between intracellular calcium and Imp α3 that are different from those involved in NCT of TDP-43. Thus, we screened available calcium-dependent regulators using RNAi lines, as previously described (Park et al., 2020a), to identify specific mediators of NCT of ATXN3tr-27Q (Fig. 3C). We expressed RNAi line of calcium-dependent regulators in C4da neurons expressing ATXN3tr-27Q and examined whether nuclear accumulation of ATXN3tr-27Q is affected by KD of these regulators during pupal stage. For NCT of TDP-43, a previous study identified CalpA as one of critical mediators between intracellular calcium and Imp α3 (Park et al., 2020a). Notably, our genetic screening identified that CBP is primarily involved in NCT of ATXN3tr-27Q (Fig. 3D). Then, we examined whether genetic manipulation of CBP can alter neuronal phenotypes caused by nuclear accumulation of ATXN3tr-27Q. KD of
CBP is known as a HAT (histone acetyltransferase) that regulates expression profiles of genome through post-translational modification of histones (Bannister and Kouzarides, 1996). Notably, it is also known that CBP can acetylate target proteins other than histones, such as a subset of Importins (Importin α7 and Rch1) (Bannister et al., 2000) and HNF4 (hepatocyte nuclear factor 4) (Soutoglou et al., 2000). To understand how CBP mediates calcium-dependent NCT of ATXN3tr-27Q in C4da neurons, we first asked whether CBP-dependent acetylation of histones is involved in this process. To this end, we knocked down several histone deacetylases (HDACs) that counteract function of CBP on histone modification (Gaub et al., 2010; Saha and Pahan, 2006) and examined subcellular localization of ATXN3tr-27Q at the larval stage. We found that KD of
Then, to determine whether CBP has a regulatory function on Imp α3, we examined the effect of
Notably, a previous study reported that CBP acetylates a subset of Imp α in cultured mammalian cells (Bannister et al., 2000). Although a previous study reported that CBP does not acetylate the mammalian homolog of
Next, we decided to verify that CBP-mediated acetylation of Imp α3 described above (Fig. 4) is involved in the calcium-dependent NCT of ATXN3tr-27Q in C4da neurons. To this end, we first overexpressed either CBP or CBP.F2161A in C4da neurons expressing ATXN3tr-27Q and examined whether enzymatic activity of CBP is important for the subcellular localization of ATXN3tr-27Q. Overexpression of CBP.F2161A acting as a dominant-negative form indeed prevented nuclear translocation of ATXN3tr-27Q during pupal stage, while overexpression of CBP induced nuclear translocation of ATXN3tr-27Q (Figs. 5A and 5B). Then, we explored whether the status of CBP-mediated acetylation in Imp α3 is associated with NCT of ATXN3tr-27Q by comparing the effects of overexpressed Imp α3 with overexpressed Imp α3.K17R on subcellular localization of ATXN3tr-27Q in C4da neurons. We found that overexpression of Imp α3.K17R prevented nuclear translocation of ATXN3tr-27Q during pupal stage (Figs. 5C and 5D), consistent with the above results using overexpression of CBP.F2161A (Figs. 5A and 5B). Taken together, these results demonstrate that CBP-mediated acetylation of
In this study, we demonstrate that CBP-mediated acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3tr-27Q in
To figure out the target-selective nature of calcium-dependent regulatory mechanisms underlying NCT of these disease-related proteins, we need to also understand how CBP-dependent acetylation affects molecular features of Imp α. IBB domain containing the known acetylation site of Imp α is required for the interaction with Imp β (Bannister et al., 2000). According to a previous study, the acetylation of Imp α is known to increase the binding affinity to Imp β. From these previous results, we speculate that the enhanced formation of heterodimer between Imp α and Imp β may be responsible for target selectivity distinguishing between TDP-43 and ATXN3. Future studies on the molecular details underlying the target selectivity of CBP-dependent acetylation of Imp α are highly demanded.
In this study, we demonstrate that nuclear accumulation of ATXN3tr-27Q can induce ectopic neuronal toxicity (e.g., aberrant neuronal remodeling during metamorphosis and morphological changes of dendrites at the adult stage) in
We showed here that reducing intracellular calcium concentration can prevent ectopic neuronal toxicity induced by nuclear accumulation of ATXN3tr-27Q. In our study, we primarily used the genetic manipulation of intracellular calcium level (Figs. 2A and 2B), but we also showed that the use of BAPTA can lead to the effects in the regulation of NCT of ATXN3tr-27Q (Supplementary Fig. S2C) comparable to the effects of genetic manipulation. This implies that pharmacochemical approach to prevent ectopic neuronal toxicity induced by mislocalized proteins under the control of intracellular calcium level may be feasible. For the functional homeostasis of neurons, there are distinct mechanisms regulating intracellular calcium levels that involve concerted actions of plasma membrane calcium channels and intracellular calcium reservoirs such as ER and mitochondria (Bagur and Hajnoczky, 2017; Bootman and Bultynck, 2020). Given that ectopic neuronal toxicity associated with calcium-dependent protein mislocalization can be a contributing factor for either the neuronal disorders or aging-dependent neuronal impairments in humans, diverse chemicals regulating intracellular calcium level through different mode-of-actions need to be tested for their effectiveness and side effects. For example, there exist mitochondria-specific chemicals (MIT-001/NecroX-7 and NecroX-5) that show a strong regulatory effect on the intracellular calcium level without inducing serious side effects (Hwang et al., 2018; Park et al., 2017; Thu et al., 2012), which can be good candidates for the potential future application to humans. We believe that our study will pave a new avenue toward understanding the exact nature of calcium-dependent NCT of proteins in neurons and be useful to develop an effective therapeutic intervention targeting neuronal toxicity associated with dysregulated NCT of proteins.
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (2022R1A4A2000703 and 2021R1A2C1003817) and the Korea Brain Research Institute (KBRI) Research Program (22-BR-03-02), funded by the Ministry of Science and ICT, Republic of Korea, and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) and Korea Dementia Research Center (KDRC), funded by the Ministry of Health & Welfare and Ministry of Science and ICT, Republic of Korea (HU21C0027).
J.H.C., E.S.K., and S.B.L. wrote the manuscript. J.H.C., M.G.J., and N.Y.L. performed experiments. J.H.C., M.G.J., and N.Y.L. analyzed the data. E.S.K., J.H.P., C.G.C., S.H.K., and S.B.L. provided expertise and feedback. S.B.L. supervised the research.
The authors have no potential conflicts of interest to disclose.
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